Pluripotent Stem Cell Protocol Handbook€¦ · stem cell research continues to grow rapidly. This growth has been a catalyst for many new researchers starting to work with stem cells

Pluripotent Stem Cell Protocol Handbook

ContentsIntroduction 4

1 Reprogramming of human fibroblasts 7

2 Selection of iPSC clones for feeder-dependent culture 21

3 Preparation of feeder layer 25

4 Feeder-dependent culture of human induced pluripotent stem cells (hiPSCs) 29

5 Transition of mature iPSCs to feeder-free culture system 39

6 Culture, expansion, and cryopreservation of pluripotent stem cells in feeder-free culture system 45

7 Ordering information 57

4 5

This handbook is a collection of protocols to support

pluripotent stem cell research and will guide you through the

following workflow:

Application Product

Reprogramming of human adult fibroblasts CytoTune-iPS 2.0 Sendai Reprogramming Kit

Selection of iPSC clones to be propagated in feeder-dependent cultures

Stem cell antibody kits for live-cell imaging

Preparation of feeder layer Mouse (ICR) Inactivated Embryonic Fibroblasts

Feeder-dependent culture of human iPSCs KnockOut Serum Replacement – Multi-Species

Transition of mature iPSCs to feeder-free culture system Essential 8 Adaptation Kit

Culture, expansion, and cryopreservation of pluripotent stem cells in feeder-free culture system

Essential 8 Medium PSC Cryopreservation Kit

Timeline for the generation of an iPSC lineThe entire reprogramming process from expanded fibroblast culture to stable induced pluripotent stem cell (iPSC) line typically takes about eight weeks but will vary significantly with genetic background. Colony selection around Day 21 is facilitated by using a conjugated antibody suitable for live staining. Passage 1 is complete after replating on to inactivated murine embryonic fibroblasts (iMEFs). Continuing to culture the cells on iMEFs with regular passaging enables the iPSC line to become fully established by Passage 10, at which time it is ready for transition to feeder-free culture. Banking and full characterization of the line is also recommended at Passage 10.

Platecells

Changemedium

Switch to iPSCmedium

Emergingcolonies

iPSC colonies ready for transferReplace spent medium

Plate transduced cellson MEF culture dishes

Performtransduction

Day: –2 0 1 2 4 6 7 8 10 12 24 P1−P10 P10 (~5 weeks) Transition to Essential 8 Medium

Fibroblast medium

iMEF feeder cells

KnockOut Serum Replacement – Multi-Species Essential 8 Medium

Vitronectin

Introduction

Start your PSC research off right

Because of the immense potential of PSCs for regenerative medicine and clinical applications, the field of stem cell research continues to grow rapidly. This growth has been a catalyst for many new researchers starting to work with stem cells and many new labs bringing stem cells into their workflows. While many newer technologies have simplified individual steps of the stem cell workflow, it can still be daunting to determine where to start a whole project and what products will be needed. Gibco™ iPSC Lab Starter Kits are ideal for these researchers and include all the reagents necessary for the PSC workflow, from reprogramming with donor-derived fibroblasts to PSC expansion in feeder-free media conditions.

Use the checklist of reagents included in the iPSC Lab Starter Kits as a point of reference for where to start when beginning with PSCs.

The protocols included in this handbook support the iPSC Lab Starter Kits and serve as a guide to using the included media and reagents. Look for this icon throughout for tips related to products included in the iPSC Lab Starter Kits.

For additional support on working with PSCs, download the Gibco™ pluripotent stem cell handbook at thermofisher.com/pschandbook

Resource Center

http://thermofisher.com/pschandbook

6 7

Reprogramming of human fibroblastsCytoTune-iPS 2.0 Sendai Reprogramming Kit

1

iPSC Lab Starter Kit with Reprogramming (Cat. No. A32020)

Product Quantity* Cat. No.**

Reprogramming

Reducing Agent (2-Mercaptoethanol , 55 mM) 50 mL 21985-023

Attachment Factor Protein (1X) 2 x 100 mL S-006-100

Collagenase Type IV 1 vial 17104-019

CytoTune-iPS 2.0 Sendai Reprogramming Kit 1 kit A16517

DMEM/F-12 with GlutaMAX-I Supplement 5 x 500 mL 10565-018

Fetal Bovine Serum, Embryonic Stem Cell Qualified , US Origin 2 x 100 mL 16141-061

FGF-Basic (AA 1–155) Recombinant Human Protein 4 vials PHG0264

Mouse (ICR) Inactivated Embryonic Fibroblasts 11 vials A24903

DMEM, high glucose, GlutaMAX-I Supplement 40 x 500 mL 10569-044

KnockOut Serum Replacement – Multi-Species 3 x 100 mL A31815-02

MEM Non-Essential Amino Acids Solution (100X) 100 mL 11140-050

TRA-1-60 Alexa Fluor 488 Conjugate Kit for Live-Cell Staining 1 kit A25618

PSC feeder-free transition and expansion

DPBS, no calcium, no magnesium 20 x 500 mL 14190250

Essential 8 Adaptation Kit 1 kit A25935

Essential 8 Medium 4 x 500 mL A1517001

Geltrex LDEV-Free, hESC-Qualified, Reduced Growth Factor Basement Membrane Matrix

2 x 5 mL A1413302

Versene Solution 100 mL 15040066

Vitronectin (VTN-N) Recombinant Human Protein, Truncated 3 x 1 mL A14700

iPSC Lab Starter Kit (Cat. No. A32021)

Product Quantity* Cat. No.**

PSC feeder-free transition and expansion

DPBS, no calcium, no magnesium 20 x 500 mL 14190250

Essential 8 Medium 6 x 500 mL A1517001

Geltrex LDEV-Free, hESC-Qualified, Reduced Growth Factor Basement Membrane Matrix

2 x 5 mL A1413302

Versene Solution 2 x 100 mL 15040066

Vitronectin (VTN-N) Recombinant Human Protein, Truncated 4 x 1 mL A14700

* Quantity provided in the kit. ** To obtain more as stand alone reagent.

Introduction

8 9

1 Reprogramming of human fibroblasts CytoTune-iPS 2.0 Sendai Reprogramming Kit 1

Safety features of the system

Non-transmissible CytoTune 2.0 Sendai reprogramming vectorsSeV vectors used in this kit consist of viral proteins NP, P, M, F (activated), HN, and L, and the SeV genome RNA, from which the F gene is deleted. Because SeV infects cells by attaching itself to cell surface receptor sialic acid, present on the surface of many cell types of different species, the vectors are able to transduce a wide range of cells. However, they are no longer capable of producing infectious particles from infected cells, because the viral genome lacks the F gene. In addition, the presence of temperature sensitivity mutations in the amino acid sequence of several SeV proteins (SeV/TSΔF, SeV/TS12ΔF, and SeV/TS15ΔF) renders the vectors easily removable from transduced cells.

Note: SeV vectors used in this kit were developed by DNAVEC Corporation (http://www.dnavec.co.jp) and their rights for commercial use are the property of DNAVEC Corporation.

Inoculating animals with transduced cellsAlthough the CytoTune 2.0 Sendai reprogramming vectors are non-transmissible, cells that have been exposed to the virus should be tested with PCR or antibody staining to help ensure the absence of the virus before being inoculated into animals. Animals that have already been infected with wild type SeV may be able to make infectious CytoTune-2.0 Sendai virus.

Biosafety Level 2Although human cells are not the natural host for the SeV, and the virus is nonpathogenic to humans, appropriate care must be taken to prevent the potential mucosal exposure to the virus. This product must be used under Biosafety Level 2 (BL-2) containment with biological safety cabinet and laminar flow hood, and with appropriate personal safety equipment to prevent mucosal exposure/splash. In the event that the virus comes into contact with skin or eyes, decontaminate by flushing with plenty of water and consult a physician. For more information on BL-2 guidelines, refer to Biosafety in Microbiological and Biomedical Laboratories, 5th ed., published by the Centers for Disease Control, which is available for downloading at: www.cdc.gov/od/ohs/biosfty/bmbl5/bmbl5toc.htm

CytoTune-iPS 2.0 Sendai Reprogramming Kit

The Invitrogen™ CytoTune™-iPS 2.0 Sendai Reprogramming Kit uses vectors based on a modified, non-transmissible form of Sendai virus (SeV) to safely and effectively deliver and express key genetic factors necessary for reprogramming somatic cells into iPSCs. In contrast to many available protocols, which rely on viral vectors that integrate into the genome of the host cell, the CytoTune-iPS 2.0 Sendai Reprogramming Kit uses vectors that are non-integrating and remain in the cytoplasm (i.e., they are zero-footprint, Figure 1.1). In addition, the host cell can be cleared of the vectors and reprogramming factor genes by exploiting the cytoplasmic nature of SeV and the temperature sensitivity mutations introduced into the key viral proteins.

The CytoTune-iPS 2.0 Sendai Reprogramming Kit contains three SeV-based reprogramming vectors, and are optimized for generating iPSCs from human somatic cells. The reprogramming vectors in this kit have been engineered to increase biological and environmental safety (“Safety features of the system” on page 9).

Figure 1.1. Comparison of the life cycles of non-integrating SeV vectors and other, integrating vectors.

CytoTune 2.0 reprogramming vectorsThe table below lists the CytoTune 2.0 reprogramming vectors included in the CytoTune-iPS 2.0 Sendai Reprogramming Kit. The reprogramming vectors include the four Yamanaka factors, Oct 3/4, Sox2, Klf4, and c-Myc, shown to be sufficient for efficient reprogramming [1].

CytoTune Sendai vector Cap color Factor GenBank ID

CytoTune 2.0 KOS Clear

Human Klf4 BC029923.1

Human Oct3/4 NM_002701.4

Human Sox2 NM_003106.2

CytoTune 2.0 hc-Myc White Human c-Myc K02276.1

CytoTune 2.0 hKlf4 Red Human Klf4 BC029923.1

AAAAA

AAAAA

AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

Endoplasmicreticulum (ER)

Target protein

Target protein

Other vectorsSendai virus vector

NPP

L

F

HN

NucleusCytoplasm

Transcription

Replication

Replication

http://www.dnavec.co.jp

http://www.cdc.gov/od/ohs/biosfty/bmbl5/bmbl5toc.htm

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Before you begin

Guidelines for generating iPSCs• To maintain sterile culture conditions, carry out all of the procedures in this handbook using sterile

laboratory practices in a laminar flow hood.

• You can use the CytoTune-iPS 2.0 Sendai Reprogramming Kit to reprogram a wide range of cell types in proliferative and quiescent states. However, the reprogramming efficiency may vary between different cell types (~0.01–1%).

• For successful reprogramming, transduce your cells using all three reprogramming vectors.

Note: For successful reprogramming, all four Yamanaka factors (i.e., Oct3/4, Sox2, Klf4, and c-Myc) need to be expressed in your host cell.

• Cells that have already been infected with Sendai virus are refractive to further infection by Sendai virus. Therefore, you cannot transduce cells with CytoTune 2.0 reprogramming vectors when they have already been transduced with other Sendai vectors such as the Invitrogen™ CytoTune™-EmGFP Sendai Fluorescence Reporter or vice versa.

• One CytoTune-iPS 2.0 Sendai Reprogramming Kit of three tubes supplies sufficient reagents to transduce a minimum of 1.5 x 106 cells at MOI (multiplicity of infection) = 5-5-3 (i.e., KOS MOI = 5, hc-Myc MOI = 5, hKlf4 MOI = 3).

• The titer of each CytoTune 2.0 Sendai reprogramming vector is lot-dependent. For the specific titer of your vectors, refer to the Certificate of Analysis (CoA) available on our website. Go to thermofisher.com/coa and search for the CoA by product lot number, which is printed on the vial.

• Viral titers can decrease dramatically with each freeze/thaw cycle. Avoid repeated freezing and thawing of your reprogramming vectors. Viral titer is not guaranteed for kits that have been refrozen or thawed.

• Prior to starting, ensure that the media are equilibrated to 37°C and appropriately gassed.

Positive controlFor a positive control, we recommend performing a reprogramming experiment with human neonatal foreskin fibroblast cells (strain BJ; ATCC No. CRL2522). Note that experimental conditions may vary among target cells and need to be optimized for each cell type. The example given in the following protocol does not guarantee the generation of iPSCs for all cell types.

CytoTune-EmGFP Sendai Fluorescence ReporterThe CytoTune-EmGFP Sendai Fluorescence Reporter (Cat. No. A16519) is a control vector carrying the Emerald Green Fluorescent Protein (EmGFP) gene. The control vector allows you to determine whether your cell line of interest is amenable or refractive to transduction by the Sendai reprogramming vectors, including the vectors from the original Invitrogen™ CytoTune™-iPS Sendai Reprogramming Kits. We recommend testing your cell lines of interest using the CytoTune-EmGFP Sendai Fluorescence Reporter before starting your reprogramming experiments.

Note: You cannot transduce cells with CytoTune reprogramming vectors that have already been transduced with the CytoTune-EmGFP Sendai Fluorescence Reporter or vice versa. If you wish to use the CytoTune-EmGFP Sendai Fluorescence Reporter during reprogramming, you must add it to the cells at the same time as the reprogramming vectors.

For detailed instructions on using the CytoTune-EmGFP Sendai Fluorescence Reporter, visit thermofisher.com/cytotunegfp

Protocol: Reprogramming fibroblasts

The major steps required for reprogramming human neonatal foreskin fibroblast cells using the CytoTune-iPS 2.0 Sendai Reprogramming Kit to generate iPSCs cultured on MEF feeder cells are shown below. Note that the timeline is provided as a guideline for experimental planning; actual timeline can vary based on the cell type and experimental conditions.

