Stempeutics Research Pvt Ltd, 3rd Floor, Manipal Hospitals Whitefield Pvt. Ltd., #143, EPIP Industrial Area, ITPL Main Road, Bangalore, Karnataka, 560 048, India.
Sci Rep. 2024 Jun 2;14(1):12654. doi: 10.1038/s41598-024-62544-8.
Mesenchymal stromal cells (MSC) from adult bone marrow are the most commonly used cells in clinical trials. MSCs from single donors are the preferred starting material but suffer from a major setback of being heterogeneous that results in unpredictable and inconsistent clinical outcomes. To overcome this, we developed a method of pooling MSCs from different donors and created cell banks to cater clinical needs. Initially, the master cell banks (MCBs) were created at passage 1 (P1) from the bone marrow MSCs isolated from of nine different donors. At this stage, MCBs from three different donors were mixed in equal proportion and expanded till P3 to create working cell banks. Further, the pooled cells and individual donor MSCs were expanded till P5 and cryopreserved and extensively characterised. There was a large heterogeneity among the individual donor MSCs in terms of growth kinetics (90% Coefficient of variation (CV) for cell yield and 44% CV for population doubling time at P5), immunosuppressive ability (30% CV at 1:1 and 300% CV at 1:10 ratio), and the angiogenic factor secretion potential (20% CV for VEGF and71% CV for SDF-1). Comparatively, the pooled cells have more stable profiles (60% CV for cell yield and 7% CV for population doubling time at P5) and exhibit better immunosuppressive ability (15% CV at 1:1 and 32% CV at 1:10 ratio ) and consistent secretion of angiogenic factors (16% CV for VEGF and 51% CV for SDF-1). Further pooling does not compromise the trilineage differentiation capacity or phenotypic marker expression of the MSCs. The senescence and in vitro tumourigenicity characteristics of the pooled cells are also similar to those of individual donor MSCs. We conclude that pooling of MSCs from three different donors reduces heterogeneity among individual donors and produces MSCs with a consistent secretion and higher immunosuppressive profile.
间充质基质细胞（MSC）来源于成人骨髓，是临床试验中最常用的细胞。来自单一供体的 MSCs 是首选的起始材料，但存在严重的异质性问题，导致临床结果不可预测且不一致。为了克服这一问题，我们开发了一种从不同供体中汇集 MSC 的方法，并创建了细胞库以满足临床需求。最初，我们从 9 位不同供体的骨髓 MSC 中分离出 MSC，在第 1 代（P1）建立主细胞库（MCB）。在这个阶段，将来自 3 个不同供体的 MCB 以相等的比例混合并扩增至第 3 代（P3），以创建工作细胞库。此外，还将汇集的细胞和单个供体 MSC 扩增至第 5 代（P5）并进行冷冻保存和广泛的特征鉴定。单个供体 MSC 之间存在较大的异质性，表现在细胞产量的生长动力学方面（90%的细胞产量变异系数（CV）和 44%的 P5 倍增时间 CV）、免疫抑制能力（1:1 时为 30%CV，1:10 时为 300%CV）和血管生成因子分泌潜力（VEGF 为 20%CV，SDF-1 为 71%CV）。相比之下，汇集的细胞具有更稳定的特征（P5 时的细胞产量 CV 为 60%，倍增时间 CV 为 7%），并且表现出更好的免疫抑制能力（1:1 时为 15%CV，1:10 时为 32%CV）和稳定的血管生成因子分泌（VEGF 为 16%CV，SDF-1 为 51%CV）。进一步的汇集不会影响 MSC 的三系分化能力或表型标志物表达。汇集细胞的衰老和体外致瘤性特征也与单个供体 MSC 相似。我们得出结论，从 3 个不同供体中汇集 MSC 可以减少单个供体之间的异质性，并产生具有一致分泌和更高免疫抑制特性的 MSC。
