Evaluation of Sericin as a Fetal Bovine Serum-Replacing Cryoprotectant During Freezing of Human Mesenchymal Stromal Cells and Human Osteoblast-Like Cells

Abstract

A reliable, cryoprotective, xeno-free medium suitable for different cell types is highly desirable in regenerative medicine. There is danger of infection or allergic reaction with the use of fetal bovine serum (FBS), making it problematic for medical applications. The aim of the present study was to develop an FBS-free cryoprotective medium for human mesenchymal stromal cells (hMSCs; primary cells) and immortalized human osteoblasts (SAOS-2 cell line). Furthermore, we endeavored to eliminate or reduce the presence of dimethyl sulfoxide (DMSO) in the medium. Sericin, a sticky protein derived from the silkworm cocoon, was investigated as a substitute for FBS and DMSO in the freezing medium. Cell viability (24 hours after thawing, both hMSC and SAOS-2) and colony-forming ability (2 weeks after thawing, only for hMSCs) were both determined. The FBSfree medium with 1% sericin in 10% DMSO was found to be a suitable freezing medium for primary hMSCs, in contrast to immortalized human osteoblasts. Surprisingly, the storage of hMSCs in a cultivation medium with only 10% DMSO also provided satisfactory results. Any drop in DMSO concentration led to significantly worse survival of cells, with little improvement in hMSC survival in the presence of sericin. Thus, sericin may substitute for FBS in the freezing medium for primary hMSCs, but cannot substitute for DMSO.

Silkworm Sericin: Properties and Biomedical Applications

“…Verdanova et al [125] used sericin to develop serum-free media as well as to replace the medium cryoprotectant DMSO in human mesenchymal stromal cells (hMSCs) and immortalized human osteoblasts (SAOS-2 cell line). The supplementation of 1% sericin in 10% DMSO, as BSA substituent, proved to be efficient cryoprotection media for hMSCs cells, since they maintained their cell viability and ability to form colonies, which was not observed in SAOS-2 cells.…”

Adult multipotent stromal cell cryopreservation: Pluses and pitfalls

“…FBS collected at different gestational stages is a common culture medium ingredient that provides growth factors, nutrients, and hormones for cell proliferation and adhesion . It is also thought to act as a CP through protection of cell proteins and stabilization of osmotic pressure . In addition to ethical, zoonosis, and xenogeneic protein concerns, variation in composition among FBS lots contributes to inconsistent cell culture performance .…”

“…There was no difference in post‐thaw cell viability, morphology, or growth kinetics among different freezing media, and 95% autologous serum with 5% DMSO was recommended for short‐term (2‐3 days) cryopreservation . Serum‐free MSC cryopreservation medium has been shown to have similar or superior post‐cryopreservation outcomes compared to FBS‐containing media . Increasing availability of FBS‐free freezing media may be important to improving consistency in MSC pre‐cryopreservation and post‐cryopreservation characteristics.…”

“…40 It is also thought to act as a CP through protection of cell proteins and stabilization of osmotic pressure. 40,48 In addition to ethical, zoonosis, and xenogeneic protein concerns, variation in composition among FBS lots contributes to inconsistent cell culture performance. 47 A recent finding that cryopreserved canine adipose-derived multipotent stromal cells (ASCs) have increased CD44 expression compared to fresh cells was attributed to FBS in the freezing medium.…”

Cryopreservation of Human Mesenchymal Stem Cells for Clinical Applications: Current Methods and Challenges

“…To replace the use of FBS, xeno-free cryomedium (e.g., sericin and 5% human albumin solution plus 5% DMSO) 10,88 have been proposed. Moreover, our recent study also demonstrates that adipose-derived MSCs preserved in cryomedium containing only 5% DMSO were able to maintain their phenotype, stemness, proliferative and differentiation potential, and high cell viability in a way comparable to 10% DMSO.…”