ISSCR Corning Innovation Showcase Stem Cell Culture, Differentiation, and Scale Up

Transcription

1 Corning Innovation Showcase Stem Cell Culture, Differentiation, and Scale Up Novel Technologies Enabling Research and Cell Processing Applications Paula Flaherty Technology Manager Deepa Saxena, Ph.D. Senior Development Scientist ISSCR 2015 June 26, 2015 ISSCR 2015

2 Today s Topics Introduction: Corning Life Sciences Corning cell culture surfaces technologies Industry trend: Animal-free Corning PureCoat ECM mimetic cultureware Collagen I peptide Fibronectin peptide Scale up for cell manufacturing: Examples Corning hpsc culture substrates for expansion and differentiation ISSCR

3 Corning Incorporated Highlights Founded: 1851 Headquarters: Corning, New York Employees: ~35,000 worldwide 2014 Sales: $10B Corning is one of the world s leading innovators in materials science. For more than160 years, Corning has applied its unparalleled expertise in specialty glass, ceramics, and optical physics to develop products that have created new industries and transformed people s lives. Corning succeeds through sustained investment in R&D, a unique combination of material and process innovation, and close collaboration with customers to solve tough technology challenges. R&D Investment: ~10% of sales Fortune 500 Rank (2015): 297 3

4 Corning: Leader in Cell Culture Surface Innovation A Century of Surface Technology Glass + Plastic + Biological + Synthetic 1907 Glass Surfaces used to culture mammalian cells 1953 Development of plastic 100 mm petri dish for U.S. Army 1985 Matrigel matrix, defined ECMs In vivo-like cell growth and differentiation 2010 Synthemax Synthetic VN peptide surface 2012 PureCoat ECM Mimetic Fibronectin and Collagen I Synthetic peptide surfaces Heat-resistant Pyrex glass TC-treated Polystyrene 1959 TC treatment and gamma irradiation 1987 BioCoat surface Convenient and consistent pre-coated cultureware 1990s Primaria and CellBIND Chargedbased modification of plastic surfaces 2000s PureCoat surfaces Amine and Carboxyl Ultra-Low Attachment Hydrogel-coated low bind surface 2014 Corning rlaminin-521 Animal-free pluripotent stem cell surface 4

5 Cell Manufacturers are Rapidly Progressing from Scientific Knowledge to Process Engineering Science and Technology Develop fundamental understanding of product, process, and material Engineering/Manufacturing Develop and control advanced product and process technology Critical Product Quality and Process Controls has been identified as a major technology need for cell manufacturers* Cell manufacturing is moving towards less animal products in their processes Reducing the risk of adventitious agent contamination Enabling control of cell manufacturing process Reducing variability of raw materials Reducing testing required lot-to-lot Progressing from fundamental science to engineering Enabling optimization of process Supporting predictive productivity Facilitating downstream purification *Cell Manufacturing Consortium, US National Institute of Standards and Technology (NIST), Advanced Manufacturing Technology, Identifying Industry Needs,

6 Complete and Customizable Cell Culture Environments Customizable solutions for all stages of cell and tissue culture research Corning Matrigel matrix Purified biological, xeno-free, and synthetic ECMs ECM mimetic cultureware Multi-layer flasks Corning CellSTACK Coated microcarriers cgmp media manufacturing Serum Serum-free media supplements and growth factors 6

7 Advanced Surfaces and Extracellular Matrices Vialed Extracellular Matrices (ECMs) Corning Matrigel matrix Defined ECMs Animal-derived Xeno-free Recombinant ECMs rlaminin-521 (Human) Synthetic ECMs Synthemax Corning PuraMatrix Custom ECM formulations Biological ECM Pre-coated Cultureware Corning BioCoat Plates Inserts Flasks Coverslips/slides Dishes Custom coatings Synthetic ECM Mimetic Pre-coated Cultureware Corning PureCoat Fibronectin peptide Collagen I peptide rlaminin-521 coming soon Synthemax (custom) Vitronectin peptide Enhanced Tissue Culturetreated Corning CellBIND surface (-) charge Corning PureCoat Amine (+) charge Carboxyl (-) charge Corning Primaria (+) and (-) mixed charge Ultra-Low Attachment Hydrogel-coated Functional Attachment Functional Attachment Functional Attachment Charge General cell culture, HTS, specialized applications HTS, general cell culture Specialized applications: cell culture enablement General cell culture, HTS, specialized applications 7

