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Cell Reprogramming
ServiceDescriptionTiming
Fibroblasts on Feeder Layer Deriving induced pluripotent stem cells from human fibroblast origin after exposure to sendai virus with reprogramming vectors and cultured with KSR medium on irradiated MEF feeder layer plates. We provide 4 to 5 clones per cell line Two to three months
Fibroblasts on Feeder Free Condition Deriving induced pluripotent stem cells from human fibroblast origin after exposure to sendai virus with reprogramming vectors and cultured with mTeSR medium on Matrigel coated plates. We provide 4 to 5 clones per cell line Two to three months
PBMC on Feeder Layer Deriving induced pluripotent stem cells from human PBMC origin after exposure to sendai virus with reprogramming vectors and cultured with KSR medium on irradiated MEF feeder layer plates. We provide 4 to 5 clones per cell line Two to three months
PBMC on Feeder Free Condition Deriving induced pluripotent stem cells from human PBMC origin after exposure to sendai virus with reprogramming vectors and cultured with mTeSR medium on Matrigel coated plates. We provide 4 to 5 clones per cell line Two to three months
Reprogramming on Feeder Layers
After culturing either human fibroblasts or PBMCs, cells are seeded and exposed to vectors from the CytoTune®-iPS 2.0 Sendai Reprogramming Kit (ThermoFisher Scientific). Cells are then reseeded onto MEF feeder layers and cultured with KSR medium for 20 to 25 days until iPSC colonies arise. Colonies are then selected and passaged 5 to 10 times until cells meet our standards of pluripotency and quality control. Service takes about 2 to 3 months to complete.
a) Schematic of the process to reprogram PBMCs to hiPSCs on MEF feeder layers including representative images at specific time points.
b) Schematic of the process to reprogram fibroblasts to hiPSCs on MEF feeder layers including representative images at specific time points.
Reprogramming on Feeder Free Condition
After culturing either human fibroblasts or PBMCs, cells are seeded and exposed to vectors from the CytoTune®-iPS 2.0 Sendai Reprogramming Kit (ThermoFisher Scientific). Cells are then reseeded onto plates coated with matrigel (Corning) and cultured in refined feeder-free reprogramming medium until iPSC colonies arise. Colonies are then selected and passaged 5 to 10 times until cells meet our standards of pluripotency and quality control. Service takes about 2 to 3 months to complete.
a) Schematic of the process to reprogram PBMCs to hiPSCs on feeder free conditions including representative images at specific time points.
b) Schematic of the process to reprogram fibroblasts to hiPSCs on feeder free conditions including representative images at specific time points.
Pluripotency and Quality Control
Once iPSC cultures undergo its 5th passage, cells are collected and fixed for pluripotent analysis via immunofluorescence and flow cytometry.
a) Immunofluorescent images of an hPSC line highlighting gene expression known to be involved in pluripotency. Targets include OCT4, NANOG, TRA-1-60 and TRA-1-81
b) Flow cytometry analysis of an iPSC clone with hPSC and isotype control antibodies targeting the expression of OCT3/4 and NANOG
iPSC clones are continually passaged until the sendai virus is removed from the culture. Sendai presence is detected using immunofluorescence and PCR assays
a) Immunofluorescent images targeting the presence of Sendai virus in fibroblasts pre- and post-viral infection as well as expression of OCT4 in later passages of the derived iPSC clones from this transduction.
b) PCR analysis detecting sendai specific genes in increasing passages of hiPSC line