Oliver-Vila Irene, Sesma-Herrero Eduardo, Belda Francisco, Seriola Anna, Ojosnegros Samuel
Serabiotics, S.L., Barcelona, Spain.
Institute for Bioengineering of Catalonia (IBEC), The Barcelona Institute of Science and Technology (BIST), Barcelona, Spain.
Cytotherapy. 2025 Apr;27(4):552-561. doi: 10.1016/j.jcyt.2025.01.003. Epub 2025 Jan 11.
BACKGROUND/AIMS: Human mesenchymal stromal cells (hMSC) are multipotent adult cells commonly used in regenerative medicine as advanced therapy medicinal products. The expansion of these cells in xeno-free supplements is highly encouraged by regulatory agencies due to safety concerns. However, the number of supplements with robust performance and consistency for hMSC expansion are limited. Here, we evaluate a xeno-free human plasma-derived protein supplement (Plastem, Grifols) for the expansion and functional evaluation of hMSCs.
hMSC from bone marrow, adipose tissue and umbilical cord were obtained from two suppliers and cultured in Dulbecco's modified Eagle's medium (DMEM/F-12) supplemented with fetal bovine serum 10% (FBS), human platelet lysate 5% (hPL) or Plastem 10%+ hPL0.5%. Cell proliferation was evaluated after culturing hMSC for 13 days with trypan blue exclusion. hMSC immunophenotype was assessed by flow cytometry of surface markers expression. Multipotentiality assay determined the ability of hMSC to differentiate into osteogenic, chondrogenic and adipogenic lineages after 21 days, by using specific staining. Immunomodulatory properties of hMSC were analyzed by measuring suppression of human peripheral blood mononuclear cell (PBMC) proliferation in co-culture with hMSC.
Plastem 10% + hPL 0.5% supported robust and sustained hMSC growth with a similar efficiency to the reference supplement FBS 10%. hMSC cultured with the xeno-free supplement presented a similar morphology comparable to FBS-supplemented cells and maintained typical expression of markers: positive (>95%) for CD90, CD73 and CD105; and negative (<5%) for CD45, CD14, CD19, CD34 and HLA-DR. Likewise, hMSC showed potent, in vitro differentiation potential into osteogenic, chondrogenic and adipogenic lineages, outperforming the results obtained with traditional reference supplements in several instances. They retained their immunomodulatory properties, inhibiting the proliferation of phytohemagglutinin (PHA)-stimulated PBMCs with a notable enhancement of the immunomodulatory capacity of hMSCs compared to conventional reference supplements.
Plastem allowed hMSC expansion while preserving phenotype and showed remarkable differentiation and immunomodulatory properties, supporting its use for cell therapy manufacturing processes as a robust, xeno-free alternative to FBS and hPL. Moreover, Plastem can be manufactured at an industrial level, making it a scalable solution for widespread application.
背景/目的：人间充质基质细胞（hMSC）是多能成体细胞，作为先进治疗药品常用于再生医学。出于安全考虑，监管机构大力鼓励在无动物源成分的培养基中扩增这些细胞。然而，性能强劲且能始终如一地用于hMSC扩增的培养基数量有限。在此，我们评估一种无动物源成分的人血浆源性蛋白培养基（Plastem，基立福公司）用于hMSC的扩增及功能评估。
从两家供应商处获取来自骨髓、脂肪组织和脐带的hMSC，并在补充有10%胎牛血清（FBS）、5%人血小板裂解液（hPL）或10%Plastem + 0.5%hPL的杜氏改良 Eagle 培养基（DMEM/F-12）中培养。用台盼蓝拒染法评估hMSC培养13天后的细胞增殖情况。通过表面标志物表达的流式细胞术评估hMSC免疫表型。多能性检测通过特异性染色确定hMSC在21天后分化为成骨、成软骨和成脂谱系的能力。通过测量与hMSC共培养时人外周血单个核细胞（PBMC）增殖的抑制情况来分析hMSC的免疫调节特性。
10%Plastem + 0.5%hPL能支持hMSC强劲且持续的生长，效率与参考培养基10%FBS相似。用无动物源成分培养基培养的hMSC呈现出与补充FBS的细胞相似的形态，并维持标志物的典型表达：CD90、CD73和CD105阳性（>95%）；CD45、CD14、CD19、CD34和HLA-DR阴性（<5%）。同样，hMSC在体外表现出向成骨、成软骨和成脂谱系的强大分化潜能，在多个实例中优于传统参考培养基的结果。它们保留了免疫调节特性，抑制植物血凝素（PHA）刺激的PBMC增殖，与传统参考培养基相比，hMSC的免疫调节能力显著增强。
Plastem能实现hMSC的扩增，同时保留其表型，并展现出卓越的分化和免疫调节特性，支持其作为FBS和hPL的强大无动物源替代物用于细胞治疗生产过程。此外，Plastem可进行工业化生产，使其成为一种可广泛应用的可扩展解决方案。