Reprogramming timelineDay –2: Plate human fibroblasts with a passage number of 5 or lower into at least two wells of a 6-well plate in fibroblast medium so that they are 50–80% confluent on the day of transduction (Day 0).

Note: The kit supplies sufficient virus to transduce cells in at least 5 wells of a 6-well plate.

Day 0: Transduce the cells using the CytoTune 2.0 Sendai reprogramming vectors at the appropriate MOI. Incubate the cells overnight.

Day 1: Replace the medium with fresh complete fibroblast medium to remove the CytoTune 2.0 Sendai reprogramming vectors.

Day 2–6: Replace the spent medium every other day.

Day 5 or 6: Prepare MEF culture dishes.

Day 7: Plate transduced cells on MEF culture dishes in fibroblast medium.

Day 8: Change the medium to iPSC medium.

Day 9–21: Replace spent medium every day and monitor the culture vessels for the emergence of iPSC colonies. When iPSC colonies are ready for transfer, perform live staining, and pick and transfer undifferentiated iPSCs onto fresh MEF culture dishes for expansion.

Media for reprogramming fibroblasts (feeder-dependent)For optimal reprogramming of human neonatal foreskin fibroblast cells using the CytoTune-iPS 2.0 Sendai Reprogramming Kit to generate iPSCs cultured on MEF feeder cells, use the following media at the designated stages of the reprogramming experiment:

• Fibroblast medium: Plating cells prior to transduction, expansion, posttransduction recovery of cells, plating of transduced cells on MEF culture dishes

• iPSC medium: Expansion of transduced cells on MEF culture dishes, live staining and picking of iPSCs


http://www.thermofisher.com/coa

http://thermofisher.com/cytotunegfp

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Materials needed:Cells and vectors• CytoTune-iPS 2.0 Sendai Reprogramming Kit (Cat. No. A16517)

Note: For successful reprogramming, you need all three tubes of reprogramming vectors.

• Human fibroblast cells to reprogram

Note: If cells are not already procured, see cells available below:

– Gibco™ Human Dermal Fibroblasts, neonatal (Cat. No. C0045C)

– Gibco™ Human Dermal Fibroblasts, adult (Cat. No. C0135C)

• Optional: Human neonatal foreskin fibroblast cells (strain BJ; ATCC No. CRL2522) as a positive reprogramming control

• Gibco™ Mouse (ICR) Inactivated Embryonic Fibroblasts (Cat. No. A24903)

Media and reagents• Gibco™ Dulbecco’s Modified Eagle Medium (DMEM), high glucose, GlutaMAX™-I Supplement (Cat. No.

10569-010)

• Gibco™ DMEM/F-12 with GlutaMAX™-I Supplement (Cat. No. 10565-018)

• Gibco™ Fetal Bovine Serum (FBS), Embryonic Stem Cell Qualified, US Origin (Cat. No. 16141-079)

• Gibco™ KnockOut™ Serum Replacement – Multi-Species (Cat. No. A31815-02) or Gibco™ KnockOut™ Serum Replacement (Cat. No. 10828010)

• Gibco™ MEM Non-Essential Amino Acids Solution (100X) (Cat. No. 11140-050)

• Gibco™ FGF-Basic (AA 1–155) Recombinant Human Protein (Cat. No. PHG0264)

• Gibco™ 2-Mercaptoethanol (55 mM) (Cat. No. 21985-023)

• Optional: Gibco™ Penicillin-Streptomycin (10,000 U/mL) (Cat. No. 15140-122)

• Gibco™ Attachment Factor Protein (1X) (Cat. No. S-006-100)

• Gibco™ TrypLE™ Select Enzyme (1X) (Cat. No. 12563 -011) or 0.05% Trypsin-EDTA solution (0.05%) (Cat. No. 25300-054)

• Gibco™ Dulbecco’s Phosphate Buffered Saline (DPBS) no calcium, no magnesium (Cat. No. 14190250)

Reprogramming protocolThe following protocol has been optimized to transduce one well of human neonatal foreskin fibroblast cells (strain BJ; ATCC No. CRL2522) as a positive control. We recommend that you optimize the protocol for your cell type, and add an appropriate number of conditions and wells to utilize the entire volume of virus.

Day –2: Prepare the cells for transduction1. Two days before transduction, plate human neonatal foreskin fibroblast cells onto at least two wells of a

6-well plate at the appropriate density to achieve between 2 x 105 and 3 x 105 cells per well on the day of transduction (Day 0). One of the wells will be used to count cells for viral volume calculations. Note: Each CytoTune-iPS 2.0 Sendai Reprogramming Kit supplies sufficient virus to transduce cells in at least 5 wells of a 6-well plate. We recommend using the entire volume of virus. Note: We recommend about 50–80% confluency on the day of transduction. Because overconfluency results in decreased transduction efficiency, we recommend replating your cells to achieve 50–80% confluency if your cells have become overconfluent during culturing.

2. Culture the cells for two more days, ensuring the cells have fully adhered and extended.

Day 0: Perform transduction3. On the day of transduction, warm 1 mL of fibroblast medium in a water bath for each well to be transduced.

4. Harvest the cells from one well to perform a cell count. These cells will not be transduced, but will be used to estimate the cell number in the other well(s) plated in Step 1.

5. Remove the cells from this well using 0.5 mL of TrypLE Select reagent or 0.05% Trypsin-EDTA solution following the procedure recommended by the manufacturer and incubating at room temperature. When the cells have rounded up (1–3 minutes later), add 1 mL of fibroblast medium into each well, and collect the cells in a 15 mL conical centrifuge tube.

6. Count the cells using the desired method (e.g., Invitrogen™ Countess™ Automated Cell Counter), and calculate the volume of each virus needed to reach the target MOI using the live-cell count and the titer information on the CoA.

Volume of virus (μL) =MOI (CIU/cell) × Number of cells

Titer of virus (CIU/mL) × 10–3 (μL/mL)

Note: We recommend initially performing the transductions with MOIs of 5, 5, and 3 (i.e., KOS MOI = 5, hc-Myc MOI = 5, hKlf4 MOI = 3). These MOIs can be optimized for your application. Note: The titer of each CytoTune 2.0 reprogramming vector is lot-dependent. For the specific titer of your vectors, go to thermofisher.com/coa and search for the CoA by product lot number, which is printed on the vial. Avoid refreezing and thawing of the reprogramming vectors since viral titers can decrease dramatically with each freeze/thaw cycle.


http://www.thermofisher.com/coa

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7. Remove one set of CytoTune 2.0 Sendai tubes from the –80°C storage. Thaw each tube one at a time by first immersing the bottom of the tube in a 37°C water bath for 5–10 seconds, and then removing the tube from the water bath and allowing it to thaw at room temperature. Once thawed, briefly centrifuge the tube and place it immediately on ice.

8. Add the calculated volumes of each of the three CytoTune 2.0 Sendai tubes to 1 mL of fibroblast medium, pre-warmed to 37°C. Ensure that the solution is thoroughly mixed by pipetting the mixture gently up and down. Complete the next step within 5 minutes.

9. Aspirate the fibroblast medium from the cells, and add the reprogramming virus mixture prepared in Step 8 to the well containing the cells. Incubate the cells overnight in a 37°C incubator with a humidified atmosphere of 5% CO2.

Day 1: Replace medium and culture cells10. 24 hours after transduction, replace the medium with fresh fibroblast medium.

Note: Depending on your cell type, you should expect to see some cytotoxicity 24–48 hours posttransduction, which can affect >50% of your cells. This is an indication of high uptake of the virus. We recommend that you continue culturing your cells and proceed with the protocol.

11. Culture the cells for 6 more days, changing the spent medium with fresh fibroblast medium every other day. Note: Depending on your cell type, you may observe high cell density before Day 5. We do not recommend passaging your cells onto MEF culture dishes before 7 days posttransduction. You may replace spent medium daily with fresh fibroblast medium if cultures become very dense.

Day 5 or 6: Prepare MEF culture dishes12. One to two days before passaging the transduced fibroblasts onto MEF feeder cells, prepare 100 mm MEF

culture dishes (see Chapter 3 “Preparation of feeder layer” on page 25 for more information).

Day 7: Plate transduced cells on MEF culture dishes13. Seven days after transduction (Step 6, page 13), fibroblast cells are ready to be harvested and plated on

MEF culture dishes. Remove the medium from the fibroblasts, and wash cells once with DPBS.

14. To remove the cells from the 6-well plate, use 0.5 mL of TrypLE Select reagent or 0.05% Trypsin-EDTA solution following the procedure recommended by the manufacturer and incubate at room temperature. When the cells have rounded up (1–3 minutes later), add 2 mL of fibroblast medium into each well, and collect the cells in a 15 mL conical centrifuge tube. Note: Because the cells can be very sensitive to trypsin at this point, minimize trypsin exposure time and incubate the cells at room temperature.

15. Centrifuge the cells at 200 x g for 4 minutes, aspirate the medium, and resuspend the cells in an appropriate amount of fibroblast medium.

16. Count the cells using the desired method (e.g., Countess Automated Cell Counter), and seed the MEF culture dishes with 5 x 104 to 2 x 105 cells per 100 mm dish and incubate overnight in a 37°C incubator with a humidified atmosphere of 5% CO2. Note: We recommend plating 5 x 104, 1 x 105, and 2 x 105 cells per 100 mm dish. Depending on your cell type, you may need to plate most of your cells on the same plate to ensure sufficient numbers of colonies. Note: Set aside any remaining cells for RNA extraction to be used as a positive control in the RT-PCR detection of the SeV genome (see page 18).

Day 8 to 28: Feed and monitor the cells17. 24 hours later, change the medium to iPSC medium (see page 31 for recipe), and replace the spent

medium every day thereafter.

18. Starting on Day 8, observe the plates every other day under a microscope for the emergence of cell clumps indicative of reprogrammed cells (see “Visual identification—Identifying and picking iPSC colonies” on page 16). Note: For BJ fibroblasts, we normally observe colony formation on Day 12 posttransduction. However, depending on your cell type, you may need to culture for up to 4 weeks before seeing colonies.

19. Three to four weeks after transduction, colonies should have grown to an appropriate size for transfer. The day before transferring the colonies, prepare MEF culture plates using 12- or 24-well plates coated with 1X Attachment Factor Protein. Note: We typically harvest colonies closer to three weeks to avoid differentiation.

20. When colonies are ready for transfer, perform live staining using TRA-1-60 or TRA-1-81 antibodies for selecting reprogrammed colonies (see “Picking iPSC colonies in feeder-dependent conditions” on page 23).

21. Manually pick colonies and transfer them onto MEF plates (see “Picking iPSC colonies in feeder-dependent conditions” on page 23).


16 17

Guidelines for generating vector-free iPSCs• The time needed to derive vector-free iPSCs may vary depending on culture and passage conditions.

In the case of human neonatal foreskin fibroblast cells (strain BJ), it takes about 1–2 months after gene transduction to obtain iPSCs free of CytoTune 2.0 Sendai reprogramming vectors.

• To obtain virus-free clones faster, we recommend that you perform single-colony subcloning for the first few passages (minimum 5) instead of bulk or pooled-clone passaging.

• To perform single-colony subcloning, pick from a single colony to transfer to another 6-well plate (Passage 1). From Passage 1, pick a single colony and transfer to another 6-well plate (Passage 2) and so forth. We recommend subcloning for 5 passages and then testing for virus-free iPSCs.

35

Generating Vector-Free iPSCs

Generating Vector-Free iPSCs

Guidelines for generating vector-free iPSCs

• The time needed to derive vector-free iPSCs may vary depending on culture and passage conditions. In the case of human neonatal foreskin fibroblast cells (strain BJ), it takes about 1–2 months after gene transduction to obtain iPSCs free of CytoTune® 2.0 Sendai reprogramming vectors.

• To obtain virus-free clones faster, we recommend that you perform single colony subcloning for the first few passages (minimum 5) instead of bulk or pooled-clone passaging.

• To perform single colony subcloning, pick from a single colony to transfer to another 6-well plate (Passage 1). From Passage 1, pick a single colony and transfer to another 6-well plate (passage 2) and so forth. We recommend subcloning for 5 passages and then testing for virus free iPSCs.

Materials required • Dulbecco’s PBS (DPBS) without Calcium and Magnesium (Cat. no. 14190)

• Rabbit anti-SeV antibody (MBL International Corporation, Woburn, MA; Cat. no PD029)

• Alexa Fluor® 488 goat anti-rabbit IgG (H+L) antibody (Cat no. A11034) or

Alexa Fluor® 594 goat anti-rabbit IgG (H+L) antibody (Cat no. A11037)

• TRIzol® LS reagent (Cat. no. 10296-010)

• SuperScript® VILO™ cDNA Synthesis Kit (Cat. no. 11754-050)

• AccuPrime™ SuperMix I (Cat. no. 12342-010)

Protocol for generating vector-free iPSCs

1. When passaging iPSC colonies, prepare duplicate plates; one for immunostaining and one for further passaging.

2. Perform immunostaining on one plate using anti-SeV antibodies (see below).

3. If any colonies stain positive, perform cell cloning on the other duplicate plate.

4. Repeat immunostaining with anti-SeV antibodies on the cloned colonies until all colonies in a plate are negative.

5. If all colonies are negative for anti-SeV antibodies, passage the cells and confirm the absence of the CytoTune® 2.0 Sendai reprogramming vectors by RT-PCR (see page 36).

