Cryopreservation of Osteoblast-Like Cells: Viability and Differentiation with Replacement of Fetal Bovine Serum in vitro

Abstract

In reconstructive medicine, the clinical use of cryopreservation techniques depends on the absence of infectious agents such as prions. Therefore, we investigated the viability and differentiation of human osteoblast-like cells during replacement of fetal bovine serum in vitro. The aim of the present study is to replace the potentially infectious supplement fetal bovine serum during the cryopreservation procedure in order to perform future clinical trials. We used a cryopreservation technique with Me₂SO for human osteoblast-like cells of iliac cancellous bone. In the cell culture of cryopreserved and fresh osteoblast-like cells, we substituted Dulbecco’s modification of Eagle’s medium (DMEM)/Ham’s F12 plus 1% penicillin/streptomycin with autologous serum, human serum albumin and Biseko® for fetal bovine serum. For the fourth treatment group, we removed fetal bovine serum without replacing it. DMEM/Ham’s F12 plus 1% penicillin/streptomycin with fetal bovine serum served as the control group. After 4, 7, 14 and 21 days of culture for the cryopreserved and noncryopreserved cells, we performed cell counting, a WST-1 test, ELISA for collagen type I, and osteocalcin. The activity of alkaline phosphatase was also measured. The best results were obtained for the group with autologous serum as a supplement after thawing, exceeding the other groups with regard to proliferation rate. Most viable cells were observed with no replacement before freezing and after thawing of the cells. With regard to differentiation, the cultures with autologous serum after thawing of the cells showed little concentration of the differentiation markers, probably due to early contact inhibition of the cells in vitro. With regard to effort and outcome, the most promising group for cryopreservation was the one with DMEM/Ham’s F12 plus 1% penicillin/streptomycin alone before freezing, especially when osteoblast-like cells were cultured in medium with autologous serum after thawing. This is important, as this in vitro setting resembles the in vivo situation when cryopreserved bone is transplanted. These findings indicate that, for clinical purposes, fetal bovine serum can be removed for cryopreservation of iliac cancellous bone with minor loss of viability.