Standardizing Cell Banking Practices for Research and ... · •Media evaluation International Stem Cell Banking Initiative (ISCF) • Int. Stem Cell Forum/ Glyn Stacey –U.K. Stem

Waisman Clinical Biomanufacturing FacilityUniversity of Wisconsin - Madison

Standardizing Cell Banking Practices for

Research and Clinical Applications

Derek J. Hei, [email protected]

Technical Director

Waisman Clinical Biomanufacturing Facility

University of Wisconsin - Madison


Stem Cell Research at the

University of Wisconsin - Madison

UW Stem Cell and Regenerative Medicine Center

– Basic and translational research through to clinical trials

– Human ES cells, iPS cells, Neural Progenitor Cells, Mesenchymal Stem Cells

WiCell Research Institute

– Core support services for UW stem cell investigators

– Derivation of new cell lines (hESC, iPS cells)

– Hosted National Stem Cell Bank with UW


– Development support for translational researchers

– Clinical production and QC testing under cGMPS


Technical hurdles -• Culture methods – animal-free

• Genetic stability of cell lines

• Cryopreservation methods

• Selective differentiation methods

• Residual undifferentiated cells

• Rejection of transplanted cells

Establish well-characterized cell banks

Develop standardized Quality Control test methods

Develop processes for large-scale clinical production

Compliance with FDA regulations (current Good Manufacturing Practice guidelines)

Moving HESC Therapies to the Clinic – The Challenges


Setting Standards for hESC Research

Cell lines (reference standards)

Cell culture methods – undifferentiated hESCs

Differentiation methods

Cell characterization and Quality Control methods

Regulatory issues for clinical applications

Potential impact –

– Positive – allows comparison across studies and between labs

– Negative – may drive science toward early, sub-optimal

methods and technologies


The NSCB mission –• Bank and distribute NIH approved hESC lines

• Characterization of hESC lines

• Technology transfer and technical support for research community

Restricted to banking NIH approved hESC lines• Prior to July 2009 – 21 lines (Cellartis, ESI, Novocell, Technion, UCSF, WiCell)

• Genetically modified human ES cell lines

• NIH Guidelines for Human Stem Cell Research – July 2009

NSCB data moved to WiscBank (www.wiscbank.com)

New cell lines – motivation to deposit with national banking

system?

The future of hESC banking in the U.S. remains unclear

National Stem Cell Bank Program

http://www.wiscbank.com/


The NSCB Banking Process

MCBs – full QC testing including complete adventitious agent panel

Distribution Lots – basic QC testing panel -• Identity (STR)

• Karyotype (G-band)

• microbial/fungal contamination, mycoplasma

• hESC marker expression (flow cytometry)

Characterization Studies – impact of culture on genetic stability,

gene expression, differentiation potential

www.nationalstemcellbank.org

http://www.nationalstemcellbank.org/


Master Cell Bank Characterization

Identity – Short Tandem Repeat (STR) - PowerPlex 1.2, HLA typing, SNP testing

Bacterial/fungal contamination, mycoplasma

Karyotype – G-band

Flow cytometry – SSEA-1/3/4; Oct-3/4, TRA-1-60/81

Post thaw recovery (count/viability)

Growth characteristics – doubling time

Comparative Genome Hybridization

Gene Expression Profiling

Adventitious agent testing – human, murine, bovine, porcine (ICH)


NSCB/WiscBank Website –Cell ordering, COAs, Technical Support

www.nationalstemcellbank.org

www.wiscbank.com

http://www.nationalstemcellbank.org/



NSCB/WiscBank Website – Technical Support

www.wiscbank.org

http://www.wiscbank.org/


HESC Characterization Studies

Performed on Distribution Lots to provide information on typical hESC growth characteristics

Testing at 5 passage intervals to P = +20 from distribution lot

Characterization tests -

Growth characteristics

HESC marker expression by multi-color flow cytometry

(Kathy Schell, UW Cancer Center)

Karyotype by G-banding (Karen Montgomery, WiCell)

Gene expression profiling (Nimblegen)

Comparative Genome Hybridization (Nimblegen)

Differentiation potential (UW investigators)


