Post on 23-May-2020
Paula FlahertyTechnology ManagerDeepa Saxena, Ph.D.Senior Development ScientistISSCR 2015 June 26, 2015
ISS
CR
2015
Corning Innovation ShowcaseStem Cell Culture, Differentiation, and Scale‐Up
Novel Technologies Enabling Research and Cell Processing Applications
2© 2015 Corning Incorporated
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
ISS
CR
2015
3© 2015 Corning Incorporated
Corning Incorporated HighlightsFounded:1851
Headquarters:Corning, New York
Employees:~35,000 worldwide
2014 Sales:$10B
R&D Investment:~10% of sales
Fortune 500 Rank (2015): 297
• 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.
4© 2015 Corning Incorporated
Corning: Leader in Cell Culture Surface InnovationA Century of Surface Technology
1915 Heat-resistant Pyrex® glass
1907Glass Surfaces used to culture
mammalian cells
1910 1920 1930 1940 1950 1960 1970 1980 1990 20102000
2010Synthemax®
Synthetic VN peptide surface
2012
1959 TC treatment and gamma irradiation
TC-treated Polystyrene
1985Matrigel® matrix, defined ECMs
In vivo-like cell growth and differentiation
1987BioCoat™ surface
Convenient and consistent pre-coated cultureware
2012PureCoat™ ECM
Mimetic Fibronectin and Collagen I
Synthetic peptide surfaces
1990sPrimaria™
and CellBIND®
Charged-based modification of plastic surfaces
2000sPureCoat surfacesAmine and CarboxylUltra-Low AttachmentHydrogel-coated low bind surface
1953Development of
plastic 100 mm petri dish for U.S. Army
Glass + Plastic + Biological + Synthetic
2015
2014Corning
rLaminin-521Animal-free pluripotent
stem cell surface
5© 2015 Corning Incorporated
Cell Manufacturers are Rapidly Progressing from Scientific Knowledge to Process Engineering
“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, 05.08.15
Science and TechnologyDevelop fundamental understanding
of product, process, and material
Engineering/ManufacturingDevelop and control advanced
product and process technology
6© 2015 Corning Incorporated
Complete and Customizable Cell Culture Environments
• Corning® Matrigel® matrix• Purified biological, xeno-free, and
synthetic ECMs• ECM mimetic cultureware
• cGMP media manufacturing• Serum• Serum-free media supplements and
growth factorsCorning CellSTACK®
Multi-layer flasks
Coated microcarriers
Customizable solutions for all stages of cell and tissue culture research
7© 2015 Corning Incorporated
Advanced Surfaces and Extracellular Matrices
Vialed Extracellular Matrices (ECMs)Corning® Matrigel® matrix Defined ECMs
Animal-derivedXeno-free
Recombinant ECMsrLaminin-521 (Human)
Synthetic ECMs SynthemaxCorning PuraMatrix™
Custom ECM formulations
Synthetic ECM Mimetic Pre-coated Cultureware
Corning PureCoat™Fibronectin peptideCollagen I peptiderLaminin-521 coming soon
• Synthemax (custom)Vitronectin peptide
Biological ECM Pre-coated CulturewareCorning BioCoat™
• Plates• Inserts• Flasks• Coverslips/slides• Dishes
•Custom coatings
Enhanced Tissue Culture-treatedCorning CellBIND® surface
(-) chargeCorning PureCoat
Amine (+) chargeCarboxyl (-) charge
Corning Primaria™(+) and (-) mixed charge
Ultra-Low Attachment Hydrogel-coated
Functional Attachment ChargeFunctional Attachment Functional Attachment
General cell culture, HTS, specialized applications HTS, general cell culture Specialized applications:
cell culture enablement General cell culture, HTS, specialized applications
Corning® PureCoat™ ECM Mimetic Cultureware
Synthetic peptide covalently attached on a surface
ISS
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2015
9© 2015 Corning Incorporated
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®
10© 2015 Corning Incorporated
Corning® PureCoat™ ECM Mimetic CulturewareManufacturing and Quality
• Manufactured under cGMP per the requirements of FDA 21 CFR 820
• Manufacturing facility ISO 9001, ISO 13485, and ISO 14001 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
End-user
No animal components in final product
No animal components used in production of raw materials
11© 2015 Corning Incorporated
Synthetic and Defined Cultureware Suitable for Expansion of a Variety of Cell Types
Corning® PureCoat™
ECM MimeticExamples of Cell Types Expanded in Defined Cell Culture Environments
Fibronectin Peptide Human BM-derived mesenchymal stem cells
Human endothelial progenitor cells
Human adipose-derived stem cells
CHO cells
Vero cells
Collagen I Peptide Human keratinocytes
Human corneal cells
Human adipose-derived stem cells
Human endothelial progenitor cells
Human cord blood-derived mesenchymal stem cellsVero cells
Corning® PureCoat™ ECM Mimetic CulturewareCollagen I peptideSynthetic surface for cell types that require Collagen I coating IS
SC
R 2015
13© 2015 Corning Incorporated
Corning® PureCoat™ Collagen I Peptide Supported Human Keratinocyte Culture and Functionality
• Human neonatal keratinocytes
• Culture in animal-free medium EpiLife® with S7 supplements
• Control: Rat tail collagen, and ECM matrix recommended with the medium
Collagen I Mimetic Coating MatrixRat Tail Collagen I
Comparable growth on 3 surfaces
Cumulative population doubling
02468
101214
1 2 3 4
Passage number
Popu
latio
n do
ublin
g
Collagen I mimeticRat tail collagen ICoating matrix
Time 0 2 hr
3 hr 4 hr
Division-arrested cells migrated to heal scratch wound
Comparable cell morphology
Corning® PureCoat™ ECM Mimetic CulturewareFibronectin peptide Synthetic surface for cell types that require Fibronectin coating IS
SC
R 2015
15© 2015 Corning Incorporated
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
AdipogenicOsteogenic
Comparable morphology and growth on Fibronectin mimetic and human-origin matrix.