Continued on next page

Protocol: Generating vector-free iPSCs

Materials needed:• Dulbecco’s Phosphate Buffered Saline (DPBS), no calcium, no magnesium (Cat. No. 14190144)

• Rabbit anti-SeV antibody (MBL International Corporation, Woburn, MA; Cat. No. PD029)

• Invitrogen™ Alexa Fluor™ 488 goat anti-rabbit IgG (H+L) secondary antibody (Cat. No. A11034) or Alexa Fluor™ 594 goat anti-rabbit IgG (H+L) secondary antibody (Cat. No. A11037)

• Invitrogen™ TRIzol™ Reagent (Cat. No. 15596-026)

• Invitrogen™ SuperScript™ VILO™ cDNA Synthesis Kit (Cat. No. 11754-050)

• Invitrogen™ AccuPrime™ SuperMix I (Cat. No. 12342-010)

Protocol1. When passaging iPSC colonies, prepare duplicate plates; one for immunostaining and one for

further passaging.

2. Perform immunostaining on one plate using anti-SeV antibody (see below).

3. If any colonies stain positive, perform cell cloning on the other duplicate plate.

4. Repeat immunostaining with anti-SeV antibody on the cloned colonies until all colonies in a plate are negative.

5. If all colonies are negative for anti-SeV antibody, passage the cells and confirm the absence of the CytoTune 2.0 Sendai reprogramming vectors by RT-PCR (see page 18).


Visual identification—Identifying and picking iPSC colonies

By Day 21 posttransduction, the cell colonies on the MEF culture dishes will have become large and compact, covering the majority of the surface area of the culture dish. However, only a fraction of these colonies will consist of iPSCs, which exhibit a hESC-like morphology characterized by a flat cobblestone-like appearance with individual cells clearly demarcated from each other in the colonies (refer to Figure 1.2, below). Therefore, we recommend that you perform live staining with TRA-1-60 or TRA-1-81 antibodies that recognize undifferentiated hESCs (see “Picking iPSC colonies in feeder-dependent conditions” on page 23).

Note: Although colonies of “transformed” cells may emerge as early as 7 days after transduction, most of these colonies will not be correctly “reprogrammed” cells. iPSCs usually emerge a little later (around day 14 posttransduction), resemble embryonic stem cells in morphology, and express the cell surface markers TRA-1-60 or TRA-1-81.

Morphology of reprogrammed cellsThe images below show the morphology of human neonatal foreskin fibroblast cells (strain BJ) that were reprogrammed into iPSCs using the CytoTune-iPS 2.0 Sendai Reprogramming Kit.

Figure 1.2. Human neonatal foreskin fibroblast cells (strain BJ) were transduced using the CytoTune-iPS 2.0 Sendai Reprogramming Kit and allowed to proliferate on MEF feeder layers in iPSC medium. The images were obtained using a 5X objective.

Fibroblasts before transduction (5X) 2 days posttransduction 10 days posttransduction

14 days posttransduction (5X) 18 days posttransduction 21 days posttransduction

18 19

Immunocytochemistry with anti-SeV antibody1. Wash cells once with DPBS.

2. Fix the cells in 4% paraformaldehyde for 5 minutes at room temperature.

3. Wash cells twice with DPBS.

4. Add the anti-SeV antibody diluted in 0.1% Triton™ X-100 in DPBS to the cells and incubate for 1 hour at 37°C.

5. Remove the antibody solution. Wash the cells 3 times with DPBS.

6. Add the secondary antibody diluted in 0.1% Triton X-100 in DPBS to the cells and incubate for 1 hour at 37°C.

7. Remove the secondary antibody solution from the dish. Wash the cells 3 times with DPBS.

8. Visualize the cells under a fluorescence microscope.

RT-PCR protocol for detecting the SeV genome and transgenes1. Extract the total RNA from 5 x 106 iPSCs using TRIzol Reagent following the instructions provided with the

reagent. As a positive control, use cells set aside during the reprogramming procedure.

2. Carry out a reverse transcription reaction using 1 μg of RNA (from step 1) and the SuperScript VILO cDNA Synthesis Kit following the instructions provided with the kit. Note: Because the CytoTune 2.0 Sendai reprogramming vectors are based on SeV, which is an RNA virus, reverse transcription is required for detecting the presence of the SeV genome in your reprogrammed cells.

3. Carry out the PCR using 10 μL of cDNA from the reverse transcription reaction (Step 2, above) and AccuPrime SuperMix I with the parameters below. For the RT-PCR primer sequences and the expected product size, refer to the table on page 19.

Step Temperature Time Cycles

Denaturation 95°C 30 seconds

30–35Annealing 55°C 30 seconds

Elongation 72°C 30 seconds

4. Analyze the PCR products using 2% agarose gel electrophoresis. Note: If you still detect CytoTune 2.0 Sendai virus in your iPSC lines after more than 10 passages, and have performed RT-PCR to show that hKlf4 is absent from your cells (this vector does not have the temperature sensitivity mutation), then you can perform a temperature shift to remove the hc-Myc vector. CytoTune 2.0 Sendai hc-Myc tends to persist in the cells longer than the other CytoTune 2.0 Sendai reprogramming vectors. However, because this vector contains a temperature sensitivity mutation, you can enhance its removal and obtain complete absence of Sendai virus by incubating your cells at 38°C to 39°C for 5 days.

RT-PCR primer setsThe table below shows the RT-PCR primer set used for detecting the SeV genome and transgenes in cells reprogrammed using the CytoTune 2.0 Sendai reprogramming vectors.

Target Primer sets Product size

SeVForward: GGA TCA CTA GGT GAT ATC GAG C*

181 bpReverse: ACC AGA CAA GAG TTT AAG AGA TAT GTA TC*

KOSForward: CAC CGC TAC GAC GTG AGC GC

528 bpReverse: ACC TTG ACA ATC CTG ATG TGG

Klf4Forward: TTC CTG CAT GCC AGA GGA GCC C

410 bpReverse: AAT GTA TCG AAG GTG CTC AA*

c-MycForward: TAA CTG ACT AGC AGG CTT GTC G*

532 bpReverse: TCC ACA TAC AGT CCT GGA TGA TGA TG

* Primer contains SeV genome sequences. Pairing of these primers with transgene-specific primers allows specific detection of transgenes carried by the CytoTune 2.0 Sendai reprogramming vectors.


20 21

Troubleshooting

Problem Possible cause Solution

Cytotoxic effects observed after transduction

Viral load too high Decrease the volume of CytoTune 2.0 vector or increase the starting cell number.

Too many colonies on the plate

Too many cells plated Decrease the number of cells plated after transduction.

No iPSC colony formation

Insufficient amount of virus used

• Check the volume of the CytoTune 2.0 vector and the starting cell number. Changing the MOI may improve the results.

• We suggest initially increasing the MOI of Klf4 to improve efficiency (e.g., KOS MOI = 5, c-Myc MOI = 5, Klf4 MOI = 6). If efficiencies are still too low, increase the MOI of KOS and c-Myc, while maintaining a 1:1 ratio between the two (e.g., KOS MOI = 10, c-Myc MOI = 10, Klf4 = 6).

• Not all cell types will be reprogrammed with the same efficiency. Check the levels of protein expression in your cell type using Applied Biosystems™ TaqMan® Protein Assays.

• Do not refreeze or aliquot virus. Viral titer is not guaranteed for kits refrozen or thawed.

Too few iPSC colonies compared to BJ fibroblasts

Cell type not efficiently reprogrammable

• Not all cell types will have the same reprogramming efficiency. Increase the number of the cells plated.

• If reprogramming a new cell type, use the CytoTune-EmGFP Sendai Fluorescence Reporter to assess uptake of Sendai virus (page 10).

iPSC colonies look differentiated

iPSC colonies transferred to MEF dishes too late

Perform staining earlier and transfer iPSC colony to fresh feeder cells.

Difficult to obtain vector-free iPSCs

Cell type cannot efficiently eliminate the CytoTune 2.0 Sendai reprogramming vector

• Some cell strains may need a longer time to eliminate the CytoTune 2.0 Sendai vectors and become vector-free compared to other strains. Perform repeated single-colony subcloning until you obtain negative cells as determined by immunocytochemistry with anti-SeV antibody.

• It may be easier to obtain SeV-negative colonies if single-colony subcloning is performed by transferring a portion of a colony with a glass pipette.

• The rate with which iPSC colonies eliminate the CytoTune 2.0 Sendai vectors may increase if the cells are incubated for 5 days at 38–39°C after you have confirmed by RT-PCR that the Klf4 vector is absent from your cells and only KOS and/or c-Myc vectors remain (see Note on page 18).

Reference1. Takahashi K, Tanabe K, Ohnuki M et al. (2007) Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131(5):861–872.

For additional technical support information and frequently asked questions, visit thermofisher.com/cytotune

Selection of iPSC clones for feeder-dependent cultureStem cell antibody kits for live-cell imaging

2

1 Reprogramming of human fibroblasts

http://thermofisher.com/cytotune

22 23

2 Selection of iPSC clones for feeder-dependent culture Stem cell antibody kits for live-cell imaging 2

Table 2.1. Recommended volumes to use during the staining protocol.

Culture format No. of tests* Staining volume Amount of 50X antibody to add

96-well plate 200 50 μL/well 1 μL




6-well plate 10 1 mL/well 20 μL

35 mm dish 10 1 mL/dish 20 μL



4-well chamber slide 25 400 μL/well 8 μL

8-well chamber slide 50 200 μL/well 4 μL

* When using the suggested staining volume, this kit contains sufficient reagents for the indicated number of tests.

Picking iPSC colonies in feeder-dependent conditions1. Place the culture dish containing the reprogrammed cells under an inverted microscope and examine

the colonies under 10X magnification. Successful antibody staining can very specifically distinguish reprogrammed colonies from just plain transformed counterparts.

2. Mark the colony to be picked on the bottom of the culture dish. Note: We recommend picking at least 10 distinct colonies by the end of each reprogramming experiment and expanding them in separate 24-well iMEF culture plates (see below). Refer to Chapter 3 for more information on the preparation of iMEF culture plates.

3. Transfer the culture dish to a sterile cell culture hood (i.e., biosafety cabinet) equipped with a stereomicroscope.

4. Using a 25 gauge 1½ inch needle, cut the colony to be picked into 5–6 pieces in a grid-like pattern.

5. Using a 200 μL pipette, transfer the cut pieces to a freshly prepared 6-well culture dish containing iPSC medium.

6. Incubate the MEF culture plate containing the picked colonies in a 37°C incubator with a humidified atmosphere of 5% CO2.

7. Allow the colonies to attach to the culture plate for 48 hours before replacing the spent medium with fresh iPSC medium. After that, change the medium every day. Refer to Chapter 4 for the proper maintenance and expansion of reprogrammed iPSC clones on MEFs.

Stem cell antibody kits

One of the fastest and most reliable methods for identifying a reprogrammed colony is live staining with TRA-1-60 antibody that recognizes undifferentiated iPSCs and enables the identification of reprogrammed cells from a variety of human cell types. The Invitrogen™ TRA-1-60 Alexa Fluor™ 488 Conjugate Kit for Live-Cell Imaging (Cat. No. A25618) pairs together Alexa Fluor™ dye–conjugated anti-TRA-1-60 antibody with a superior imaging medium, Gibco™ FluoroBrite™ DMEM (Cat. No. A1896701), which enhances fluorescence signals while preserving cell health during live-cell imaging workflows. These reagents are specially formulated and sterile-filtered to meet the demands of live-cell imaging applications: they contain no cytotoxic preservatives (e.g., no sodium azide) and are tested to confirm that endotoxin levels are low and that they are free of common microbial contaminants.

Protocol: Live-cell imaging

Materials needed:• Note: Alternate conjugations available include:

– Invitrogen™ TRA-1-60 Alexa Fluor™ 555 Conjugate Kit for Live-Cell Imaging (Cat. No. A24879)

– Invitrogen™ TRA-1-60 Alexa Fluor™ 594 Conjugate Kit for Live-Cell Imaging (Cat. No. A24882)

– Invitrogen™ CD44 Alexa Fluor™ 488 Conjugate Kit for Live-Cell Imaging (Cat. No. A25528)

See Table 2.1 (on the next page) for recommended volumes.

ProtocolCaution: Use aseptic technique when handling live-cell imaging reagents to prevent contamination.

1. Centrifuge the dye-conjugated antibody solution (e.g., 2 minutes at 10,000 x g) and only use the supernatant.

Note: This step minimizes transferring protein aggregates that may have formed during storage, thereby reducing nonspecific background staining.

2. Add a 1:50 volume of the dye-conjugated antibody directly to the cell culture medium of the cells to be stained (see Table 2.1 on the next page for guidance). Mix by gentle swirling.

3. Incubate for 30 minutes at 37°C.

4. Remove the staining solution and gently wash the cells 2–3 times with FluoroBrite DMEM.

5. For optimal results, observe and image the cells immediately (i.e., within 30 minutes).

Note: To continue culturing the cells, replace the FluoroBrite DMEM with fresh cell culture medium and return the cells to the 37°C incubator.

24 25

Example data

Figure 2.1. Induced pluripotent stem cells growing on a mouse fibroblast feeder layer were stained with dye-labeled antibodies for cell surface markers TRA-1-60 (red, a positive pluripotency marker) and CD44 (green, a fibroblast marker that also serves as a negative pluripotency marker).

Stem Cell Antibody Kits for Live Cell Imaging | 3

Figure 1 Induced pluripotent stem cells growing on a mouse fibroblast feeder layer were stained with dye-labeled antibodies for cell surface markers TRA-1-60 (Red, a positive pluripotency marker) and CD44 (Green, a fibroblast marker that also serves as a negative pluripotency marker).

Example data

Product list Current prices may be obtained from our website or from our Customer Service Department.

