Standardizing Cell Banking Practices for Research and Clinical Applications Derek J. Hei, Ph.D. hei@waisman.wisc.edu Technical Director
Stem Cell Research at the 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
Moving HESC Therapies to the Clinic The Challenges 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)
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
National Stem Cell Bank Program 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
The NSCB Banking Process www.nationalstemcellbank.org 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
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
NSCB/WiscBank Website Technical Support 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
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 -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 Applications Goals 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 Waisman Clinical Biomanufacturing Facility Julie Johnson Carol Emler Diana Drier Lisa Marie Byrne John Welp Tim Sparks Laurie Larson. WiCell Research Institute Robert Drape Tenneille Ludwig Jessica Martin Dan Felkner /NSCB Banking Team Karen Montgomery Jeff Jones Erik Forsberg PACT Peiman Hematti Tim Hacker Amish Raval Marlowe Eldridge University of Wisconsin Jamie 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