Cells maintained multipotency after expansion on Fibronectin mimetic.
Cells expressed ISCT established marker profile.
Successfully tested with additional xeno-free media.
16© 2015 Corning Incorporated
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
• Controls: Corning rat tail Collagen I, human Fibronectin
Cumulative population doubling of ECFCs
0
2
4
6
8
10
12
2 3 4Passage number
Pop
ulat
ion
doub
ling
Collagen I Mimetic Rat tail collagen IFibronectin Mimetic Human fibronectin
ECFCs demonstrated typical morphology, functionality and differentiation after expansion on Corning PureCoat™ ECM mimetic cultureware.
Collagen I Mimetic Fibronectin Mimetic
Mor
phol
ogy
DiI-
Ac-
LDL
upta
ke
Tube
form
atio
n
ECM mimetic Fibronectin and Collagen I both supported ECFC® expansion under low-serum conditions.
Corning® stemgro® hMSC Culture Environment
ISS
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2015
18© 2015 Corning Incorporated
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 hMSCsCorning Cat . No.40-410-KIT
stemgro/CellBIND
10% FBS/TC‐treated Competitor/CellBIND
19© 2015 Corning Incorporated
Surface Markers, Karyotype and Differentiation After 7 Passages in Corning® stemgro® hMSC Medium
Medium/Surface Passage # CD73 CD90 CD105 CD14 CD45
Input Cells p0 95.2 95.0 96.8 0.0 0.0
stemgro/CellBIND® p5 98.5 99.2 96.7 0.0 0.0
Competitor/CellBIND p5 88.4 93.0 95.1 0.0 0.0
Adipogenic- Oil Red O Osteogenic Alizarin Red Chondrogenic Alcian Blue
Scale-up and Manufacturing
ISS
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2015
21© 2015 Corning Incorporated
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/cm2 in T-175, 3-Layer and 5-Layer ECM mimetic Multi-Flasks
Scale-up
0
5000
10000
15000
20000
25000
30000
35000
T-175 3-Layer 5-Layer
Cel
l yie
ld /c
m s
q.
MSCsKeratinocytes
Cost/ sq. cm.
Cost effective
Equivalent cell yield
Uniform cell growth
22© 2015 Corning Incorporated
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.
400 μm400 μm
020406080
100120
CS2L T175 T75
% Viability, 9 Days in Culture
0.00E+00
2.00E+07
4.00E+07
6.00E+07
8.00E+07
1.00E+08
T-75 T-175 ML5 CS2 CS10
Cell Yield/Vessel Type
Viability of MSCs cultured on Fibronectin mimetic Corning CellSTACK 2-layer, T-175, and T-75 flasks were comparable.
Cell yields from T-75, T-175, 5-Layer Multi-Flask (n=3), Corning CellSTACK (n=3), and 10-Layer demonstrated scalable yields.
23© 2015 Corning Incorporated
MSCs Maintained Characteristic Immunophenotypeand Multi-lineage Differentiation Capability
MSCs were positive for CD13, CD29, CD44, CD73, CD90, CD105, and negative for CD45, CD80, and HLA-DR.
MSCs were differentiated into adipogenic, osteogenic, and chondrogenic lineages.
Adipogenic (Oil red O) Chondrogenic (Alcian Blue)Osteogenic (von kossa)
Poster Presentation IIT-10009 6/25/15
24© 2015 Corning Incorporated
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%.
25© 2015 Corning Incorporated
hASCs Maintained Characteristic Immunophenotypeand Multi-lineage Differentiation Capability
Poster Presentation IIT-10010 6/25/15
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 mesenchymallineages
26© 2015 Corning Incorporated
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)
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