Solutions to eradicate stem cell culture obstacles & barriers using diverse surface coating technology Hyunju Lee Regional Product manager
Solutions to eradicate stem cell culture
obstacles & barriers using diverse
surface coating technology
Hyunju Lee
Regional Product manager
2
Problem of current cell culture..
•Cell’s viability: Attachment & detachment?
•Limitations in clinical applications: Animal component free?
•Reproducibility of cell culture?
•Effective scale-up for Bioproduction?
We can overcome it using surface coating technology of
NUNC!!
Obstacles & barriers of cell culture
3
Understanding diverse cell culture surface
• Dry coating Energy treatment
-Nunclon delta
-Nunclon Vita
• Wet coating ECM components or Special Polymer coating
-ECM coating
-Special polymer coating- Upcell / Hydrocell
4
Understanding diverse cell culture surface
• Dry coating Energy treatment
-Nunclon delta
-Nunclon Vita
• Wet coating ECM components or Special Polymer coating
-ECM coating
-Special polymer coating- Upcell / Hydrocell
5
NUNCLON Delta
(Energy treatment)
For normal adherent cell
6
Nunclon delta surface
(%)100
0
No.
of
att
ached c
ells
Hydrophilic Hydrophobic
Silicon coating
Polystyrene
Optim
um
zone
NUNCLON DELTA
Corona Treatment
Nunclon Delta culture treated surface
•Hydrophilic surface
•Perfect for adherent cell cultures
•A proven cell culture surface for 25 years
Untreated Polystyrene surface
•Hydrophobic surface
•Suit for suspension cell’s proliferation and
growth
7
ECM Coating
(Wet)
For fastidious adherent cell
8
What is ECM?
• Cells are connected to Basal Lamina or ECM through Cell Junction
Epitherial tissue
Muscle tissue
Connective tissue
Small Intestine
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ECM Coating
Media and FBS can
construct ECM
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ECM coating
Poly-D-Lysine/ Collagen/CC2 coating
•Uniform coating creates a positive
charge on the surface
•Cells with Low adherence or growth
•CC2 surface : Mimics Poly-D-Lysine
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Special energy treatment
Nunclon Vita
For ADCF stem cell culture
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INNOVATION
Surfaces for stem cell culture
Surfaces: Most stem cells require extra help from the culture surface
to promote their attachment and growth. Traditionally, to maintain and
expand undifferentiated ESCs, one has to rely on feeder cells or ECM
coating.
• Feeder Layers
• Matrigel coating
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INNOVATION
Normal Surfaces for stem cell culture <Feeder layer>
Feeder Layers –a monolayer of fibroblast cells (e.g.
MEF, hFF) to promote fastidious cell culture.
Often the cells of the feeder layer are irradiated or
otherwise treated so that they will not proliferate. In
most cases the feeder cells produce growth factors or
cytokines to facilitate stem cell growth. ESCs
Feeder layer
New Feeder layer
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INNOVATION
Normal Surfaces for stem cell culture <Matrigel>
Matrigel – is the trade name by BD Biosciences for a gelatinous protein mixture
secreted by mouse sarcoma cells. This mixture resembles the complex
extracellular environment found in many tissues and is used as a substrate for
cell culture. Culture dishes can be purchased from BD with Matrigel-coating, or
they can be coated in labs by end users.
Matrigel contains animal components that are not suitable for clinical
applications.
Human ESCs and
iPSCs cultured on
Matrigel surface.
(phase contract, 20X)
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Problem: Existing methods for cultivating human pluripotent stem cells
rely heavily on components such as ECM (Matrigel) coatings and feeder
cells.
Such methods have a number of drawbacks:
- add variability / less reproducible
- are time & labor-intensive; pricey
- have limitations in clinical applications
- not well adaptable to scalable cell expansion.
Solution: NunclonTM VitaTM is a unique energy-treated polystyrene
surface that enables growth of most cell lines directly on the surface
without using matrix coatings or feeder cells.
Which problem does Nunclon Vita solve ?
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The Nunclon Vita Surface treatment
Polystyrene Polystyrene with functional
binding groups
Plasma Treatment
Hydrophobic Hydrophilic
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hESC Culture on Nunclon Vita Surface
• No matrix coatings or feeder layers saving time and labor
• hESC can be maintained for over 10 passages in culture
• hiPS cells can be expand for at least 3 passages
• Need conditioned media & ROCK inhibitor
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INNOVATION
Media and Supplements
Conditioned Media – Media that have been
enriched by feeder cells. These cells
"condition" the media by releasing growth
factors, cytokines, chemicals, etc. into the
supernatants. CM are obtained by sterile
filtration of the supernatants.
There are many kinds of CM depending on
the cells that “condition” them. The actual
components of CM are usually unknown.
Labs make their own CM and freeze them
down for future use. This is a labor-intensive
and time-consuming process. CM can also
be purchased with a premium price.
Basic
fibroblast
growth factor
Irradiated
feeder
MEF
Media
Basic
fibroblast
growth factorES cell
colony
Matrigel-
coated
surface
Conditioned
Media (CM)
Filter and
store CM
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INNOVATION
Media and Supplements
Defined Media – also called “chemically defined medium” in which the
chemical nature of the major ingredients and their amounts are known.
They are sometimes referred to as synthetic media.