NSCB Quality Control –Comparative Genome

Hybridization

Chromosome 12

NimbleGen 385K whole genome array

Complementary to G-Band

Resolution: G-Band = 2-10 Mb; CGH = 4-20 kB

Sensitivity: G-Band = 5-10%, CGH > 40-60%

Utilize opposite sex hESC as reference (H1 male, H9 female)

Identify Copy Number Variants (CNVs) and cross-check with normal CNV database (Database of Genomic Variants, http://projects.tcag.ca/variation)

Verify findings using FISH analysis

Data – WiscBank website (www.wiscbank.com), GEO (www.ncbi.nlm.nih.gov/geo)

Karen Montgomery - WiCell


http://www.ncbi.nlm.nih.gov/geo


International Collaborations for hESC Banking

Stem cell registries –• hESCReg – European hESC Registry

• UMass International Stem Cell Registry

• Compile databases of critical information – publications, cell

characterization data…

International Stem Cell Initiative• Cell line characterization

• Media evaluation

International Stem Cell Banking Initiative (ISCF)• Int. Stem Cell Forum/ Glyn Stacey – U.K. Stem Cell Bank

• Consensus guidance for supply of hESC for research (Stem Cell Rev

and Rep (2009) 5:301-314)

• Consensus guidance for banking of clinical-grade hESCs – under

development


Cell Banking Standards for Clinical Production

U.S. Food and Drug Administration –• Current Good Manufacturing Practice (cGMP) guidelines – 21 CFR

210, 211, 610

• Human Cellular and Tissue-Based Products (HCT/Ps) – 21 CFR

1271 (May 2005)

Donor eligibility

Current Good Tissue Practice guidelines

• Guidance documents – future?

EMEA – Guideline on human cell-based medicinal products

(EMEA/CHMP/410869/2006)

International Conference on Harmonisation• Harmonization for U.S. FDA, EMEA, Japan

• Viral Safety Evaluation for Cells of Human or Animal Origin (Q5A)

• Derivation and Characterisation of Cell Substrates (Q5D)


Human ES Cell Banking for

Clinical Applications

Waisman Clinical Biomanufacturing Facility-WiCell

Regulatory compliance

• Current Good Manufacturing Practice (cGMP) guidelines

• HCT/Ps, Good Tissue Practice guidelines (21 CFR 1271)

• Full cGMP batch records for media and MCB production

• Quality Control Procedures – full documentation, assay qualification

• IND support – WCBF DMF, cell line DMF (future)

Key technical issues

• Scalable cell bank production without the use of murine feeders

• Address potential pathogen contamination issues from prior derivation and

culture practices

• Aseptic processing

• Differentiation control


Human ES Cell Banking for

Clinical ApplicationsGoals

• Address key technical issues

• Establish reference standard cell banks for clinical applications

• Shorten timeline and cost from discovery to clinical trials

Current cell banks -• WA01, WA09 MCBs produced to date

• Full adventitious agent testing (ICH Q5A guidelines) including testing for

human, murine, bovine and porcine pathogens

• Provide portion of MCB and produce WCB for each investigator

Feeder independent culture – cGMP mTeSR1 media and growth

factors (FGF-2) produced by WCBF

Future –feeder independent derivation of new cell lines


WCBF, NSCB, WID Seed Grant, and PACT Teams

WiCell Research InstituteRobert Drape

Tenneille Ludwig

Jessica Martin

Dan Felkner /NSCB Banking Team

Karen Montgomery

Jeff Jones

Erik Forsberg

University of WisconsinJamie Thomson, Guokai Chen

Tim Kamp, Jianhua Zhang, Chad Koonce

Jon Odorico, Torey Browning, Carly Kibbe

Igor Slukvin, Jessica Dias

Su-Chun Zhang, Ben Thiede

Kathy Schell, Erik Puffer

Sean Palecek, Samira Azarin


Julie JohnsonCarol EmlerDiana DrierLisa Marie ByrneJohn WelpTim SparksLaurie Larson….

PACTPeiman Hematti Tim HackerAmish RavalMarlowe Eldridge

Standardizing Cell Banking Practices for Research and ... · •Media evaluation International Stem Cell Banking Initiative (ISCF) • Int. Stem Cell Forum/ Glyn Stacey –U.K. Stem

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