The effect of serum types on Chondrogenic differentiation of adipose-derived stem cells

Abstract

Background: Fetal bovine serum (FBS) is the most essential supplement in culture media for cellular proliferation, metabolism, and differentiation. However, due to a limited supply and subsequently rising prices, a series of studies have investigated a biological feasibility of replaceable serums to substitute FBS. Along with the increasing interests to manufacture stem cell-based cellular products, optimizing the composition of culture media including serums and exogenous growth factors (GFs) is of importance. In this experiment, the effect of bovine serum (BS) and newborn calf serum (NCS) on proliferation and chondrogenic differentiation capacity of human adipose derived stem cells (ADSCs) was evaluated, especially in the chondrogenically supplemented culture condition.

Methods: ADSCs were chondrogenically cultured with FBS, BS, and NCS for 14 days. For the acceleration of in vitro chondrogenesis, exogenous insulin-like growth factor and transforming growth factor-β3 were added. Viability and proliferation of ADSCs were evaluated using Live/Dead fluorescence staining and DNA amount, respectively. To investigate a chondrogenic differentiation, a series of assays were performed including a quantification of glycosaminoglycan deposition, alcian blue staining, and RT-PCR analysis for type II collagen, aggrecan and Sox-9 genes.

Results: The results demonstrated that proliferation of ADSCs was facilitated in FBS condition as compared with other serum types. For chondrogenic marker gene expression, serum substitutes enhanced Sox-9 expression level on day 14. The deposition of glycosaminoglycan was more facilitated in BS condition regardless of additional chondrogenic GFs.

Conclusion: It could be presumably speculated that serum types and exogenous supplements of GFs could also be important parameters to optimize culture media composition, especially in order to maintain the enhanced levels of both proliferation and chondrogenic differentiation of ADSCs during expansion.

Keywords: Adipose derived stem cell; Chondrogenic differentiation; Fetal bovine serum; Serum substitutes.

Scheme 1

Schematic procedures for serum adaptation and expansion of ADSCs in a chondrogenic culture condition

Fig. 1

Viability and distribution of ADSCs stained using Live/Dead assay on day 7 (a) and day 14 (b) in different serum and growth factor conditions. Scale bar = 1000 μm

Fig. 2

Proliferation of ADSCs, determined using dsDNA amounts, on day 7 and 14 (n = 4). # indicates a significant difference (p < 0.05) as compared with FBS group within the same growth factor condition on day 7 while * indicates a significant difference (p < 0.05) as compared with FBS group within the same growth factor condition on day 14

Fig. 3

Chondrogenic marker gene expression using RT-PCR on day 7 (a) and 14 (b) (n = 3). The relative expression ratio of collagen type II to collagen type I was presented in (c). * indicates a significant difference (p < 0.05) as compared with FBS group within the same growth factor condition

Fig. 4

GAG deposition (a) and histological observation of alcian blue staining (b) on day 14. $ indicates a significant difference as compared with GM group. * indicates a significant difference as compared with FBS group within the same growth factor. # indicates a significant difference as compared with FBS group with growth factors. (*^$#p < 0.05)