Cat. no. Product name Unit sizeA25528 CD44 Rat anti-human/mouse mAb, AlexaFluor® 488 Conjugate Kit for Live Cell Imaging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 kitA25618 TRA-1-60 Mouse anti-human mAb, AlexaFluor® 488 Conjugate Kit for Live Cell Imaging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 kitA24879 TRA-1-60 Mouse anti-human mAb, Alexa Fluor® 555 Conjugate Kit for Live Cell Imaging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 kitA24882 TRA-1-60 Mouse anti-human mAb, Alexa Fluor® 594 Conjugate Kit for Live Cell Imaging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 kit

Related productsA24881 PSC 4-Marker Immunocytochemistry Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 kitA25526 PSC (OCT4, SSEA4) Immunocytochemistry Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 kitA25525 PSC (SOX2, TRA-1-60) Immunocytochemistry Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 kitA25538 3-Germ Layer Immunocytochemistry Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 kitA1896701 FluoroBrite™ DMEM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 500 mLA14353 Alkaline Phosphatase Live Stain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50 μLA15871 TaqMan® hPSC Scorecard™ Kit, FAST 96 well . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 platesA18945 Gibco® Human Episomal iPSC Line . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 vialA16517 CytoTune®-iPS 2.0 Sendai Reprogramming Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 packA14703 Episomal iPSC Reprogramming Vectors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 kitA15960 Epi5™ Episomal iPSC Reprogramming Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 kitA1517001 Essential 8® Medium. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 500 mLA14700 Vitronectin (VTN-N) Recombinant Human Protein, Truncated . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 mLA24354 Human Neural Stem Cell Immunocytochemistry Kit. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 testsA1647801 PSC Neural Induction Medium. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 500 mL

Preparation of feeder layerInactivated Mouse Embryonic Fibroblasts

3

2 Selection of iPSC clones for feeder-dependent culture

26 27

Inactivated Mouse Embryonic Fibroblasts 3

Note: It is not necessary to wash the culture surface before adding cells or medium.

3. You can use the coated vessels immediately or store them at room temperature for up to 24 hours.

Prepare MEF Medium1. Prepare 500 mL of complete iMEF medium by mixing the following components (pre-warmed in a 37°C,

5% CO2 incubator):

Component Amount

DMEM, high glucose 444.5 mL

FBS, ESC-qualified 50 mL

MEM Non-Essential Amino Acids Solution, 10 mM 5 mL

2-Mercaptoethanol 0.5 mL

Thaw MEFs1. Remove the cryovial containing inactivated MEFs from the liquid nitrogen storage tank.

2. Briefly roll the vial between hands to remove frost, and swirl it gently in a 37°C water bath.

3. When only a small ice crystal remains in the vial, remove it from water bath. Spray the outside of the vial with 70% ethanol before placing it in the cell culture hood.

4. Pipet the thawed cells gently into a 50 mL conical tube.

5. Add 10 mL of pre-warmed MEF medium dropwise to the cells while gently swirling the conical tube. Gently mix by pipetting up and down.

Note: Adding the medium slowly helps the cells to avoid osmotic shock.

6. Transfer entire cell suspension to a 15 mL conical tube and centrifuge at 200 x g for 5 minutes.

7. Aspirate the supernatant and resuspend the cell pellet in an appropriate volume of pre-warmed MEF medium.

8. Use an appropriate volume of the cell suspension to determine the viable cell number using your method of choice.

Plate MEFs1. Aspirate the gelatin solution from the AF-coated culture vessels.

2. Add the appropriate amount of MEF medium into each culture vessel (refer to the table on the next page).

3. Into each of these culture vessels, add the appropriate amount of MEF suspension (refer to the table on the next page).

3 Preparation of feeder layer

Mouse (ICR) Inactivated Embryonic Fibroblasts

Inactivated Mouse Embryonic Fibroblasts (iMEFs) are used as feeder layers for culturing PSCs, including mouse and human PSCs, in their undifferentiated state. Gibco™ iMEFs are isolated from ICR mouse embryos under sterile conditions, expanded for up to two passages, and then mitotically inactivated by γ-irradiation. The growth-arrested feeder layer supports the PSC culture by providing nutrients, growth factors, and matrix components, and it enables PSCs to survive and proliferate more readily in culture.

Before you begin

Guidelines for using MEFs as feeder layers to culture mouse and human pluripotent stem cells (PSCs):

• All solutions and equipment that come in contact with the cells must be sterile. Always use proper aseptic technique and work in a laminar flow hood.

• Make sure to prepare MEF feeder layer at least one day before culturing ESCs.

• After thawing, transfer MEFs into pre-warmed medium.

• Plate MEFs on culture vessels coated with 1X Attachment Factor (AF) solution.

• Use MEF dishes or plates within 3–4 days of preparation.

Protocol: Setting up MEFs as feeder layer

Materials needed:


• Gibco™ Dulbecco’s Modified Eagle Medium (DMEM), high glucose, GlutaMAX-I Supplement (Cat. No. 10569 -044)




• Gibco™ MEM Non-Essential Amino Acids Solution (MEM NEAA Solution) (100X) (100 mL) (Cat. No. 11140-050)

• Gibco™ MEM Non-Essential Amino Acids Solution (MEM NEAA Solution) (100X) (20 x 100 mL) (Cat. No. 11140-076)

Coat culture vessels with Attachment Factor Protein1. Cover the whole surface of each culture vessel with AF solution and incubate the vessels for 30 minutes

at 37°C or for 2 hours at room temperature. Refer to the recommended coating volumes in the table on page 28.

2. Using sterile technique in a laminar flow hood, completely remove the AF solution from the culture vessel by aspiration.

28 29

Note: The appropriate cell density should be optimized for the specific application. We recommend 3.0 x 104 MEFs/cm2 as a good starting point.

4. Move the culture vessels in several quick back-and-forth and side-to-side motions to disperse the cells across the surface of the vessels.

5. Incubate the cells in a 37°C incubator with a humidified atmosphere of 5% CO2.

6. Use the MEF culture vessels within 3–4 days after plating.

Vessel size AF coating volume Media volume Number of MEFs

96-well plate 0.1 mL 0.1 mL 1.0 x 104/well

24-well plate 0.3 mL 0.5 mL 6.0 x 104/well

12-well plate 0.5 mL 1 mL 1.1 x 105/well

6-well plate 1 mL 2 mL 2.9 x 105/well

60 mm dish 3 mL 5 mL 5.9 x 105

100 mm dish 9 mL 10 mL 1.8 x 106

25 cm2 flask 3 mL 5 mL 7.5 x 105

75 cm2 flask 9 mL 15 mL 2.3 x 106

Expected resultsThe bright-field image below shows Mouse (ICR) Inactivated Embryonic Fibroblasts plated at the recommended density on culture dishes coated with 1X AF solution. The image was taken with a 10X objective.

ReferencesEvans MJ, Kaufman MH (1981) Establishment in culture of pluripotent cells from mouse embryos. Nature 292:154–156.

Thomson JA, Itskovitz-Eldor J, Shapiro SS et al. (1998) Embryonic stem cell lines derived from human blastocysts. Science 282:1145–1147.

Williams RL, Hilton DJ, Pease S et al. (1988) Myeloid leukaemia inhibitory factor maintains the developmental potential of embryonic stem cells. Nature 336:684–687.

For additional technical information such as Safety Data Sheets (SDS), Certificates of Analysis, visit www.lifetechnologies.com/support. For further assistance, email [email protected] © 2014 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. DISCLAIMER: LIFE TECHNOLOGIES CORPORATION AND/OR ITS AFFILIATE(S) DISCLAIM ALL WARRANTIES WITH RESPECT TO THIS DOCUMENT, EXPRESSED OR IMPLIED, INCLUDING BUT NOT LIMITED TO THOSE OF MERCHANTABILITY, FITNESS FOR A PARTICULAR PURPOSE, OR NON-INFRINGEMENT. TO THE EXTENT ALLOWED BY LAW, IN NO EVENT SHALL LIFE TECHNOLOGIES AND/OR ITS AFFILIATE(S) BE LIABLE, WHETHER IN CONTRACT, TORT, WARRANTY, OR UNDER ANY STATUTE OR ON ANY OTHER BASIS FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING BUT NOT LIMITED TO THE USE THEREOF.

www.lifetechnologies.com 11 June 2014

Vessel Size AF Coating

Volume Volume of

Media Number of MEFs

96-well plate 0.1 mL 0.1 mL 1.0 × 104/well

24-well plate 0.3 mL 0.5 mL 6.0 × 104/well

12-well plate 0.5 mL 1 mL 1.1 × 105/well

6-well plate 1 mL 2 mL 2.9 × 105/well

60-mm dish 3 mL 5 mL 5.9 × 105

100-mm dish 9 mL 10 mL 1.8 × 106

25-cm2 flask 3 mL 5 mL 7.5 × 105

75-cm2 flask 9 mL 15 mL 2.3 × 106

Expected Results The bright field image below shows Mouse (ICR) Inactivated Embryonic Fibroblasts plated at the recommended density on culture dishes coated with 1× AF solution. The image was taken with a 10× objective.

Related Products Product Cat. no.

Dulbecco’s Modified Eagle Medium (D-MEM), high glucose

A10569

Dulbecco’s PBS (DPBS) without Calcium and Magnesium

14190

Fetal Bovine Serum (FBS), ES-Cell Qualified A16141

Attachment Factor S-006-100

Explanation of Symbols and Warnings The symbols present on the product label are explained below:

Temperature limitation Use by Batch code Catalog number Manufacturer

References Evans, M., Kaufman, M. (1981) Establishment in culture of

pluripotent cells from mouse embryos. Nature 292, 154–156.

Thomson, J.A., Itskovitz-Eldor, J., Shapiro, S.S., Waknitz, M.A., Swiergiel, J.J., Marshall, V.S., Jones, J.M. (1998) Embryonic stem cell lines derived from human blastocysts. Science 282, 1145–1147.

Williams, R.L., Hilton, D.J., Pease, S., Willson, T.A., Stewart, C.L., Gearing, D.P., Wagner, E.F., Metcalf, D., Nicola, N.A., Gough, N.M. (1988) Myeloid leukaemia inhibitory factor maintains the developmental potential of embryonic stem cells. Nature 336, 684–687.

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Important Licensing Information This product may be covered by one or more Limited Use Label Licenses. By use of this product, you accept the terms and conditions of all applicable Limited Use Label Licenses.

Made in USA 7335 Executive Way Frederick, MD 21704

3 Preparation of feeder layer

Feeder-dependent culture of human induced pluripotent stem cells (hiPSCs)KnockOut Serum Replacement – Multi-Species

4

30 31

KnockOut Serum Replacement – Multi-Species 4

Preparing media and materials10 μg/mL FGF-Basic solution (1,000 μL)1. To prepare 1 mL of 10 μg/mL FGF-Basic solution, aseptically mix the following components:

Component Amount

FGF-Basic 10 μg

DPBS, no calcium, no magnesium 980 μL

10% KnockOut SR - Multi-Species 10 μL

2. Aliquot and store at –20°C for up to 6 months.

1 mg/mL Collagenase Type IV solution1. Add DMEM/F-12 to Collagenase Type IV to make a 10 mg/mL stock solution. Gently vortex to suspend and

filter sterilize the solution. This solution can be aliquoted and frozen at –20°C until use.

2. Make a working solution of 1 mg/mL Collagenase Type IV in DMEM/F-12. The working solution can be used for 2 weeks if properly stored at 2°C to 8°C (store in aliquots to avoid repeated warming).

iMEF Medium (for 500 mL of complete medium)See Chapter 3 “Preparation of feeder layer” on page 25.

Pluripotent Stem Cell (PSC) Culture Medium (for 100 mL complete medium)1. To prepare 100 mL of complete PSC Culture Medium, aseptically mix the following components:

Component Amount

DMEM/F-12 79 mL

KnockOut SR – Multi-Species 20 mL

MEM Non-Essential Amino Acids Solution, 10 mM 1 mL

FGF-Basic (10 μg/mL)* 40 μL

2-Mercaptoethanol, (55 mM) 100 μL

* Add FGF-Basic at the time of medium change (final concentration 4 ng/mL).

2. Complete PSC Culture Medium (without FGF-Basic) can be stored at 2°C to 8°C for up to 4 weeks. FGF-Basic must be added to a final concentration of 4 ng/mL at time of medium change.

Preparing MEF dishesSee Chapter 3 “Preparation of feeder layer” on page 25 for more on preparing MEF dishes.

4 Feeder-dependent culture of human induced pluripotent stem cells (hiPSCs)

KnockOut™ Serum Replacement – Multi-Species

iPSCs have traditionally been maintained on a layer of inactivated MEF feeder cells with medium containing a rich supplement like Gibco™ KnockOut™ Serum Replacement (SR). KnockOut SR is a more defined fetal bovine serum (FBS) substitute that supports more robust and better-quality PSC growth compared to FBS. This robust growth is particularly valuable when culturing newly derived iPSC clones. PSCs can be maintained under feeder-dependent conditions with KnockOut SR for many passages without compromising the proliferation or pluripotency and differentiation potential of the cells.

Resource Center

Included in the Gibco iPSC Lab Starter Kit with Reprogramming is KnockOut Serum Replacement (Cat. No. 10828010). KnockOut Serum Replacement is the same formulation as KnockOut Serum Replacement – Multi-Species and the protocols are the same.

Protocol: Feeder-dependent culture of human induced pluripotent stem cells

Materials needed:• Gibco™ Dulbecco’s Modified Eagle Medium (DMEM), high glucose, GlutaMAX™-I Supplement (Cat. No.