8 Corning PureCoat ECM Mimetic Cultureware Synthetic peptide covalently attached on a surface ISSCR 2015

9 Corning PureCoat ECM Mimetic Cultureware Animal-free, synthetic peptide (Collagen I peptide or Fibronectin peptide) covalently attached on a surface Support attachment and expansion of cell types that require Collagen I or Fibronectin ECM coating Pre-coated, ready to use Stable at room temperature for 18 months Manufactured in a GMP compliant facility US Class I medical device Sterile by aseptic processing Compatible with a wide range of dissociation reagents and serum-free, xeno-free, and animal-free media Available in 24-well, 6-well, T-75, T-175, 3-Layer or 5-Layer Multi-Flask format, and custom Corning CellSTACK 9

10 Corning PureCoat ECM Mimetic Cultureware Manufacturing and Quality Manufactured under cgmp per the requirements of FDA 21 CFR 820 Manufacturing facility ISO 9001, ISO 13485, and ISO certified Animal-free manufacturing process Quality control confirms the following: Nonpyrogenic Nontoxic Functional activity Sterile Regulatory documentation available ISO Certificates Certificate of Compliance Certificate of Analysis Lot-specific QC results No animal components used in production of raw materials No animal components in final product End-user 10

11 Synthetic and Defined Cultureware Suitable for Expansion of a Variety of Cell Types Corning PureCoat ECM Mimetic Fibronectin Peptide Collagen I Peptide Examples of Cell Types Expanded in Defined Cell Culture Environments Human BM-derived mesenchymal stem cells Human endothelial progenitor cells Human adipose-derived stem cells CHO cells Vero cells Human keratinocytes Human corneal cells Human adipose-derived stem cells Human endothelial progenitor cells Human cord blood-derived mesenchymal stem cells Vero cells 11

12 Corning PureCoat ECM Mimetic Cultureware Collagen I peptide Synthetic surface for cell types that require Collagen I coating ISSCR 2015

13 Corning PureCoat Collagen I Peptide Supported Human Keratinocyte Culture and Functionality Human neonatal keratinocytes Collagen I Mimetic Rat Tail Collagen I Coating Matrix Culture in animal-free medium EpiLife with S7 supplements Control: Rat tail collagen, and ECM matrix recommended with the medium Comparable cell morphology Population doubling Cumulative population doubling Collagen I mimetic Rat tail collagen I Coating matrix Passage number Time 0 2 hr 3 hr 4 hr Comparable growth on 3 surfaces Division-arrested cells migrated to heal scratch wound 13

14 Corning PureCoat ECM Mimetic Cultureware Fibronectin peptide Synthetic surface for cell types that require Fibronectin coating ISSCR 2015

15 Xeno-free Human Mesenchymal Stem Cell Culture on Fibronectin Mimetic Surface Human bone marrow-derived MSCs in MesenCult -XF medium Control: Human-origin coating matrix Fibronectin Mimetic Human-origin matrix Cells expressed ISCT established marker profile. Osteogenic Adipogenic Comparable morphology and growth on Fibronectin mimetic and human-origin matrix. Successfully tested with additional xeno-free media. Cells maintained multipotency after expansion on Fibronectin mimetic. 15

16 Human Endothelial Colony Forming Cell (ECFCs ) Culture on ECM Mimetic Collagen I and Fibronectin Peptide Culture for 4 passages in EGM -2 BulletKit containing 2% FBS (Lonza) DiI-acetylated-LDL uptake Differentiation on Corning BioCoat angiogenesis system: endothelial cell tube formation Morphology Collagen I Mimetic Fibronectin Mimetic Controls: Corning rat tail Collagen I, human Fibronectin 12 Cumulative population doubling of ECFCs Collagen I Mimetic Fibronectin Mimetic Rat tail collagen I Human fibronectin DiI-Ac-LDL uptake Population doubling Passage number ECM mimetic Fibronectin and Collagen I both supported ECFC expansion under low-serum conditions. Tube formation ECFCs demonstrated typical morphology, functionality and differentiation after expansion on Corning PureCoat ECM mimetic cultureware. 16

