Abstract
Human embryonic stem cells (hESCs), due to their self-renewal capacity and pluripotency, are an important source of cells for regenerative medicine, cell therapy, and clinical transplantation. However, there are still many immediate obstacles that need to be addressed before their practical application. In this study, we focus on three avenues for improvement: cultivation, differentiation, and cryopreservation. First, we replaced common Matrigel with a synthetic peptide-acrylate surface (Synthemax®) in defined mTeSR™1 medium to expand undifferentiated hESCs. We confirmed that the cells still expressed pluripotency markers, had the ability to differentiate into three germ layers, and maintained a normal karyotype after 10 passages of subculture. Next, we reported an efficient protocol for deriving nearly 86% definitive endoderm cells from hESCs under serum-free conditions. We were also able to obtain 25% insulin-producing cells within 21 days by following a simple three-step protocol. Moreover, the results of immunocytochemical and quantitative gene expression analyses showed that the efficiency of induction was not significantly different between the Synthemax® surface and the Matrigel-coated surface. Thus, Synthemax® could be a stable substrate for the long-term culture of hESCs, and the differentiated insulin-producing cells could be a therapeutic resource for diabetic patients in the future. Last, we used the Cell Alive System (CAS), which combines a programmed freezer with an oscillating magnetic field to reduce cryo-injury during the freezing process. The hESC clumps suspended in freezing medium were divided into three groups: (i) cells frozen by a conventional freezing container, Mr. Frosty, and kept in a -80°C freezer (MF); (ii) cells frozen to -32°C by CAS, and then transferred to a -80°C freezer (CAS); and (iii) cells frozen to -32°C by CAS, and then transferred to a pre-cooled Mr. Frosty and kept in a -80°C freezer (CAS-MF) overnight. All cryovials were placed in liquid nitrogen for one week, and hESCs were then thawed and cultured on feeder cells for 7 days. The results of alkaline phosphatase (AP) staining showed that the attachment efficiency of the cells cryopreserved by CAS and CAS-MF was significantly higher (29.0% and 44.0%) than that achieved using the MF method (7.0%). Furthermore, we confirmed that cells cryopreserved using CAS-MF could be subcultured while expressing pluripotency markers, could differentiate into the three germ layers, and could maintain a normal karyotype. These results demonstrate that the use of CAS-MF offers an efficient method for hESC banking.