10569-010)


• Gibco™ DMEM/F-12 with GlutaMAX™-I Supplement (Cat. No. 10565-018)

• KnockOut Serum Replacement – Multi-Species (Cat. No. A31815-02)

• Gibco™ MEM Non-Essential Amino Acids Solution (100X) (Cat. No. 11140-050)



• Gibco™ FGF-Basic (AA 1–155) Recombinant Human Protein (Cat. No. PHG0264)

• Gibco™ Collagenase Type IV (Cat. No. 17104-019) for enzymatic passaging or Gibco™ StemPro™ EZPassage™ Disposable Stem Cell Passaging Tool (Cat. No. 23181-010) for mechanical passaging

• Cell Scraper (Falcon™, Cat. No. 353085)

• Gibco™ Dulbecco’s Phosphate Buffered Saline (DPBS), no calcium, no magnesium (Cat. No. 14190144)


• 37°C water bath

• Appropriate tissue culture plates and supplies

32 33

Enzymatic passaging using collagenaseYou may passage cells via the enzymatic method as described below, or mechanically as described in the following section.

1. Aspirate the MEF medium from a dish containing inactivated MEFs and add pre-warmed PSC Culture Medium to the dish, 3–4 hours before plating iPSCs.

2. Label the new MEF dish with the cell line name, the new passage number, the date, the split ratio, and user initials. Return the plate to the incubator.

3. Under a dissecting microscope, remove differentiated colonies from the dish to be passaged.

4. Aspirate the spent medium from the dish with a Pasteur pipette.

5. Add Collagenase Type IV (1 mg/mL) solution to the dish containing iPSCs. Adjust the volume of Collagenase Type IV for various dish sizes (e.g., 35 mm dishes require 1 mL of Collagenase Type IV).

6. Incubate the dish(es) for 30–60 minutes in a 37°C, 5% CO2 incubator. Note that the incubation times may vary among different batches of collagenase; therefore, examination of the colonies is needed to determine the appropriate incubation time. Note: As an alternative to Collagenase Type IV, you may use Gibco™ Dispase II (Cat. No. 17105-041) at a concentration of 2 mg/mL and incubate the dish(es) for 2–3 minutes in a 37°C, 5% CO2 incubator.

7. Stop the incubation when the edges of the colonies are starting to pull away from the plate (see Figure 4.2).

Enzymatic Passaging Using Collagenase

You may passage cells via the enzymatic method as described below, or mechanically as described in the following section.


2. Label the new MEF dish with the cell line name, the new passage number, the date, the split ratio, and user initials. Return the plate to the incubator.

3. Under a dissecting microscope, remove differentiated colonies from the dish to be passaged.

4. Aspirate the spent medium from the dish with a Pasteur pipette.

5. Add Collagenase Type IV (1 mg/mL) solution to the dish containing iPSCs. Adjust the volume of Collagenase Type IV for various dish sizes (e.g., 35-mm dishes require 1 mL of Collagenase IV).

6. Incubate the dish(es) for 30–60 minutes in a 37°C, 5% CO2 incubator. Note that the incubation times may vary among different batches of collagenase; therefore, examination of the colonies is needed to determine the appropriate incubation time.

Note: As an alternative to Collagenase Type IV, you may use Dispase at a concentration of 2 mg/mL and incubate the dish(es) for 2–3 minutes in a 37° C, 5% CO2 incubator.

7. Stop the incubation when the edges of the colonies are starting to pull away from the plate (see Figure 4).

Figure 4 PSC colony pulling away from iMEF layer after treatment with enzyme

8. Aspirate the Collagenase Type IV Solution with a Pasteur pipette. Remove the collagenase carefully without disturbing the attached cell layer.

9. Add PSC Culture Medium to each dish. Use a 5-mL pipette to gently blow the cells off the surface of the dish while pipetting up and down. Make sure to pipet gently to minimize the formation of bubbles.

10. After the iPSCs have been removed from the surface of the well, pool the contents of the wells into a 15-mL conical tube.

11. Using a 5-mL pipette, add PSC Culture Medium to the dish to wash and collect any residual cells. Pipet up the medium and cells, and then add the collected cells to the 15-mL tube.

12. Pipet cells up and down gently a few times in the 15-mL tube to further break up cell colonies. Pipet carefully to reduce foaming.

Note: Avoid making a single cell suspension.

13. Centrifuge at 200 × g for 5 minutes, and then aspirate the supernatant from the iPSC pellet.

14. Resuspend the pellet with an appropriate amount of PSC Culture Medium (refer to Table 2). This is dependent on the split ratio and the number of dishes used.

15. Mix the cell suspension well with a 10-mL pipette. Be careful not to break up the colonies too much or cause bubbles in the media.

Figure 4.2. PSC colony pulling away from iMEF layer after treatment with enzyme.

8. Aspirate the Collagenase Type IV solution with a Pasteur pipette. Remove the collagenase carefully without disturbing the attached cell layer.

9. Add PSC Culture Medium to each dish. Use a 5 mL pipette to gently blow the cells off the surface of the dish while pipetting up and down. Make sure to pipet gently to minimize the formation of bubbles.


Passaging iPSCsWhen to split cellsIn general, split cells when one of the following occurs:

• The MEF feeder layer is one week old.

• iPSC colonies are becoming too dense or too large.

• Increased differentiation occurs.

Guidelines for passaging newly derived iPSC clones• In the first 5 to 8 passages, newly reprogrammed clones are passaged at a 1:1 split ratio approximately

every week. As the clones expand in higher passages, the new clones are treated more similarly to established iPSC lines, with split ratios typically between 1:2 and 1:4 every few days.

• From P1 to P5, colonies are fragile and few, requiring mechanical passaging. A 25 gauge 1½ inch needle can be used to cut each colony into a few pieces. A 200 μL pipette can be used to transfer the cut pieces to a freshly prepared 6-well culture dish containing iPSC medium.

• From P6 onwards, clones slowly become more amenable to enzymatic passaging. The first few times that a clone is passaged enzymatically, make sure to have a backup culture that is passaged mechanically in case the clone is not yet ready.

Guidelines for passaging established iPSC lines• The split ratio can vary, though it is generally between 1:2 and 1:4. Occasionally, cells will grow at a different

rate and the split ratio will need to be adjusted. A general rule is to observe the last split ratio and adjust the ratio according to the appearance of the iPSC colonies.

• If the cells look healthy and colonies have enough space, split using the same ratio. If they are overly dense and crowding, increase the ratio. If the cells are sparse, decrease the ratio. Cells will need to be split every 4–10 days based upon appearance.

• iPSCs do well in iMEF plates that have been conditioned with PSC Culture Medium. It is common practice to condition new feeder plates before passaging iPSCs onto them.

Figure 4.1. PSC colonies ready to be passaged. Note the large colony and the close proximity of the colonies to each other.

Figure 2 iPSCs cultured on mitotically inactivated MEF feeder layer in PSC Culture Medium containing KSR

Passaging iPSCs

When to Split Cells

In general, split cells when one of the following occurs:

• The MEF feeder layer is 2 weeks old.



Split Ratio

• The split ratio can vary, though it is generally between 1:2 and 1:4. Occasionally, cells will grow at adifferent rate and the split ratio will need to be adjusted. A general rule is to observe the last split ratio andadjust the ratio according to the appearance of the iPSC colonies.

• If the cells look healthy and colonies have enough space, split using the same ratio. If they are overly denseand crowding, increase the ratio. If the cells are sparse, decrease the ratio. Cells will need to be split every4–10 days based upon appearance.

• iPSCs do well in iMEF plates that have been conditioned with PSC Culture Medium. It is common practiceto condition new feeder plates before passaging iPSCs into them.

Figure 3 PSC colonies ready to be passaged. Note the large colony and the close proximity of the colonies to each other.

PSC colony

MEF


34 35

Mechanical passaging using the StemPro EZPassage Disposable Cell Passaging Tool1. Replace the medium in the dish containing the cells with fresh PSC Culture Medium.

2. Under a laminar flow hood, open the package containing the EZPassage tool and remove the tool.

3. Hold the culture vessel in one hand and pull (roll) the EZPassage tool across the entire dish in one direction. Apply gentle but firm pressure so that the entire roller blade touches the dish and maintains uniform pressure during the rolling action.

4. Keep rolling the EZPassage tool parallel to the first pass until the entire dish has been covered.

5. Rotate the culture dish 90°, and then repeat the rolling process as described above.

6. When you are finished, discard the EZPassage tool and do not reuse. Use a cell scraper to lift cell clusters off the plate, if necessary.

7. Using a serological pipette, rinse the dish with medium so that the cut colonies are suspended in the medium.

8. Transfer the medium containing the colonies to a 15 mL sterile tube.

9. Seed the cell colonies on dishes plated with mitotically inactivated MEFs at an appropriate density.

10. Place the plates into a 37°C, 5% CO2 incubator. Shake the plates gently to evenly spread out cells.

Figure 4.3. PSC colony after being cut with the StemPro EZPassage Disposable Cell Passaging Tool.

16. Add appropriate volume of cell suspension to each dish. Return the dish to the incubator.

17. Move the dish(es) in several quick, short, back-and-forth and side-to-side motions to disperse cells across the surface of the dishes.

18. Incubate cells overnight to allow colonies to attach. Replace spent medium daily.

Note: While cells are attaching, be careful when opening and closing the incubator doors to avoid disturbing the even distribution of cells on the surface of the wells.

Mechanical Passaging Using the StemPro® EZPassage™ Disposable Cell Passaging Tool

1. Replace the medium in the dish containing the cells with fresh PSC Culture Medium.

2. Under a laminar flow hood, open the package containing the EZPassage™ tool and remove the tool.

3. Hold the culture vessel in one hand and pull (roll) the EZPassage™ tool across the entire dish in one direction. Apply gentle but firm pressure so that the entire roller blade touches the dish and maintains uniform pressure during the rolling action.

4. Keep rolling the EZPassage™ tool parallel to the first pass until the entire dish has been covered.

5. Rotate the culture dish 90°, and then repeat rolling the cell layer as described above.

6. When you are finished, discard the EZPassage™ tool and do not reuse. Use a cell scraper to lift cell clusters off the plate, if necessary.

7. Using a serological pipette, rinse the dish with medium so that the cut colonies are suspended in the medium.

8. Transfer the medium containing the colonies to a 15-mL sterile tube.

9. Seed the cell colonies on dishes plated with mitotically inactivated MEFs at an appropriate density.

10. Place the plates into a 37°C, 5% CO2 incubator. Shake the plates gently to evenly spread out cells.

Figure 5 PSC colony after being cut with the StemPro® EZPassage™ Disposable Cell Passaging Tool


10. After the iPSCs have been removed from the surface of the well, pool the contents of the wells into a 15 mL conical tube.

11. Using a 5 mL pipette, add PSC Culture Medium to the dish to wash and collect any residual cells. Pipet up the medium and cells, and then add the collected cells to the 15 mL tube.

12. Pipet cells up and down gently a few times in the 15 mL tube to further break up cell colonies. Pipet carefully to reduce foaming. Note: Avoid making a single cell suspension.

13. Centrifuge at 200 x g for 5 minutes, and then aspirate the supernatant from the iPSC pellet.

14. Resuspend the pellet with an appropriate amount of PSC Culture Medium (refer to Table 4.1 on page 38). This is dependent on the split ratio and the number of dishes used.

15. Mix the cell suspension well with a 10 mL pipette. Be careful not to break up the colonies too much or cause bubbles in the medium.

16. Add appropriate volume of cell suspension to each dish. Return the dish to the incubator.

17. Move the dish(es) in several quick, short, back-and-forth and side-to-side motions to disperse cells across the surface of the dishes.

18. Incubate cells overnight to allow colonies to attach. Replace spent medium daily. Note: While cells are attaching, be careful when opening and closing the incubator doors to avoid disturbing the even distribution of cells on the surface of the wells.


36 37

Thawing and plating iPSCsOptional: If a clone has been frozen and needs to be placed back in culture, use the following protocol to thaw and plate the iPSCs.


2. Label the dish containing inactivated MEF cells with the passage number from the vial, the date, and user initials.

3. Remove the vial of iPSCs from liquid nitrogen storage using metal forceps. Note: If the vial is exposed to ambient temperatures for more than 15 seconds between removal and thawing, transfer the vial into a container containing a small amount of liquid nitrogen.

4. Roll the vial between your gloved hands until the outside is free of frost. This should take ~10–15 seconds.

5. Immerse the vial in a 37°C water bath without submerging the cap. Swirl the vial gently.

6. When only an ice crystal remains, remove the vial from the water bath.

7. Spray the outside of the vial with 70% ethanol and place it in a laminar flow hood.

8. Pipet cells gently into a sterile 50 mL conical tube using a 5 mL sterile pipette.

9. Slowly add 10 mL of PSC Culture Medium drop-wise to cells in the 50 mL conical tube. While adding the medium, gently move the tube back and forth to mix the iPSCs. This reduces osmotic shock to the cells.

10. Rinse the vial with 1 mL of PSC Culture Medium and add to the 50 mL conical with cells.

11. Transfer cell suspension to a 15 mL conical tube and centrifuge the cells at 200 x g for 5 minutes.

12. Aspirate and discard the supernatant.

13. Resuspend the cell pellet in sufficient volume of PSC Culture Medium according to Table 4.3 on the next page by gently pipetting the cells up and down in the tube a few times.

14. Aspirate the spent PSC Culture Medium from the MEF dish and slowly add the thawed colonies onto the dish. Place dish gently into the 37°C, 5% CO2 incubator and move the dish in several quick, short, back-and-forth and side-to-side motions to disperse cells across the surface of the dishes.

