Cryopreservation of stem cells, especially embryonic stem cells, is problematic because of low post-thaw cell survival rates and spontaneous differentiation following recovery. In this investigation, mouse embryonic stem cells (mESCs) were encapsulated in arginine-glycine-aspartic acid-serine
(RGDS)-coupled calcium alginates (1.2 wt%), allowed to attach to the substratum and then cryopreserved in 10% vol. dimethyl sulfoxide (DMSO) solution at a slow cooling rate of 1°C/min. RGDS coupling to alginate was confirmed by Transmission Fourier Transform Infra-Red spectroscopy (T-FTIR)
and quantified by using ninhydrin-Ultraviolet/Visible light (ninhydrin-UV/VIS) assay. Flow cytometry data showed that mESCs cryopreserved in RGDS-alginate beads had a higher expression of stem cell markers compared with cells cryopreserved in suspension or cells cryopreserved in unmodified
alginates. Cell viability after thawing was assessed using trypan blue exclusion assay and monitored using Alamar blue assay for 6 hours. It was shown that post-thaw cell survival rate was significantly higher for cells encapsulated in RGDS-modified alginate (93±2%, mean and standard
error) than those in suspension (52±2%) or in unmodified alginates (62±3%). These results showed that cells encapsulated and attached to a substratum have better survival rate and stem cell marker expression 24 hours after cryopreservation than those in suspension. Encapsulation
in RGDS-alginate was optimized for peptide concentration, cryoprotective agent loading time and cooling rate. The best result was obtained when using 12.5 mg peptide/g alginate, 30 minutes loading time and 1C/min cooling rate.
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Document Type: Research Article
Publication date: September 1, 2011
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CryoLetters is a bimonthly international journal for low temperature sciences, including cryobiology, cryopreservation or vitrification of cells and tissues, chemical and physical aspects of freezing and drying, and studies involving ecology of cold environments, and cold adaptation
The journal publishes original research reports, authoritative reviews, technical developments and commissioned book reviews of studies of the effects produced by low temperatures on a wide variety of scientific and technical processes, or those involving low temperature techniques in the investigation of physical, chemical, biological and ecological problems.