CONCLUSION IDENTIFICATION OF OPTIMAL CONDITIONS FOR GENERATING MSCs FOR PRECLINICAL TESTING: COMPARISON OF THREE COMMER- CIAL SERUM-FREE MEDIA AND LOW-SERUM GROWTH MEDIUM Yelica López, Elizabeth Trevino, Mark L Weiss Kansas State University, Department of Anatomy and Physiology, Manhattan, KS 66506 Our goal is to move human mesenchymal stromal cells derived from Wharton’s jelly )WJ-MSCs) into clinical trials. One important step is to generate an SOP to produce GMP-grade MSCs for preclinical evaluation. Here, three commercially prepared serum-free (SF) media and a well-defined 2% serum growth medium (SGM) were compare to determine expansion, phenotypic stability, and multipotency. Xeno free conditions were desired because a single medium is sim- pler and eliminates exposure to animal products. SF xeno free MSC NutriStem® medium produced by Biological Industries (BI), StemCell Technologies (SC) and Invitrogen (IV) were compared with our SGM. WJ-MSCs were cultured at 10000 cell/cm2 in standard conditions of 5% CO2, 21% oxygen. Additionally, three attachment solutions as recommended by the manufacturers were used for SF conditions (note that expansion in SGM did not require an attachment solution). After initial isolation,WJ-MSCs were split into four media conditions and expanded till passage 5 (p5) and the following parameters were evaluated: expansion, posi- tive and negative surface marker expression, differentiation potential and colony forming unit-fibroblast (CFU-F) assay. WJ-MSCs cultured in MSC NutriStem® medium showed significantly greater proliferation compared to other SF media and to SGM. SF expansion did not impact expression of CD73, CD90, CD105, HLA-ABC (all positive), or CD34, CD45, HLA-DR (all negative). WJ-MSCs differ- entiated efficiently after expansion in MSC NutriStem®. CFU-F assay revealed no significant difference in colony forming efficiency between MSC NutriStem® and SGM. We conclude that for expansion of WJ-MSCs in SF conditions using MSC NutriStem® and substrate provided optimal cell expansion compared to two other SF formulations and SGM. WJ-MSCs maintained their phenotypic surface marker profile of MSCs and their multipotency as demonstrated by osteocytic, chondrocytic and adipocytic lineage differentiation. Once WJ-MSCs are isolated in MSC NutriStem® medium, preclinical validation testing will begin. ABSTRACT OBJECTIVE •• •compared three commercially avail- able serum-free media to support WJ- MSCs expansion, differentiation and maintenance of CFU-F to our gold- standard medium that contains 2% FBS • NutriStem® a new serum-free, xeno- free medium was tested METHODS METHODS RESULTS FITC Isotype PE Isotype 99.57% 0.43% 50.17% 50.63% HLA-ABC HLA-DR 0.91% 99.11% 99.57% 0.42% CD45 2.Immunophenotyping MSC NutriStem alone 3.43% 1. Morphology 2% FBS StemPro Mesencult Dead cells SUMMARY REFERENCES While more research is required, we conclude that using MSC NutriStem® xeno-free, serum-free medium and sub- strate provided superior expansion of WJ-MSCs compared to other SF formu- lations and SGM. ACKNOWLEDGMENTS VV • Human WJ-MSCs were obtained from 8 umbilical cords • The proliferation rate, viability, stemmness (estimated from CFU-F), and tri-lineage differentiation capaci- ties were compared in 4 different growth media • Proliferation and viability were evalu- ated from P1 to P5, differentiation, flow cytometry and CFU-F assay were per- formed at P5 - López Y et al. Evaluating the impact of oxygen concen- tration and plating density on human Wharton’s jelly- derived mesenchymal stroma cells. TOTERMJ 4: 2011. - Seshareddy K et al. Method to isolate mesenchymal like cells from Wharton’s Jelly of umbilical cord. Methods Cell Biol 86: 101-119, 2008. MSC NutriStem BI 2% FBS StemPro InVitrogen Mesencult Stem Cell P1 •Trypan blue exclusion test • Proliferation P5 WJ-MSC primary isolation C CFU-F assay Differentiation 100 cells/cm² 50 cells/cm² 10 cells/cm² 10000 cells/cm² INTRODUCTION • WJ-MSCs are an attractive resource for cell therapy • We would prefer for clinical evalua- tions that the cells be cultured in xeno- free conditions • We compared three commercially available serum-free media for expan- sion of WJ-MSCs with our gold- standard medium 0 500000 1000000 1500000 2000000 2500000 3000000 Cell Number StemPro 2% FBS MSC NutriStem Mesencult 1. MSC NutriStem outperformed the other media used for WJ-MSC expansion 2. No differences in CFU-F were found between MSC NutriStem and gold- standard medium at passage 5 3. MSC NutriStem and the gold-standard medium supported tri-lineage differentia- tion with no apparent differences between them. 4. WJ-MSCs stained positively for MSC surface markers from all groups. Authors declare no competing interests. Work supported by Biological Industries. MSC NutriStem is a research product and not yet registered with the FDA. MSC NutriStem MSC NutriStem + Inducers SEATTLE 2012 18TH SCT ANNUAL MEETING
