Lauvrud Anne Therese, Giraudo Maria Vittoria, Wiberg Rebecca, Wiberg Mikael, Kingham Paul J, Brohlin Maria
Department of Medical and Translational Biology, Umeå University, SE-901 87 Umeå, Sweden.
Department of Diagnostics and Intervention, Umeå University, SE-901 87 Umeå, Sweden.
Regen Ther. 2024 Oct 10;26:901-910. doi: 10.1016/j.reth.2024.09.013. eCollection 2024 Jun.
Before performing cell therapy clinical trials, it is important to understand how cells are influenced by different growth conditions and to find optimal xeno-free medium formulations. In this study we have investigated the properties of adipose tissue-derived stem cells (ASCs) cultured under xeno-free conditions.
Human lipoaspirate samples were digested to yield the stromal vascular fraction cells which were then seeded in i) Minimum Essential Medium-α (MEM-α) supplemented with 10 % (v/v) fetal bovine serum (FBS), ii) MEM-α supplemented with 2 % (v/v) human platelet lysate (PLT) or iii) PRIME-XV MSC expansion XSFM xeno-free, serum free medium (XV). Flow cytometry for ASCs markers CD73, CD90 and CD105 together with the putative pericyte marker CD146 was performed. Growth rates were monitored over multiple passages and adipogenic differentiation performed at early and expanded passage culture. Growth factor gene expression was analyzed and an angiogenesis assay performed.
Cells in FBS and PLT grew at similar rates whereas the cells cultured in XV medium proliferated significantly faster up to 60 days in culture. All cultures were >98 % positive for CD73, CD90 and CD105, whereas CD146 expression was significantly higher in XV cells. Adipogenic differentiation was most pronounced in cells which had been cultured in XV medium whilst cells grown in PLT were inferior compared with cells from the FBS cultures. gene expression was highest in cells cultured in PLT whilst cells grown in XV medium showed 10-fold lower expression compared with FBS cells. In contrast, gene expression was 90-fold greater in cells cultured in XV medium compared with those cultured in FBS. Conditioned medium from XV cultured cells showed the most angiogenic activity, inducing the greatest endothelial cell network formation and maturation.
Culture under different conditions alters the ASCs characteristics. Since cells cultured in XV medium showed the best adipogenic and angiogenic profile this might be a preferred medium formulation for preparing cells required for reconstructive surgical applications such as cell-assisted fat grafting.
在进行细胞治疗临床试验之前，了解细胞如何受到不同生长条件的影响并找到最佳的无动物源培养基配方非常重要。在本研究中，我们研究了在无动物源条件下培养的脂肪组织来源干细胞（ASC）的特性。
将人抽脂样本消化以获得基质血管成分细胞，然后将其接种于：i）补充有10%（v/v）胎牛血清（FBS）的α-最小必需培养基（MEM-α）；ii）补充有2%（v/v）人血小板裂解物（PLT）的MEM-α；或iii）PRIME-XV MSC无动物源、无血清培养基（XV）。对ASC标志物CD73、CD90和CD105以及假定的周细胞标志物CD146进行流式细胞术检测。在多次传代过程中监测生长速率，并在早期传代培养和扩增传代培养时进行成脂分化。分析生长因子基因表达并进行血管生成测定。
FBS和PLT中的细胞生长速率相似，而在XV培养基中培养的细胞在培养长达60天时增殖明显更快。所有培养物中CD73、CD90和CD105的阳性率均>98%，而CD146在XV细胞中的表达明显更高。在XV培养基中培养的细胞中，成脂分化最为明显，而在PLT中生长的细胞与FBS培养的细胞相比则较差。在PLT中培养的细胞中生长因子基因表达最高，而在XV培养基中生长的细胞与FBS细胞相比表达低10倍。相比之下，在XV培养基中培养的细胞中生长因子基因表达比在FBS中培养的细胞高90倍。来自XV培养细胞的条件培养基显示出最强的血管生成活性，诱导最大程度的内皮细胞网络形成和成熟。
在不同条件下培养会改变ASC的特性。由于在XV培养基中培养的细胞显示出最佳的成脂和血管生成特性，这可能是制备用于重建性手术应用（如细胞辅助脂肪移植）所需细胞的首选培养基配方。
