Derivation of new human embryonic stem cell lines (Yazd1-3) and their vitrification using Cryotech and Cryowin tools: A lab resources report

Background: Cell banking of initial outgrowths from newly derived human embryonic stemcells (hESCs) requires an efficient freezing method. Vitrification is used for the preservation ofgametes and early embryos in assisted reproduction techniques (ART). Moreover, vitrificationwas applied for cryopreservation of hESCs using open pulled straws.Objective: To derive and characterize new hESC lines and then use Cryotech and Cryowin toolsfor their vitrification.Materials and Methods: Human ESC lines were generated in a microdrop culture system usingmouse embryonic fibroblasts (MEFs) as the feeder layer; this was later scaled up using bothMEFs and Yazd human foreskin fibroblasts batch 8 (YhFF#8). To bank the cell lines, master cellbanks of 100 Cryotech and Cryowin tools were produced for each individual cell line using thevitrification method; flasks of hESC lines were also cryopreserved using a conventional slowfreezingmethod.Results: The pluripotency of cell lines was assessed by their expression of pluripotencyassociatedgenes (OCT4/POU5F1, NANOG, and SOX2) and markers such as SSEA4, TRA-1-60, and TRA-2-49. Their in vitro capacity to differentiate into germ layers and germ cellsusing embryoid body (EB) formation and monolayer culture was assessed by screening theexpression of differentiation-associated genes. The chromosomal constitution of each hESCline was assessed by G-banding karyotyping.Conclusion: Cryotech and Cryowin tools used to vitrify new hESCs at an early stage of derivationis an efficient method of preserving hESCs.

کلیدواژه ها:

Derivation ، Human embryonic stem cells ، Human foreskin fibroblast ، Microdrop ، Vitrification