The most commonly used defined media for human ESC and iPSC are:
TesR by StemCell Technologies; Nutristem by Biological Industries; and
StemPro by Invitrogen. All of them are very expensive. These defined
media are usually used with Matrigel surface or feeder cells to support
human ESC and iPSC growth.
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Medium Requirement
hESCs in chemically defined
media with ROCKi (10 mM)
hESCs in hFF-conditioned
media with ROCKi (10 mM)
Conditioned medium is required for expansion of human
ES cells and iPS cells on Nunclon Vita surface
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INNOVATION
Media and Supplements
ROCK inhibitors – inhibit Rho kinase (ROCK) activity. A selective ROCK inhibitor,
Y-27632, is known to enhance human ESC recovery upon single cell dissociation
and following cryopreservation. Recently, it has been reported that Y-27632
improves survival of human ESC after cell sorting by flow cytometry.
Y-27632 is widely available through chemical and media companies (e.g. Sigma,
EMD, Stemgent, etc).
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ROCK Inhibitor Requirement
0 mM 1 mM 2 mM
10 mM4 mM
Y27632 is required for seeding and maintaining
human ES cells and iPS cells on Nunclon Vita surface
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Non-Enzymatic Harvest of hESC – Removal of ROCKi
Non-Enzymatic Release and Dissociation
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Special Wet coating
Polymer coating -Upcell
For temperature responsive cell culture
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Special polymer coating- UpCell
Extremely hydrophilic
(cell detachment)Optimal for cell
attachment
(cell adhesion)
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Special polymer coating- UpCell
“Do not need Trypsin, Scrapping”
of
Trypsin EDTA/scraping UPCELL
•Good for harvest
Macrophage, Dendritic cell &
Osteoclast culture
•Do not destroy membrane
and secreted protein.
Membrane protein & cell
junction analysis
Flow cytometry
•Better recovery of harvested
cell Stem cell culture
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Special polymer coating- UpCell
“Do not need Trypsin, Scrapping”
•Good for harvest
Macrophage, Dendritic cell &
Osteoclast culture
•Do not destroy membrane
and secreted protein.
Membrane protein & cell
junction analysis
Flow cytometry
•Better recovery of harvested
cell Stem cell culture
Comparison of E-cadherin blot by
difference in harvest
S: Physical scraping
T: Temperature harvest
D: Dispase treatment
*E-cadherin : Junction protein
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Special polymer coating- UpCell
“Do not need Trypsin, Scrapping”
•Good for harvest
Macrophage, Dendritic cell &
Osteoclast culture
•Do not destroy membrane
and secreted protein.
Membrane protein & cell
junction analysis
Flow cytometry
•Better recovery of harvested
cell Stem cell culture
Trypsin
upcell
Re-attachment kinetics of A-549 cells
10 min 60 min 120 min 300 min
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Creating stemcell sheet for clinical application
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Creating stemcell sheet for clinical application
■LSCD - Persistent epithelial breakdown, superficial corneal vasularization, chronic
discomfort and impaired vision cased by the migration of conjunctival epithelial cells and
blood vessels onto the corneal surface.
■ Issues: Donor shortage, rejection
LCSD caused by Stevens-Johnson syndrome
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Creating stem cell sheet for clinical application
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Creating stem cell tissue model
5 layers of cell-sheets
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Application of cell-sheet – multiple homo cell-sheets (2)
Pile it up!
Cardiac patch
Skeletal myoblast/MSC sheet harvested from UpCell
Stick it on!
Impaired myocardium
Dilated cardiomyopathy
100um
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Application of cell-sheet – multiple homo cell-sheets (2)-Neonatal rat Cardiomyocyte sheet
Shimizu et al., 2002
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Clinical trial
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Special Wet coating
Polymer coating -Hydrocell
For spheroid culture & Embryonic body formation
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Special coating- Hydrocell
Hydrocell
(%)
100
0
No. o
f a
tta
ch
ed
ce
lls
Hydrophilic Hydrophobic
Silicon coating
-Hyd
roC
ell
Lo
w c
ell
bin
din
g d
ish
• Cultivation of cells that are sensitive to unwanted activation and differentiation signals arising from cell adhesion
> Macrophage culture
> Basophilic leukocytes
• Single cell suspension and spheroid culture
> Neurosphere formation
> Embryoid body formation of ES cells
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Special coating- Hydrocell
50um
Neurosphere generation from dental pulp of adult rat incisor
Primary sphere culture
under +EGF, +bFGF
A: Nestin positive progenitor cells (red)
B: Tuj 1 positive neuronal cells (red)
C: S100 positive glial cells (green)
Bar: 10um
Sasaki et al., 2008
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Special coating- Hydrocell
Differentiation of neurosphere
HydroCell sphere culture 7-9 days,
followed by culture on poly-L-ornithine and
fibronectin coated chamber slides 7-14 days
50um 10um 10um
Sasaki et al., 2008
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Special coating- Hydrocell
Focus Focus
HydroCellTM
(Non adherent・round・uniform size)
Non-treated
polystyrene
(Adherent・Non-uniform size)
HydroCellTM Non-treated
polystyrene
※Cultured under 20%FBS
Embryoid body formation of ES cells
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•Cell’s viability: Attachment & detachment?
•Limitations in clinical applications: Animal component free?
•Reproducibility of stem cell culture?
•Effective scale-up for Bioproduction?
Overcome Obstacles & barriers
•Nunclon Vita
•Upcell
•Hydrocell
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Thank you very much!