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CONCLUSION
IDENTIFICATION OF OPTIMAL CONDITIONS FOR GENERATING MSCsFOR PRECLINICAL TESTING: COMPARISON OF THREE COMMER-CIAL SERUM-FREE MEDIA AND LOW-SERUM GROWTH MEDIUM
Yelica López, Elizabeth Trevino, Mark L WeissKansas State University, Department of Anatomy and Physiology, Manhattan, KS 66506
Our goal is to move human mesenchymal stromal cells derived from Wharton’sjelly )WJ-MSCs) into clinical trials. One important step is to generate an SOP toproduce GMP-grade MSCs for preclinical evaluation. Here, three commerciallyprepared serum-free (SF) media and a well-defined 2% serum growth medium(SGM) were compare to determine expansion, phenotypic stability, andmultipotency. Xeno free conditions were desired because a single medium is sim-pler and eliminates exposure to animal products. SF xeno free MSC NutriStem®medium produced by Biological Industries (BI), StemCell Technologies (SC) andInvitrogen (IV) were compared with our SGM. WJ-MSCs were cultured at 10000cell/cm2 in standard conditions of 5% CO2, 21% oxygen. Additionally, threeattachment solutions as recommended by the manufacturers were used for SFconditions (note that expansion in SGM did not require an attachment solution).After initial isolation,WJ-MSCs were split into four media conditions and expandedtill passage 5 (p5) and the following parameters were evaluated: expansion, posi-tive and negative surface marker expression, differentiation potential and colonyforming unit-fibroblast (CFU-F) assay. WJ-MSCs cultured in MSC NutriStem®medium showed significantly greater proliferation compared to other SF mediaand to SGM. SF expansion did not impact expression of CD73, CD90, CD105,HLA-ABC (all positive), or CD34, CD45, HLA-DR (all negative). WJ-MSCs differ-entiated efficiently after expansion in MSC NutriStem®. CFU-F assay revealed nosignificant difference in colony forming efficiency between MSC NutriStem® andSGM. We conclude that for expansion of WJ-MSCs in SF conditions using MSCNutriStem® and substrate provided optimal cell expansion compared to two otherSF formulations and SGM. WJ-MSCs maintained their phenotypic surface markerprofile of MSCs and their multipotency as demonstrated by osteocytic,chondrocytic and adipocytic lineage differentiation. Once WJ-MSCs are isolated inMSC NutriStem® medium, preclinical validation testing will begin.
ABSTRACT
OBJECTIVE
••
• compared three commercially avail-able serum-free media to support WJ-MSCs expansion, differentiation andmaintenance of CFU-F to our gold-standard medium that contains 2%FBS
• NutriStem® a new serum-free, xeno-free medium was tested
METHODS
METHODS RESULTS
FITC Isotype PE Isotype
99.57%0.43% 50.17%
50.63%
HLA-ABC HLA-DR
0.91%99.11% 99.57% 0.42%
CD45
2.Immunophenotyping
MSC NutriStemalone
3.43%
1. Morphology2% FBS StemPro Mesencult
Deadcells
SUMMARY
REFERENCES
While more research is required, weconclude that using MSC NutriStem®xeno-free, serum-free medium and sub-strate provided superior expansion ofWJ-MSCs compared to other SF formu-lations and SGM.
ACKNOWLEDGMENTS
VV
• Human WJ-MSCs were obtained from8 umbilical cords
• The proliferation rate, viability,stemmness (estimated from CFU-F),and tri-lineage differentiation capaci-ties were compared in 4 differentgrowth media
• Proliferation and viability were evalu-ated from P1 to P5, differentiation, flowcytometry and CFU-F assay were per-formed at P5
- López Y et al. Evaluating the impact of oxygen concen-tration and plating density on human Wharton’s jelly-derived mesenchymal stroma cells. TOTERMJ 4: 2011.
- Seshareddy K et al. Method to isolate mesenchymal likecells from Wharton’s Jelly of umbilical cord. Methods CellBiol 86: 101-119, 2008.
MSCNutriStem
BI2% FBS StemPro
InVitrogenMesencultStem CellP1
•Trypan blue exclusion test
• Proliferation
P5
WJ-MSC primary isolation
C
CFU-F assay Differentiation
100 cells/cm²
50 cells/cm²
10 cells/cm²
10000 cells/cm²
INTRODUCTION• WJ-MSCs are an attractive resourcefor cell therapy
• We would prefer for clinical evalua-tions that the cells be cultured in xeno-free conditions
• We compared three commerciallyavailable serum-free media for expan-sion of WJ-MSCs with our gold-standard medium
0
500000
1000000
1500000
2000000
2500000
3000000
Cell
Nu
mb
er
StemPro 2% FBS MSCNutriStem
Mesencult
1. MSC NutriStem outperformed the othermedia used for WJ-MSC expansion
2. No differences in CFU-F were foundbetween MSC NutriStem and gold-standard medium at passage 5
3. MSC NutriStem and the gold-standardmedium supported tri-lineage differentia-tion with no apparent differences betweenthem.
4. WJ-MSCs stained positively for MSCsurface markers from all groups.
Authors declare no competing interests.
Work supported by Biological Industries.
MSC NutriStem is a research product andnot yet registered with the FDA.
MSC NutriStem
MSC NutriStem+ Inducers
SEATTLE 201218TH SCT
ANNUAL MEETING
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