15. Incubate the cells overnight.

16. The next day, remove the spent medium with debris using a sterile serological pipette and transfer it into a prepared MEF dish. You can use this dish as a backup in case there is a problem with the main dish.


Table 4.1. Tools and enzymes for routine passaging of feeder-dependent PSCs.

Passaging tool or enzyme Description Protocol

StemPro EZPassage Disposable Stem Cell Passaging Tool (Cat. No. 23181-010)

Novel stem cell passaging device that makes bulk mechanical passaging of stem cell colonies more rapid and reproducible

Generation of uniformly cut stem cell colonies by rolling tool across plate

Collagenase Type IV (Cat. No. 17104-019) Optimized for enzymatic passaging, relatively gentle, dissociates well at physiological temperature and pH, and requires no mechanical agitation or special equipment

30–60 minutes incubation at 37°C (1 mg/mL)

Dispase II (Cat. No. 17105-041) Useful for the isolation and routine enzymatic passaging of cells. Relatively gentle, dissociates well at physiological temperature and pH, and generally maintains cell membrane integrity

2–3 minutes incubation at 37°C (2 mg/mL)

Table 4.2. Dissociation enzymes for harvesting of cells for downstream applications.

Dissociation enzyme or tool Description Protocol

Gibco™ StemPro™ Accutase™ Cell Dissociation Reagent (Cat. No. A11105-01)

Cell detachment solution for detaching primary and stem cells and maintaining high cell viability

1–2 minutes incubation at 37°C (1X, ready to use)

Gibco™ TrypLE™ Express Enzyme (1X) (Cat. No. 12604-021)

Animal origin–free, recombinant enzyme used for dissociating a wide range of adherent cells. Compared to trypsin, it is gentle on cells, easy to use, and room temperature stable

3–5 minutes incubation at 37°C (1X, ready to use)


38 39

Transition of mature iPSCs to feeder-free culture systemEssential 8 Adaptation Kit

5

17. Add fresh PSC Culture Medium to each dish according to the volumes in Table 4.3. Place both plates gently into a 37°C, 5% CO2 incubator overnight.

18. Examine cells under the microscope and replace spent medium daily from both plates. If feeding more than one plate, use a different pipette for each well to reduce risk of contamination. Colonies may not be visible for up to a week.

Table 4.3. Volume of PSC Culture Medium required.

Culture vessel Surface area Volume (mL)

6-well plate 10 cm2/well 2 mL per well

12-well plate 4 cm2/well 1 mL per well

24-well plate 2 cm2/well 0.5 mL per well

35 mm dish 10 cm2 2 mL



Figure 4.4. iPSCs cultured on mitotically inactivated MEF feeder layer in PSC Culture Medium containing KnockOut SR – Multi-Species.

Figure 2 iPSCs cultured on mitotically inactivated MEF feeder layer in PSC Culture Medium containing KSR

Passaging iPSCs

When to Split Cells


• The MEF feeder layer is 2 weeks old.



Split Ratio

• The split ratio can vary, though it is generally between 1:2 and 1:4. Occasionally, cells will grow at adifferent rate and the split ratio will need to be adjusted. A general rule is to observe the last split ratio andadjust the ratio according to the appearance of the iPSC colonies.

• If the cells look healthy and colonies have enough space, split using the same ratio. If they are overly denseand crowding, increase the ratio. If the cells are sparse, decrease the ratio. Cells will need to be split every4–10 days based upon appearance.

• iPSCs do well in iMEF plates that have been conditioned with PSC Culture Medium. It is common practiceto condition new feeder plates before passaging iPSCs into them.

Figure 3 PSC colonies ready to be passaged. Note the large colony and the close proximity of the colonies to each other.

PSC colony

MEF


40 41

Essential 8 Adaptation Kit 5

Protocol: Adapting cells from feeder-dependent to feeder-free culture system

Materials needed:• Gibco™ Essential 8™ Adaptation Kit (Cat. No. A25935)

– Gibco™ Essential 8™ Basal Medium (500 mL) – Gibco™ Essential 8™ Supplement (50X) (10 mL) – rhLaminin-521 (100 μg)

• Gibco™ TrypLE™ Select Enzyme (1X) (Cat. No. 12563-011)

Supplemental reagents needed:• Gibco™ Dulbecco’s Phosphate Buffered Saline (DPBS), calcium, magnesium (Cat. No. 14040182)

• Gibco™ Dulbecco’s Phosphate Buffered Saline (DPBS), no calcium, no magnesium (Cat. No. 14190144)

Coat culture vessels with rhLaminin-521Instructions for coating a 60 mm culture dish with rhLaminin-521 at a coating concentration of 0.5 μg/cm2 are provided below. For volumes used in other culture vessels, refer to Table 5.1 on page 43. To calculate the working concentration of rhLaminin-521 used with other coating concentrations and to determine the appropriate dilution factor, use the equations on the previous page.

1. Upon receipt, thaw the vial of rhLaminin-521 slowly at 2°C to 8°C, mix by gentle trituration, and prepare usage size aliquots in polypropylene tubes. Freeze aliquots at –30°C to –10°C or store aliquots at 2°C to 8°C for up to 3 months.

2. To coat a 60 mm dish, add 100 μL aliquot of rhLaminin-521 into a 15 mL conical tube containing 4 mL of sterile DPBS containing calcium and magnesium (Cat. No. 14040182). Gently resuspend by pipetting the rhLaminin-521 dilution up and down. Note: This results in a working concentration of 2.5 μg/mL (i.e., a 1:40 dilution).

3. Add the diluted rhLaminin-521 solution to the 60 mm dish (refer to Table 5.1 on page 43 for the recommended volumes for other culture vessels). When used to coat a 60 mm dish (20 cm2) at 4 mL/well, the final coating concentration will be 0.5 μg/cm2.

4. Incubate the plates in a 37°C, 5% CO2 incubator for 2 hours for efficient coating. Note: Alternatively, the plate can be coated at 2°C to 8°C overnight. Do not allow the culture vessel to dry. Prior to use, pre-warm the culture vessel to room temperature for at least 1 hour.

5. Immediately prior to plating of cells, aspirate the rhLaminin-521 solution and discard. It is not necessary to rinse off the culture vessel after the removal of rhLaminin-521. Cells can be passaged directly onto the rhLaminin-521–coated culture vessels.

5 Transition of mature iPSCs to feeder-free culture system

Essential 8 Adaptation Kit

While PSCs have traditionally been maintained on feeder layers, feeder-free culture systems are simpler, eliminating the need to prepare MEFs prior to culture or remove MEFs prior to downstream applications. Aside from this advantage, Gibco™ Essential 8™ Medium, in particular, offers a fully-defined formulation that minimizes lot-to-lot variability.

Transitioning from feeder-dependent to feeder-free systems can sometimes be stressful for PSCs. Recombinant human Laminin 521 (rhLaminin-521) is a recombinant human protein that provides a defined surface for feeder-free human PSC culture and has been proven to promote cellular survival under stressful conditions in the absence of small molecule inhibitors. In addition, rhLaminin-521 has been shown to support PSC growth for >20 passages without any signs of karyotypic abnormalities and maintain the ability of PSCs to differentiate into all three germ line lineages.

rhLaminin-521 is provided in the Gibco™ Essential 8™ Adaptation Kit to promote optimal PSC survival following feeder-dependent to feeder-free transitions when used in conjunction with Essential 8 Medium.

Before you begin

Guidelines for thawing and plating rhLaminin-521• Thaw rhLaminin-521 slowly at 2ºC to 8ºC. Avoid extended exposure of protein to ambient temperatures. For

long coating procedures the laminin stock solution should be kept on ice.

• Once thawed, rhLaminin-521 stock is stable for up to 3 months when stored at 2ºC to 8ºC.

• Divide thawed rhLaminin-521 into usage-size aliquots and store in a non–frost-free freezer at –30ºC to –10ºC. Avoid repeated freeze-thaw cycles.

• Plates can be coated in advance of experiments, sealed with Parafilm™ wrap, and stored at 2°C to 8°C under aseptic conditions for up to 2 weeks. Do not allow the culture surface to dry.

Working concentrationThe optimal working concentration of rhLaminin-521 is cell line dependent and must be determined empirically. We recommend using an initial coating concentration of 0.5 μg/cm2 on the culture surface. Prior to coating culture vessels, calculate the working concentration according to the formula below and dilute the stock appropriately. Refer to Table 5.1 on page 43 for culture surface area and required coating volumes.

Working conc. = Coating conc. ×Culture surface area

Dilution factor =Stock concentration (100 μg/mL)

Vol. required for surface area Working concentration

For example, to coat a 60 mm dish at a coating concentration of 0.5 μg/cm2, you will need to prepare 4 mL of diluted rhLaminin-521 solution (20 cm2/dish surface area and 4 mL of diluted rhLaminin-521/dish; see Table 5.1) at the following working concentration:

Working conc. =0.5 μg

cm2 ×20 cm2

=2.5 μg

Dilution factor =100 μg/mL

= 40X (i.e., 1:40 dilution)4 mL mL 2.5 μg/mL

42 43

16. Distribute 1 mL of the resuspended PSC clusters into the rhLaminin-521 precoated dish. Move the vessel in several quick back-and-forth and side-to-side motions to disperse the cells across its surface.

17. Incubate the cells in a 37°C, 5% CO2 incubator and passage them when they are 60%–85% confluent to maintain optimal cell health. Note: Cells cultured in Essential 8 Medium must be fed daily.

Table 5.1. rhLaminin-521 coating reagent volumes (per well or per dish).

Culture vessel (surface area) Volume of diluted rhLaminin-521 solution

6-well plate (10 cm2) 2 mL

12-well plate (4 cm2) 0.8 mL

24-well plate (2 cm2) 0.4 mL

35 mm dish (10 cm2) 2 mL

60 mm dish (20 cm2) 4 mL

100 mm dish (60 cm2) 12 mL

Table 5.2. Passaging and culture reagent volumes (per well or per dish).

Culture vessel (surface area)

Volume of DPBS for wash

Volume of Collagenase Type IV

Volume of Essential 8 Medium*

Volume of Essential 8 Medium**

6-well plate (10 cm2) 2 mL 1 mL 1 mL 2 mL

12-well plate (4 cm2) 1 mL 0.4 mL 0.4 mL 8 mL

24-well plate (2 cm2) 0.5 mL 0.2 mL 0.2 mL 0.4 mL

35 mm dish (10 cm2) 2 mL 1 mL 1 mL 2 mL



* For initial resuspension and wash. ** For final two resuspensions. Note: Split ratios may need to be optimized depending on the cell line and the percentage confluency of PSCs at the time of harvest.

Related products• Essential 8 Medium (Cat. No. A1517001)

• Gibco™ rhLaminin-521 (Cat. No. A29248)

• Gibco™ DPBS, no calcium, no magnesium (Cat. No. 14190)

• Collagenase Type IV (Cat. No. 17104-019)

• DMEM/F-12 with GlutaMAX™-I Supplement (Cat. No. 10565)

Essential 8 Adaptation Kit 5

Adapt cells to feeder-free culture in Essential 8 MediumFollow the instructions below to adapt feeder-dependent PSC cultures to feeder-free conditions in Essential 8 Medium on rhLaminin-521–coated culture vessels. The volumes given in the procedure are for 60 mm culture dishes. For volumes used in other culture vessels, refer to Tables 5.1 and 5.2 on page 43.

1. When the feeder-dependent cultures reach passaging confluency (60%–85% confluent with round colonies that are not overcrowded), the cells are ready for adaptation to feeder-free culture conditions.

2. Coat culture vessels with rhLaminin-521 per instructions noted on page 41.

3. Prepare a 1 mg/mL Gibco™ Collagenase Type IV solution in Gibco™ DMEM/F-12 with GlutaMAX™ Supplement and filter sterilize using a 0.2 μm filter.

4. Aspirate the spent medium from the culture vessel.

5. Rinse the vessel once with 4 mL of DPBS without calcium or magnesium.

6. Add 2 mL of 1 mg/mL Collagenase Type IV, pre-warmed to 37ºC.

7. Incubate the vessel for ~45 minutes in a 37°C, 5% CO2 incubator. Note: Stop the incubation when the edges of the colonies begin to curl from the plate. Do not over-incubate.

8. Add 2 mL of Essential 8 Medium and gently dislodge the colonies from the plate by washing off colonies with a 5 mL serological pipette. Repeat trituration until the desired cluster size is achieved.

9. Transfer the suspended colony clusters into a 15 mL conical tube.

10. Add 2 mL of Essential 8 Medium to dislodge the remaining colonies and transfer them to the 15 mL conical tube.

11. Let the colony fragments sediment to the bottom of the 15 mL conical tube for 5 minutes by gravity.

12. Discard the supernatant, add 4 mL of Essential 8 Medium, and gently resuspend the sedimented colony fragments by pipetting up and down 2 times.

13. Gravity sediment the clusters for 2–5 minutes.

14. While the colony fragments are sedimenting, aspirate the matrix solution from the freshly prepared rhLaminin-521–coated 60 mm dish and add 4 mL of Essential 8 Medium.

15. Aspirate the supernatant and resuspend the sedimented PSC clusters by gently pipetting them up and down 2 times in 4 mL Essential 8 Medium, taking care not to break them down further.

