Standardized qc assays and large scale expansion of pluripotent stem cells using an automated closed system.

ConclusionHere, we established a workflow encompassing stable expansion of hPSCs using a xeno-free cultivation medium, assessment of pluripotency using a defined marker combination and assessment of differentiation potential based on lineage-specific, complete media both combined with quantitative, multicolor flow cytometry analysis to characterize and quality control the cultivated hPSCs, as well as cryopreservation of hPSCs using an animal component-free, chemically defined cryopreservation media.Following this workflow, hPSCs could be stably expanded over 20 passages with persistent, high expression of pluripotency markers (>92%) TRA-1-60, SSEA-4, SSEA-5, Oct-4, Sox2 and almost no expression of differentiation maker SSEA-1, showed a homogenous morphology and also retained a stable karyotype. Quantitative, flow cytometry–based analysis reproducibly proved their differentiation potential into ectoderm, mesoderm, and endoderm. Cells cultivated under the same conditions showed a reproducible recovery after cryopreservation and thawing resulting in a 8-12 fold expansion in passage 1, high pluripotency marker expression as well as genomic stability confirmed 5 passages post thaw.Thus, the workflow shown here assures a standardized, robust expansion of hPSCs, includes characterization and quality control of the expanded cells, as well as efficient cryopreservation. The flow based QC strategy was also successfully applied for characterization of cells cultivated under standardiz...
Source: Cytotherapy - Category: Cytology Source Type: research