改进的符合 GMP 标准的方法来处理脂肪抽吸物，以无动物来源的培养基中冷冻保存基质血管部分，并扩增脂肪干细胞。

BACKGROUND: The stromal vascular fraction (SVF) derived from adipose tissue contains adipose-derived stromal/stem cells (ASC) and can be used for regenerative applications. Thus, a validated protocol for SVF isolation, freezing, and thawing is required to manage product administration. To comply with Good Manufacturing Practice (GMP), fetal bovine serum (FBS), used to expand ASC in vitro, could be replaced by growth factors from platelet concentrates. METHODS: Throughout each protocol, GMP-compliant reagents and devices were used. SVF cells were isolated from lipoaspirates by a standardized enzymatic protocol. Cells were cryopreserved in solutions containing different albumin or serum and dimethylsulfoxide (DMSO) concentrations. Before and after cryopreservation, we analyzed: cell viability (by Trypan blue); immunophenotype (by flow cytometry); colony-forming unit-fibroblast (CFU-F) formation; and differentiation potential. ASC, seeded at different densities, were expanded in presence of 10% FBS or 5% supernatant rich in growth factors (SRGF) from platelets. The differentiation potential and cell transformation grade were tested in expanded ASC. RESULTS: We demonstrated that SVF can be obtained with a consistent yield (about 185 × 10 cells/ml lipoaspirate) and viability (about 82%). Lipoaspirate manipulation after overnight storage at +4 °C reduced cell viability (-11.6%). The relative abundance of ASC (CD34CD45CD31) and endothelial precursors (CD34CD45CD31) in the SVF product was about 59% and 42%, respectively. A period of 2 months cryostorage in autologous serum with added DMSO minimally affected post-thaw SVF cell viability as well as clonogenic and differentiation potentials. Viability was negatively affected when SVF was frozen at a cell concentration below 1.3 × 10 cells/ml. Cell viability was not significantly affected after a freezing period of 1 year. Independent of seeding density, ASC cultured in 5% SRGF exhibited higher growth rates when compared with 10% FBS. ASC expanded in both media showed unaltered identity (by flow cytometry) and were exempt from genetic lesions. Both 5% SRGF- and 10% FBS-expanded ASC efficiently differentiated to adipocytes, osteocytes, and chondrocytes. CONCLUSIONS: This paper reports a GMP-compliant approach for freezing SVF cells isolated from adipose tissue by a standardized protocol. Moreover, an ASC expansion method in controlled culture conditions and without involvement of animal-derived additives was reported.

背景：源自脂肪组织的基质血管部分 (SVF) 含有脂肪来源的基质/干细胞 (ASC)，可用于再生应用。因此，需要经过验证的 SVF 分离、冷冻和解冻方案来管理产品管理。为了符合良好生产规范 (GMP)，可以用血小板浓缩物中的生长因子替代用于体外扩增 ASC 的胎牛血清 (FBS)。

方法：在每个方案中都使用符合 GMP 的试剂和设备。通过标准化的酶促方案从脂肪抽吸物中分离 SVF 细胞。将细胞冷冻保存在含有不同白蛋白或血清和二甲基亚砜 (DMSO) 浓度的溶液中。在冷冻保存前后，我们分析了：细胞活力（通过台盼蓝）；免疫表型（通过流式细胞术）；成纤维细胞集落形成单位 (CFU-F) 形成；和分化潜力。将 ASC 以不同的密度接种，在含有 10% FBS 或富含血小板生长因子 (SRGF) 的 5%上清液中进行扩增。在扩增的 ASC 中测试分化潜力和细胞转化程度。

结果：我们证明 SVF 可以以一致的产量（约 185×10 个细胞/ml 脂肪抽吸物）和活力（约 82%）获得。在 +4°C 下储存过夜后对脂肪抽吸物进行操作会降低细胞活力（-11.6%）。SVF 产物中 ASC（CD34CD45CD31）和内皮前体细胞（CD34CD45CD31）的相对丰度分别约为 59%和 42%。在自体血清中添加 DMSO 并在 2 个月内冷冻储存对解冻后 SVF 细胞活力以及克隆形成和分化潜力的影响最小。当 SVF 以低于 1.3×10 个细胞/ml 的细胞浓度冷冻时，细胞活力会受到负面影响。当冷冻期为 1 年时，细胞活力不会受到显著影响。无论接种密度如何，在 5% SRGF 中培养的 ASC 与在 10% FBS 中培养的 ASC 相比，生长速度更高。在两种培养基中培养的 ASC 均保持不变（通过流式细胞术），且无遗传损伤。5% SRGF 和 10% FBS 扩增的 ASC 均能有效地分化为脂肪细胞、成骨细胞和软骨细胞。

结论：本文报道了一种符合 GMP 的方法，用于通过标准化方案从脂肪组织中冷冻 SVF 细胞。此外，还报道了一种 ASC 扩增方法，该方法在受控培养条件下进行，不涉及动物源性添加剂。