5 Transition of mature iPSCs to feeder-free culture system

44 45

Culture, expansion, and cryopreservation of pluripotent stem cells in feeder-free culture systemEssential 8 Medium

6

46 47

Essential 8 Medium 6

Protocol: Culturing pluripotent stem cells in feeder-free medium

Materials needed:• Essential 8 Medium, consisting of Essential 8 Basal Medium and Essential 8 Supplement (50X)

(Cat. No. A1517001)

• Vitronectin (VTN-N) Recombinant Human Protein, Truncated (Cat. No. A14700) or Gibco™ Geltrex™ LDEV-Free, hESC-Qualified, Reduced Growth Factor Basement Membrane Matrix (Cat. No. A1413301)

• Gibco™ Dulbecco’s Phosphate Buffered Saline (DPBS), no calcium, no magnesium (Cat. No. 14190-144)

• Gibco™ Versene Solution (Cat. No. 15040-066) or Invitrogen™ UltraPure™ 0.5 M EDTA, pH 8.0 (Cat. No. 15575-020)

• Gibco™ Dulbecco’s Modified Eagle Medium (DMEM), high glucose, with GlutaMAX™-I Supplement (Cat. No. 10569-010)

• Gibco™ PSC Cryopreservation Kit (Cat. No. A2644601)

Resource Center

Please note, the Gibco™ iPSC Lab Starter Kits (Cat. Nos. A32020 and A32021) do not include the Gibco PSC Cryopreservation Kit. For the following cryopreservation protocol, purchase the PSC Cryopreservation Kit using Cat. No. A2644601.

• 37°C water bath

• Appropriate tissue culture plates and supplies

Prepare media and reagentsEssential 8 Medium (500 mL of complete medium)1. Thaw Essential 8 Supplement (50X) at room temperature for ~1 hour. Do not thaw frozen supplement

at 37°C.

2. To prepare 500 mL of complete Essential 8 Medium, aseptically mix the following components:

Component Amount

Essential 8 Basal Medium 490 mL

Essential 8 Supplement (50X) 10 mL

3. Complete Essential 8 Medium can be stored at 2°C to 8°C for up to 2 weeks. Note: Before use, warm complete medium required for that day at room temperature until it is no longer cool to the touch. Do not warm the medium at 37°C.

6 Culture and expansion of pluripotent stem cells in feeder-free culture system

Essential 8 Medium

Gibco™ Essential 8™ Medium is a fully defined, feeder-free medium formulated for the growth and expansion of human pluripotent stem cells (PSCs). Originally developed by Chen et al. [1] in the laboratory of James Thomson, and validated by Cellular Dynamics International, Essential 8 Medium has been extensively tested and is proven to be able to maintain pluripotency in multiple PSC lines. Unlike most feeder-free media, Essential 8 Medium does not require the presence of bovine serum albumin (BSA) or human serum albumin (HSA) that contributes to lot-to-lot variability. In addition, most serum-free media consist of more than 20 components, which may add complexity, time, and cost, while Essential 8 Medium is composed of only eight components. Complete Essential 8 Medium is prepared by adding Gibco™ Essential 8™ Supplement (50X) to Gibco™ Essential 8™ Basal Medium, which are provided with the product.

Standard physical growth conditions for human PSCs in complete Essential 8 Medium are 37°C in a humidified atmosphere of 5% CO2. Cultures are grown in complete Essential 8 Medium on vitronectin-coated, tissue culture–treated vessels and must be passaged with EDTA or Versene solution. Cells are typically passaged approximately 24 hours sooner than they would be in other feeder-free media, with passaging occurring when the cells are 85% confluent. This uncomplicated, xeno-free medium minimizes batch variability and improves feeder-free culture conditions for pluripotent stem cells. Multiple variations of Essential 8 Medium are available, including a weekend-free Gibco™ Essential 8™ Flex Medium (Cat. No. A2858501).

Before you begin

There are three major differences that you will observe with cells cultured in Essential 8 Medium on vitronectin (VTN-N) compared to other feeder-free systems:

1. Cells are typically passaged ~24 hours sooner than they would be with other feeder-free medium.

2. Passaging should take place when cells are at ~85% confluency. If cells are passaged when they are more than 85% confluent, the health of cells and final cell yield may be compromised.

3. Cells must be passaged in EDTA or Versene solution. Collagenase and dispase are not recommended.

48 49

5. Aliquot 1 mL of diluted vitronectin solution to each well of a 6-well plate (refer to Table 6.1 below for recommended volumes for other culture vessels). Note: When used to coat a 6-well plate (10 cm2/well) at 1 mL/well, the final concentration will be 0.5 μg/cm2.

6. Incubate at room temperature for 1 hour. Note: Dishes can now be used or stored at 2°C to 8°C wrapped in laboratory film for up to a week. Do not allow the vessel to dry. Prior to use, pre-warm the culture vessel to room temperature for at least 1 hour.

7. Aspirate the diluted vitronectin solution from the culture vessel and discard. It is not necessary to rinse off the culture vessel after removal of vitronectin. Cells can be passaged directly onto the vitronectin-coated culture dish. Note: Geltrex LDEV-Free, hESC-Qualified, Reduced Growth Factor Basement Membrane Matrix may be substituted for vitronectin (see “Alternate substrate protocol”, page 54).

Table 6.1. Required volume of diluted vitronectin substrate.

Culture vessel Surface area Diluted substrate volume

6-well plate 10 cm2/well 1 mL/well

12-well plate 4 cm2/well 0.4 mL/well





0.5 mM EDTA in DPBS (50 mL)1. To prepare 50 mL of 0.5 mM EDTA in DPBS, aseptically mix the following components in a 50 mL conical

tube in a biological safety cabinet:

Component Amount

DPBS, no calcium, no magnesium 50 mL

0.5 M EDTA 50 μL

Note: If using Versene Solution instead of regular EDTA, no additional dilution of the reagent is required. It is provided at a working concentration and can be stored at 4°C until use.

2. Filter sterilize the solution. The solution can be stored at room temperature for up to six months.

Coat culture vessels with vitronectin (VTN-N)1. Upon receipt, thaw the vial of vitronectin at room temperature and prepare 60 μL aliquots of vitronectin in

polypropylene tubes. Freeze the aliquots at –80°C or use immediately.

2. Prior to coating culture vessels, calculate the working concentration of vitronectin using the formula below and dilute the stock appropriately. Refer to Table 6.1, page 49, for culture surface area and volume required. The optimal working concentration of vitronectin is cell line dependent. We recommend using a final coating concentration of 0.5 μg/cm2 for human PSC culture.

Working conc. = Coating conc. ×Culture surface area

Vol. required for surface area

Dilution factor =Stock concentration (0.5 mg/mL)

Working concentration

For example, to coat a 6-well plate at a coating concentration of 0.5 μg/cm2, you will need to prepare 6 mL of diluted vitronectin solution (10 cm2/well surface area and 1 mL of diluted vitronectin/well; Table 6.1, page 49) at the following working concentration:

Working conc. =0.5 μg

×10 cm2

=5 μg

cm2 1 mL mL

Dilution factor =0.5 mg/mL

= 100X (i.e., 1:100 dilution)5 μg/mL

3. To coat the wells of a 6-well plate, remove a 60 μL aliquot of vitronectin from –80°C storage and thaw at room temperature. You will need one 60 μL aliquot per 6-well plate.

4. Add 60 μL of thawed vitronectin into a 15 mL conical tube containing 6 mL of sterile DPBS without calcium and magnesium at room temperature. Gently resuspend by pipetting the vitronectin dilution up and down. Note: This results in a working concentration of 5 μg/mL (i.e., a 1:100 dilution).

6 Culture and expansion of pluripotent stem cells in feeder-free culture system Essential 8 Medium 6

50 51

2. Aspirate the spent medium from the vessel containing PSCs with a Pasteur pipette, and rinse the vessel twice with DPBS without calcium and magnesium. Refer to Table 6.2 below for the recommended volumes.

3. Add 1X Versene solution to the vessel containing PSCs. Adjust the volume of Versene solution for various dish sizes (refer to Table 6.2 below). Swirl the dish to coat the entire cell surface. Note: 0.5 mM EDTA in DPBS may be substituted for Versene solution.

4. Incubate the vessel at room temperature for 5–8 minutes or 37°C for 4–5 minutes. When the cells start to separate and round up, and the colonies appear to have holes in them when viewed under a microscope, they are ready to be removed from the vessel. Note: In larger vessels or with certain cell lines, this may take longer than 5 minutes.

5. Aspirate the Versene solution with a Pasteur pipette.

6. Add pre-warmed complete Essential 8 Medium to the dish according to Table 6.2 below.

Table 6.2. Required volume of reagents.

Culture vesselApproximate surface area

Volume of DPBS

Volume of 1X Versene solution

Volume of complete Essential 8 Medium

6-well plate 10 cm2/well 2 mL/well 1 mL/well 2 mL/well

12-well plate 4 cm2/well 1 mL/well 0.4 mL/well 1 mL/well

24-well plate 2 cm2/well 0.5 mL/well 0.2 mL/well 0.5 mL/well

35 mm dish 10 cm2 2 mL 1 mL 2 mL



7. Remove the cells from the well(s) by gently squirting medium and pipetting the colonies up using a 5 mL serological pipette. Avoid creating bubbles. Collect cells in a 15 mL conical tube. Important: Do not scrape the cells from the dish. There may be obvious patches of cells that were not dislodged and left behind. Do not attempt to recover them through scraping. Note: Little or no extra pipetting is required to break up cell clumps after Versene treatment. Two to three triturations should be sufficient. Do not pipet vigorously or the colonies will break apart. Note: Depending upon the cell line, work with no more than one to three wells at a time, and work quickly to remove cells after adding Essential 8 Medium to the well(s). The initial effect of the Versene solution will be neutralized quickly by the medium. Some lines re-adhere very rapidly after medium addition, and must be removed 1 well at a time. Others are slower to reattach, and may be removed 3 wells at a time.

8. Aspirate residual vitronectin solution from the precoated dish.

Passage PSCsWhen to split cellsIn general, split cells when one of the following occurs:

• PSC colonies are becoming too dense or too large.

• PSC colonies are showing increased differentiation.

• The colonies cover approximately 85% of the surface area of the culture vessel, usually every 4 days. Even if the colonies are sparse and small, it is important to split the culture every 4 to 5 days.

3

5. Aliquot 1 mL of diluted vitronectin solution to each well of a 6-well plate (refer to Table 1, for recommendedvolumes for other culture vessels).

Note: When used to coat a 6-well plate (10 cm2/well) at 1 mL/well, the final concentration will be 0.5 µg/cm2.

6. Incubate at room temperature for 1 hour.

Note: Dishes can now be used or stored at 2–8°C wrapped in laboratory film for up to a week. Do not allowthe vessel to dry. Prior to use, pre-warm the culture vessel to room temperature for at least 1 hour.

7. Aspirate the diluted vitronectin solution from the culture vessel and discard. It is not necessary to rinse offthe culture vessel after removal of vitronectin. Cells can be passaged directly onto the vitronectin-coatedculture dish.

Note: Geltrex™ LDEV-Free hESC-Qualified Reduced Growth Factor Basement Membrane Matrix may besubstituted for vitronectin (see Appendix, page 8).

Table 1 Required volume of diluted Vitronectin substrate

Culture vessel Approximate surface area (cm2)

Diluted substrate volume (mL)

6-well plate 10 cm2/well 1 mL/well



35-mm dish 10 cm2 1 mL



Passage PSCs When to split cells


• PSC colonies are becoming too dense or too large.

• PSC colonies are showing increased differentiation.

• The colonies cover approximately 85% of the surface area of the culture vessel, usually every 4 days. Even ifthe colonies are sparse and small, it is important to split the culture every 4 to 5 days.

Figure 1 A. PSCs growing in Essential 8™ Medium on vitronectin 24 hours after a passage, prior to changing the medium. B. PSCs growing in Essential 8™ Medium on vitronectin that are ready for passage. C. PSCs growing in Essential 8™ Medium on vitronectin that are over-confluent.

A B C

Figure 6.1. (A) PSCs growing in Essential 8 Medium on vitronectin 24 hours after a passage, prior to changing the medium. (B) PSCs growing in Essential 8 Medium on vitronectin that are ready for passage. (C) PSCs growing in Essential 8 Medium on vitronectin that are over-confluent.

Split ratio• The split ratio can vary, though it is generally between 1:2 and 1:4 for early passages and between 1:3 and

1:12 for established cultures. Occasionally, cells will grow at a different rate and the split ratio will need to be adjusted.

• A general rule is to observe the last split ratio and adjust the ratio according to the appearance of the PSC colonies. If the cells look healthy and colonies have enough space, split using the same ratio. If they are overly dense and crowding, increase the ratio. If the cells are sparse, decrease the ratio.

Passage PSC colonies with Versene or EDTA solutionNote: Newly derived PSC lines may contain a fair amount of differentiation through passage 4. It is not necessary to remove differentiated material prior to passaging. By propagating and splitting the cells, the overall culture health should improve throughout the early passages.

Important: Enzymes such as collagenase and dispase do not work well with cells cultured in Essential 8 Medium and on vitronectin. Use of these enzymes for passaging cells results in compromised viability and attachment.

1. Prior to starting, equilibrate your vitronectin-coated dishes to room temperature in a laminar flow hood (this takes about one hour). Pre-warm the required volume of Essential 8 Medium at room temperature until it is no longer cool to the touch. Note: Do not warm medium in a 37°C water bath.


52 53

Cryopreserve PSCsFreeze PSCs1. Thaw and prechill PSC Cryomedium from the PSC Cryopreservation Kit (Cat. No. A2644601) at 2°C to 8°C.

Note: Use of Essential 8 Medium with 10% DMSO for cryopreservation instead of PSC Cryomedium is possible, but will result in lower relative performance.