17 Corning stemgro hmsc Culture Environment ISSCR 2015

18 Corning stemgro hmsc Culture Environment Serum-free, xeno-free, defined medium In combination with Corning CellBIND surface provides complete cell culture environment for hmscs Enables efficient expansion of hmscs Corning Cat. No KIT stemgro/cellbind 10% FBS/TC treated Competitor/CellBIND 18

19 Surface Markers, Karyotype and Differentiation After 7 Passages in Corning stemgro hmsc Medium Medium/Surface Passage # CD73 CD90 CD105 CD14 CD45 Input Cells p stemgro/cellbind p Competitor/CellBIND p Adipogenic- Oil Red O Osteogenic Alizarin Red Chondrogenic Alcian Blue 19

20 Scale-up and Manufacturing ISSCR 2015

21 Cost-effective Scale-up in Corning PureCoat ECM Mimetic Multi-Flask Easy and cost effective scale-up with consistent performance Uniform cell growth between the layers results in equivalent cell yield/cm 2 in T-175, 3-Layer and 5-Layer ECM mimetic Multi-Flasks Cell yield /cm sq Scale-up MSCs Keratinocytes T Layer 5-Layer Equivalent cell yield Cost/ sq. cm. Cost effective Uniform cell growth 21

22 Corning PureCoat ECM Fibronectin Mimetic Closed System Corning CellSTACKs Have Been Qualified to Manufacture MSC Treatment for Graft-versus-Host Disease Typical MSC morphology was observed for the MSCs cultured on Fibronectin mimetic surface % Viability, 9 Days in Culture 400 μm CS2L T175 T75 Viability of MSCs cultured on Fibronectin mimetic Corning CellSTACK 2-layer, T-175, and T-75 flasks were comparable. 1.00E E E E E E+00 Cell Yield/Vessel Type 400 μm T-75 T-175 ML5 CS2 CS10 Cell yields from T-75, T-175, 5-Layer Multi- Flask (n=3), Corning CellSTACK (n=3), and 10-Layer demonstrated scalable yields. 22

23 MSCs Maintained Characteristic Immunophenotype and Multi-lineage Differentiation Capability MSCs were positive for CD13, CD29, CD44, CD73, CD90, CD105, and negative for CD45, CD80, and HLA-DR. Adipogenic (Oil red O) Osteogenic (von kossa) Chondrogenic (Alcian Blue) MSCs were differentiated into adipogenic, osteogenic, and chondrogenic lineages. Poster Presentation II T /25/15 23

24 Xeno-free Culture of Human Adipose Stem Cells (hasc) in Corning stemgro hmsc Medium and Corning CellBIND Surface Cells exhibited a spindle-like morphology, and at a confluence higher than 75%, cell colonies formed a typical river-like pattern of growth. Average doubling time (h) for two sub-culturing steps of expanded cells, for hasc061 and hasc064 was similar, 51 and 54h, respectively. For hasc057 a shorter doubling time was observed, i.e., an average time of 32h. Viability of expanded cells varied from 93% to 99%. 24

25 hascs Maintained Characteristic Immunophenotype and Multi-lineage Differentiation Capability Concomitant expression of CD73, CD90 and CD105 in the absence of CD34 range between 90.0 to 97.6%. Cells expressed low hematopoietic and endothelial markers, i.e., expression of 1.3% for CD31, 2.5% for CD34 and 0.5%, for CD45. Successful differentiation into the three mesenchymal lineages Poster Presentation II T /25/15 25

26 Summary Corning PureCoat ECM mimetic cultureware: Synthetic, defined, and animal-free Compatible with multiple media Support xeno-free expansion of multiple cell types while maintaining functionality Provide a ready to use alternative to ECM protein coating where animal-free and defined conditions are desirable Demonstrated to support cell manufacturing scale Corning stemgro hmsc culture environment: Serum-free, xeno-free, defined medium In combination with Corning CellBIND surface provides complete cell culture environment for hmscs Enables efficient expansion of hmscs Has demonstrated expansion of hasc derived from xeno-free cgmp cryopreserved human stromal vascular fraction (hsvf) 26