Journal: International Journal of Molecular Sciences

Article Title: End-to-End Platform for Human Pluripotent Stem Cell Manufacturing

doi: 10.3390/ijms21010089

Figure Lengend Snippet: L7™ TFO2 hPSC xeno-free media supports the microcarrier (MC)-based expansion of RTiPSC3B and RTiPSC4i human-induced pluripotent stem cells (hiPSCs) leading to >2 × 10 6 cells/mL ( A ) and >10 fold expansion ( B ) in 17 days. Small- and large-sized MCs support the cell growth ( C ) and expansion ( D ) of RTiPSC3B in L7™ TFO2 hPSC xeno-free media. Inoculating less cells/mL does not compromise cell yield, but leads to rapid cell growth and higher cell expansion of RTiPSC4i ( E , F ) and RTiPSC3B ( G , H ), which were cultured on small- and large-sized MCs. Coating MCs with the L7™ hPSC Matrix is essential for the attachment and expansion of hiPSCs in L7™ TFO2 hPSC medium. RTiPSC3B inoculated at 0.2 × 10 6 cells/mL with small-sized microcarriers ( I ) and LiPSC18R inoculated at 0.04 × 10 6 cells/mL with large-sized microcarriers ( J ). RTiPSC4i inoculated at 0.04 × 10 6 cells/mL with large-sized coated and uncoated microcarriers. Cell growth ( K ) and fold expansion ( L ) are shown.

Article Snippet: After nucleofection, the cells were plated onto 6-well plates pre-coated with L7TM hPSC Matrix, in priming medium containing 0.5 mM sodium butyrate (ReproCELL USA, Inc., 04-0005, Beltsville, MD, USA) to enhance reprogramming efficiency.

Techniques: Cell Culture

Journal: International Journal of Molecular Sciences

Article Title: End-to-End Platform for Human Pluripotent Stem Cell Manufacturing

doi: 10.3390/ijms21010089

Figure Lengend Snippet: Human iPSCs expanded in a bioreactor have typical hiPSC morphology and express hPSC-associated markers post-harvest. ( A ) Phase contrast images of iPSCs expanded in a bioreactor plated either as single cells after cell release from MCs (left panel), or as clusters of cells on MCs (right panel). Magnification, 100×; Scale bar: 100 µm; ( B ) Immunofluorescence staining of OCT3/4, NANOG, SSEA-4, TRA-1-60 and TRA-1-81 of iPSCs expanded in a bioreactor and plated onto 2D cell culture plates. Representative images from two different cell lines are shown. Magnification, 100×; ( C ) Quantitative analysis of hPSC-associated markers OCT3/4, SSEA4, TRA-1-60 and TRA-1-81 by flow cytometry of RTiPSC3B and LiPSC18R cells expanded in a bioreactor. Data obtained from either three independent runs (RTiPSC3B) or two independent runs (LiPSC18R). Data plotted are mean ± standard deviation (SD); ( D ) Pluripotency of RTiPSC3B cells expanded in a bioreactor demonstrated by embryoid body (EB) formation followed by immunofluorescence staining for germ layer-specific markers. Magnification, 100× (ectoderm, endoderm), 200× (mesoderm). ( E ) Representative immunofluorescence staining for lineage-specific markers RTiPSC3B and LiPSC18R cell lines. Magnification, 100× (Neural stem cells, definitive endoderm), 200× (cardiomyocytes)

Article Snippet: After nucleofection, the cells were plated onto 6-well plates pre-coated with L7TM hPSC Matrix, in priming medium containing 0.5 mM sodium butyrate (ReproCELL USA, Inc., 04-0005, Beltsville, MD, USA) to enhance reprogramming efficiency.

Techniques: Immunofluorescence, Staining, Cell Culture, Flow Cytometry, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: End-to-End Platform for Human Pluripotent Stem Cell Manufacturing

doi: 10.3390/ijms21010089

Figure Lengend Snippet: Cells expanded in a bioreactor and concentrated by kSep show hPSC characteristics of morphology and marker expression. ( A ) Phase contrast images of single cells (RTiPSC3B and LiPSC18R) post concentration by kSep400, 24 h and 72 h post plating onto 2D cell culture plates. Magnification, 40×; ( B ) Immunofluorescence staining RTiPSC3B cells expanded in a bioreactor and concentrated via kSep show expression of OCT3/4, NANOG, SSEA-4 and TRA-1-60. Magnification, 100×; ( C ) Quantitative analysis of hPSC-associated markers SSEA4, TRA-1-60 and TRA-1-81 by flow cytometry of iPSCs expanded in a bioreactor and concentrated via kSep. Results are shown for RTiPSC3B and LiPSC18R lines; ( D ) Pluripotency of RTiPSC3B and LiPSC18R cells expanded in a bioreactor and concentrated by kSep were demonstrated by directed differentiation into definitive endoderm, neural stem cells and cardiomyocytes. Magnification, 100× (Neural stem cells, definitive endoderm), 200× (cardiomyocytes)

Article Snippet: After nucleofection, the cells were plated onto 6-well plates pre-coated with L7TM hPSC Matrix, in priming medium containing 0.5 mM sodium butyrate (ReproCELL USA, Inc., 04-0005, Beltsville, MD, USA) to enhance reprogramming efficiency.

Techniques: Marker, Expressing, Concentration Assay, Cell Culture, Immunofluorescence, Staining, Flow Cytometry

Journal: International Journal of Molecular Sciences

Article Title: End-to-End Platform for Human Pluripotent Stem Cell Manufacturing

doi: 10.3390/ijms21010089

Figure Lengend Snippet: Thaw to 3D inoculation: iPSCs thawed into suspension retain hPSC-characteristics. ( A ) iPSCs thawed into suspension and expanded in a bioreactor have typical iPSC morphology when plated onto 2D, either before or after release from MCs. Magnification, 40×.; ( B ) Detection of hPSC-associated markers by immunofluorescence staining in cells post-harvest and concentration by kSep400. Magnification, 100×.; ( C ) Quantitative analysis of hPSC-associated markers by flow cytometry of cells post-harvest and cells concentrated after harvest; ( D ) Direct differentiation of iPSCs thawed into suspension, expanded in a bioreactor and concentrated via kSep400. Magnification, 100× (Neural stem cells, definitive endoderm), 200× (cardiomyocytes).2.13. D to 3D Inoculation.

Article Snippet: After nucleofection, the cells were plated onto 6-well plates pre-coated with L7TM hPSC Matrix, in priming medium containing 0.5 mM sodium butyrate (ReproCELL USA, Inc., 04-0005, Beltsville, MD, USA) to enhance reprogramming efficiency.

Techniques: Suspension, Immunofluorescence, Staining, Concentration Assay, Flow Cytometry

Journal: International Journal of Molecular Sciences

Article Title: End-to-End Platform for Human Pluripotent Stem Cell Manufacturing

doi: 10.3390/ijms21010089

Figure Lengend Snippet: Quality assessment of hiPSCs expanded through a 3D seed train. ( A ) Immunofluorescence staining of hPSC-associated markers. Magnification, 100×; ( B ). Quantitative analysis of hPSC-associated markers; ( C ) Immunofluorescence staining of germ layer-specific markers, performed on embryoid bodies (EBs). Magnification, 100× (ectoderm, endoderm), 200× (mesoderm)

Article Snippet: After nucleofection, the cells were plated onto 6-well plates pre-coated with L7TM hPSC Matrix, in priming medium containing 0.5 mM sodium butyrate (ReproCELL USA, Inc., 04-0005, Beltsville, MD, USA) to enhance reprogramming efficiency.

Techniques: Immunofluorescence, Staining