Serum-free medium supplemented with high-concentration FGF2 for cell expansion culture of human ear chondrocytes promotes redifferentiation capacity
Serum-free medium supplemented with high-concentration FGF2 for cell expansion culture of human ear chondrocytes promotes redifferentiation capacity
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DOI：
10.1089/107632702760240490
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发表时间：
2002-08-01
影响因子：
--
通讯作者：
van Osch, GJVM
中科院分区：
文献类型：
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作者：
Mandl, EW;van der Veen, SW;van Osch, GJVM
对于自体软骨的组织工程，需要进行细胞扩增以获得所需的细胞数量。标准扩增培养基含有牛血清。这有几个缺点，即传播疾病的风险和血清批次差异。本研究的目的是使无血清培养基至少具有与含血清培养基相同的细胞数量扩增潜力。三个幼儿的耳软骨细胞在含血清培养基（SCM；含 10% 胎牛血清的 DMEM）或补充有 5 或 100 ng/mL 成纤维细胞生长因子-2 (FGF2) 的无血清培养基（SFM；含 ITS+ 的 DMEM）中扩增。为了促进细胞粘附到培养瓶上，每次胰蛋白酶消化后在无血清条件下用10％血清培养1天。第四次传代后，软骨细胞被包裹在藻酸盐珠中，并在补充有 10 ng/mL IGF-I 和 10 ng/mL TGFbeta-2 的 SFM（含有 ITS+、氢化可的松和 L-抗坏血酸的 DMEM）中重新分化。结果显示，使用 100 ng/mL FGF2 进行的 SFM 扩增与 SCM 中的扩增相当。无论使用哪种扩增培养基，使用含有 IGF-I 和 TGFbeta-2 的 SFM 再分化均显示出高 II 型胶原表达和高 GAG/DNA 产量。然而，软骨细胞在含有 100 ng/mL FGF2 的 SFM 中扩增导致 I 型胶原蛋白和 II-fibrau（成纤维细胞膜标记物）阳性细胞较少。本研究表明，使用无血清培养基进行软骨组织工程是可能的。在补充有高浓度 FGF2 的 SFM 中扩增未成熟耳软骨细胞可产生高细胞数量，此外，与在含 10% 血清的培养基中扩增的细胞相比，其具有更好的再分化能力。
For tissue engineering of autologous cartilage, cell expansion is needed to obtain the cell numbers required. Standard expansion media contain bovine serum. This has several disadvantages, that is, the risk of transmitting diseases and serum-batch variations. The aim of this study was to rind a serum-free medium with at least the same potential to expand cell numbers as serum-containing media. Ear chondrocytes of three young children were expanded in either serum-containing medium (SCM; DMEM with 10% fetal calf serum) or serum-free medium (SFM; DMEM with ITS+) supplemented with 5 or 100 ng/mL fibroblast growth factor-2 (FGF2). To promote cell adherence onto the culture flask, the serum-free conditions were cultured with 10% serum for 1 day after each trypsinization. After the fourth passage, the chondrocytes were encapsuled in alginate beads and redifferentiated in a SFM (DMEM with ITS+, hydrocortisone, and L-ascorbic acid) supplemented with 10 ng/mL IGF-I and 10 ng/mL TGFbeta-2. Results showed that expansion in SFM with 100 ng/mL FGF2 was comparable to expansion in SCM. Redifferentiation with SFM with IGF-I and TGFbeta-2 showed high collagen type II expression and high GAG/DNA production regardless of which expansion medium had been used. However, chondrocytes expanded in SFM with 100 ng/mL FGF2 resulted in less positive cells for collagen type I and II-fibrau (a fibroblast membrane marker). The present study shows that it is possible to use serum-free medium for tissue engineering of cartilage. Expansion of immature ear chondrocytes in SFM supplemented with high-concentration FGF2 resulted in high cell numbers, which in addition had better redifferentiation capacity than cells expanded in medium with 10% serum.