2. Harvest PSCs according to standard cell passaging protocols.

3. Centrifuge the cell suspension at 200 x g for 4 minutes.

4. Aspirate the medium, being careful not to disturb the cell pellet.

5. Add PSC Cryomedium (chilled to 2°C to 8°C) dropwise to the cells while gently rocking the tube back and forth followed by gentle resuspension of cell pellet. Note: In general, from a 100 mm dish, 8–12 vials containing 1 x 106 viable cells/mL can be generated.

6. Dispense aliquots of the suspension into cryogenic vials according to manufacturer’s specifications (e.g., 1.5 mL in a 2 mL cryovial). Note: Mix the cell suspension in PSC Cryomedium frequently to maintain a homogenous suspension. If utilizing clumped passaging methods at cell harvest, then mix cell suspension by gentle inversion to prevent breaking cells into smaller clumps.

7. Cryopreserve cells in an automated or manual controlled-rate freezing apparatus following standard procedures (approximately 1°C decrease per minute).

8. Transfer frozen cell vials to liquid nitrogen (vapor phase); we recommend storage at –200°C to –125°C.

Thaw and recover PSCs1. Quick-thaw cryopreserved PSCs in a 37°C water bath until only a small ice crystal remains.

2. Gently pipet the thawed cells up and down to create a cell suspension and transfer to a 50 mL conical tube.

3. Dilute the cell suspension with 3 mL of growth medium per mL of cryopreserved cells, adding it dropwise while gently rocking the tube back and forth to avoid osmotic shock to the cells.

4. Centrifuge cell suspension at 200 x g for 4 minutes.

5. Aspirate the medium, being careful not to disturb the cell pellet.

9. Add an appropriate volume of pre-warmed Essential 8 Medium to each well of a coated 6-well plate so that each well contains 2 mL medium after the cell suspension has been added. Refer to Table 6.2 on page 51 for volumes for other culture vessels.

10. Mix the cell suspensions from step 7 by gently inverting a few times, then transfer the appropriate volume of cell suspension into each well containing pre-warmed complete Essential 8 Medium according to the desired split ratio.

11. Move the vessel in several quick figure eight motions to disperse cells across the surface of the vessels.

12. Place dish gently into the 37°C, 5% CO2 incubator and incubate the cells overnight.

13. Feed PSCs the day after splitting. Replace spent medium daily. Note: It is normal to see cell debris and small colonies after passage.

14. Optional: To improve cell survival, you can add Gibco™ RevitaCell™ Supplement (Cat. No. A2644501) to 1X final concentration (e.g., 20 μL per 2 mL of cell suspension) for the first 24 hours post-passage.

Figure 6.2. Normal pluripotent stem cell morphology. The expected morphology of PSCs is demonstrated specifically by tightly packed colonies with defined borders and a high nucleus-to-cytoplasm ratio. The image above shows PSCs at passage 6.

5

7. Remove the cells from the well(s) by gently squirting medium and pipetting the colonies up using a 5-mL serological pipette. Avoid creating bubbles. Collect cells in a 15-mL conical tube.

IMPORTANT! Do not scrape the cells from the dish. There may be obvious patches of cells that were not dislodged and left behind. Do not attempt to recover them through scraping.

Note: Little or no extra pipetting is required to break up cell clumps after Versene treatment. Two to three triturations should be sufficient. Do not pipet vigorously or the colonies will break apart.

Note: Depending upon the cell line, work with no more than one to three wells at a time, and work quickly to remove cells after adding Essential 8™ Medium to the well(s). The initial effect of the Versene will be neutralized quickly by the medium. Some lines re-adhere very rapidly after medium addition, and must be removed 1 well at a time. Others are slower to re-attach, and may be removed 3 wells at a time.

8. Aspirate residual vitronectin solution from the pre-coated dish.

9. Add an appropriate volume of pre-warmed Essential 8™ Medium to each well of a coated 6-well plate so that each well contains 2 mL medium after the cell suspension has been added. Refer to Table 2, page 4, for volumes for other culture vessels.

10. Mix the cell suspensions from step 7 by gently inverting a few times, then transfer the appropriate volume of cell suspension into each well containing pre-warmed complete Essential 8™ Medium according to the desired split ratio.

11. Move the vessel in several quick figure eight motions to disperse cells across the surface of the vessels.

12. Place dish gently into the 37°C, 5% CO2 incubator and incubate the cells overnight.

13. Feed PSCs the day after splitting. Replace spent medium daily.

Note: It is normal to see cell debris and small colonies after passage.

14. (Optional): To improve cell survival, you can add RevitaCell™ Supplement (Cat. no. A26445) to 1X final concentration (i.e., 20 µL per 2 mL of cell suspension) for the first 24 hours post-passage.

Figure 2 Normal pluripotent stem cell morphology. The expected morphology of PSCs is demonstrated specifically by tightly packed colonies with defined borders and a high nucleus-to-cytoplasm ratio. The image below shows PSCs at passage 6.


54 55

4. Quickly cover the whole surface of each culture dish with the Geltrex solution (refer to Table 6.4 below).

5. Incubate the dishes in a 37°C, 5% CO2 incubator for 1 hour. Note: Dishes can now be used or stored at 2°C to 8°C for up to a week. Do not allow dishes to dry.

6. Aspirate the diluted Geltrex solution from the culture dish and discard. You do not need to rinse off the Geltrex solution from the culture dish after removal. Cells can now be passaged directly onto the Geltrex matrix–coated culture dish.

Table 6.4. Volume of Geltrex matrix solution required.

Culture vessel Approximate surface area Diluted substrate volume


12-well plate 4 cm2/well 750 μL/well

24-well plate 2 cm2/well 350 μL/well

35 mm dish 10 cm2 1.5 mL



Frequently asked questions1. What is Essential 8 Medium?

Essential 8 Medium is a xeno-free and feeder-free medium specially formulated for the growth and expansion of human pluripotent stem cells. Originally developed by Chen et al. [1] in the laboratory of James Thomson, and validated by Cellular Dynamics International, Essential 8 Medium has been extensively tested and demonstrated the ability to maintain pluripotency in multiple PSC lines.

2. How many components are there in Essential 8 Medium? Essential 8 Medium contains only the eight required components for culturing PSCs. The medium was developed by Chen et al. [1] to overcome the variability issues observed with mTeSR™ Medium. Essential 8 Medium is designed to have less variability due to limited components and removal of BSA from the formulation (Table 6.5) [1]. Essential 8 Medium is provided as a convenient two-component kit: 500 mL Essential 8 Basal Medium and 10 mL Essential 8 Supplement (50X).

6. Gently resuspend the cells in growth medium supplemented with RevitaCell Supplement at a 1X final concentration (e.g., 100 μL of RevitaCell Supplement in 10 mL of growth medium). Note: Note: RevitaCell Supplement contains a ROCK inhibitor. Do not add any additional ROCK inhibitors to the growth medium.

7. Transfer the cell suspension to an appropriate growth vessel and incubate for 18–24 hours in the recommended culture environment. Note: Refer to Table 6.3 below for recommended cell seeding densities.

8. Following 18–24 hour incubation, aspirate the growth medium supplemented with RevitaCell Supplement and replace it with unsupplemented growth medium (i.e., without the addition of RevitaCell Supplement) for the remainder of the culture.

Table 6.3. Recommended cell seeding densities and volumes of medium for plating (per dish or per well).

Culture vessel (surface area)

Number of viable cells added* Essential 8 Medium + 1X RevitaCell Supplement**20,000 cells/cm2 40,000 cells/cm2

6 well (10 cm2) 200,000 400,000 2 mL

12 well (4 cm2)) 80,000 160,000 1 mL

24 well (2 cm2) 40,000 80,000 0.5 mL

35 mm (10 cm2) 200,000 400,000 2 mL

60 mm (20 cm2) 400,000 800,000 4 mL

100 mm (60 cm2) 1,200,000 2,400,000 12 mL

* Time to confluency is 4–5 days for a seeding density of 20,000 cells/cm2 and 3–4 days for a seeding density of 40,000 cells/cm2. ** For resuspension.

Alternative substrate protocolCoat culture vessels with Geltrex LDEV-Free, hESC-Qualified Basement Membrane Matrix1. Thaw a 5 mL bottle of Geltrex LDEV-Free, hESC-Qualified, Reduced Growth Factor Basement Membrane

Matrix at 2°C to 8°C overnight.

2. Dilute the thawed Geltrex solution 1:1 with cold, sterile DMEM/F-12 to prepare 1 mL aliquots in tubes chilled on ice. These aliquots can be frozen at –20°C or used immediately. Note: Aliquot volumes of 1:1–diluted Geltrex solution may be adjusted according to your needs.

3. To create working stocks, dilute a Geltrex aliquot 1:50 with cold DMEM on ice, for a total dilution of 1:100. Note: An optimal dilution of the Geltrex solution may need to be determined for each cell line. Try various dilutions from 1:30 to 1:100.


56 57

Ordering information7

Table 6.5. Components of mTesR Medium and Essential 8 Medium.

Components mTeSR Medium Essential 8 Medium

DMEM/F-12 X X

L-Ascorbic acid X X

Selenium X X

Transferrin X X

NaHCO3 X X

Glutathione X

L-Glutamine X

Defined lipids X

Thiamine X

Trace elements B X

Trace elements C X

2-Mercaptoethanol X

BSA X

Insulin X X

FGF2 X X

TGFβ1 X X

Pipecolic acid X

LiCl X

GABA X

3. Is Essential 8 Medium xeno-free (human- or recombinant-origin only)? Yes. Essential 8 Medium allows for reliable and robust cultures with a xeno-free, eight-component medium.

4. Can Essential 8 Medium and VTN-N support long-term growth of PSCs? Essential 8 Medium and vitronectin have been shown to support PSC growth for >50 passages without any signs of karyotypic abnormalities, and maintain the ability of PSCs to differentiate into all three germ line lineages. As published by Chen et al. [1] in the laboratory of James Thomson, the VTN-N variant of vitronectin supports human pluripotent stem cell culture attachment and survival better than wild-type vitronectin when used in conjunction with Essential 8 Medium.

5. Does Essential 8 Medium exhibit variability from lot to lot? Essential 8 Medium has reduced variability compared to existing feeder-free culture media. Unlike other media that contain over 20 highly variable ingredients, Essential 8 Medium is produced under cGMP and has an optimized formulation and growth factor levels to help ensure maximum cell health, pluripotency, and growth, with minimal variability.

Reference1. Chen G, Gulbranson DR, Hou Z et al. (2011) Chemically defined conditions for human iPSC derivation and culture. Nat Methods 8:424–429.

6 Culture and expansion of pluripotent stem cells in feeder-free culture system

58 59

Ordering information

Product Cat. No.

iPSC Lab Starter Kit with Reprogramming A32020

iPSC Lab Starter Kit A32021

2-Mercaptoethanol (55 mM) 21985-023

AccuPrime™ SuperMix I 12342-010

Attachment Factor Protein (1X) S-006-100

Collagenase Type IV 17104-019

CytoTune-iPS 2.0 Sendai Reprogramming Kit A16517

CytoTune-EmGFP Sendai Fluorescence Reporter A16519

DMEM, high glucose, GlutaMAX-I Supplement 10569-044

DMEM/F-12 with GlutaMAX-I Supplement 10565-018

DPBS, calcium, magnesium 14040182

DPBS, no calcium, no magnesium 14190250

Essential 8 Adaptation Kit A25935

Essential 8 Flex Medium A2858501

Essential 8 Medium A1517001

Fetal Bovine Serum, Embryonic Stem Cell Qualified, US Origin 16141-061

FGF-Basic (AA 1–155) Recombinant Human Protein PHG0264

FluoroBrite™ DMEM A1896701

Geltrex LDEV-Free, hESC-Qualified, Reduced Growth Factor Basement Membrane Matrix A1413302

KnockOut Serum Replacement – Multi-Species A31815-02

KnockOut Serum Replacement 10828010*

MEM Non-Essential Amino Acids Solution (100X) (100 mL) 11140-050

MEM Non-Essential Amino Acids Solution (20 x 100mL) 11140-076

Mouse (ICR) Inactivated Embryonic Fibroblasts A24903

SuperScript VILO cDNA Synthesis Kit 11754-050

TRA-1-60 Alexa Fluor 488 Conjugate Kit for Live-Cell Imaging A25618

TRIzol Reagent 15596-026

Versene Solution 15040066

Vitronectin (VTN-N) Recombinant Human Protein, Truncated A14700

7 Ordering information

Accessory products

Product Cat. No.

Dispase II 17105-041

Human Dermal Fibroblasts, adult C0135C

Human Dermal Fibroblasts, neonatal C0045C

Penicillin-Streptomycin (10,000 U/mL) 15140-122

PSC Cryopreservation Kit A2644601

RevitaCell Supplement (100X) A2644501

rhLaminin-521 A29248

StemPro Accutase Cell Dissociation Reagent A11105-01

StemPro EZPassage Disposable Stem Cell Passaging Tool 23181-010

TrypLE Express Enzyme (1X) 12604-021

TrypLE Select Enzyme (1X) 12563-011

Trypsin-EDTA (0.05%), phenol red 25300-054

UltraPure 0.5 M EDTA, pH 8.0 15575-020

Find out more at thermofisher.com/stemcellprotocols

* For human ex vivo tissue and cell culture processing applications. Caution: When used as a medical device, Federal Law restricts this device to sale by or on the order of a physician.

For Research Use Only. Not for use in diagnostic procedures. © 2016 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. Essential 8 is a trademark of Cellular Dynamics International, Inc. Falcon is a registered trademark of Corning Incorporated. mTeSR is a trademark of STEMCELL Technologies Inc. TaqMan is a registered trademark of Roche Molecular Systems, Inc., used under permission and license. Triton is a trademark of The Dow Chemical Company or an affiliated company of Dow. COL02768 1116

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