Che Hui, Hart Melanie L, Lauer Jasmin C, Selig Mischa, Voelker Marita, Kurz Bodo, Rolauffs Bernd
Orthopedics and Sports Medicine Center, The Affiliated Suzhou Hospital of Nanjing Medical University, Suzhou Municipal Hospital, Gusu School, Nanjing Medical University, Suzhou, People's Republic of China.
G.E.R.N. Research Center for Tissue Replacement, Regeneration & Neogenesis, Department of Orthopedics and Trauma Surgery, Faculty of Medicine, Medical Center-Albert-Ludwigs-University of Freiburg, 79108 Freiburg im Breisgau, Germany.
Biomed Mater. 2025 Jan 23;20(2). doi: 10.1088/1748-605X/ada335.
Cell micro-patterning controls cell fate and function and has potential for generating therapeutically usable mesenchymal stromal cell (MSC) populations with precise functions. However, to date, the micro-patterning of human cells in a translational context has been impossible because only ruminant media supplements, e.g. fetal bovine serum (FBS), are established for use with micro-patterns (MPs). Thus, there are currently no good manufacturing practice (GMP)-compliant media available for MPs. This study tested a xenogenic-free human plasma and platelet lysate (hP + PL) medium supplement to determine its compatibility with MPs. Unfiltered hP + PL medium resulted in significant protein deposition, creating a 'carpet-like' layer that rendered MPs ineffective. Filtration (3×/5×) eliminated this effect. Importantly, quantitative comparison using droplet digital PCR revealed that human MSCs in all media types exhibited similar profiles with strong myogenic Calponin 1/Transgelin 2 (TAGLN2) and weaker osteogenic alkaline phosphatase/Runt-related transcription factor 2 marker expression, and much weaker adipogenic (lipoprotein lipase/peroxisome proliferator-activated receptor gamma) and chondrogenic (collagen type II/aggrecan) expression, with profiles being dominated by myogenic markers. Within these similar profiles, an even stronger induction of the myogenic marker TAGLN2 by all hP + PL- compared to FBS-containing media. Overall, this suggested that FBS can be replaced with hP + PL without altering differentiation profiles. However, assessing individual MSC responses to various MP types with defined categories revealed that unfiltered hP + PL medium was unusable. Importantly, FBS- and 3× filtered hP + PL media were comparable in each differentiation category. Summarized, this study recommends 3× filtered hP + PL as a xenogenic-free and potentially GMP-compliant alternative to FBS as a culture medium supplement for micro-patterning cell populations in both basic and translational research that will ensure consistent and reliable MSC micro-patterning for therapeutic use.
细胞微图案化可控制细胞命运和功能，并且具有生成具有精确功能的、可用于治疗的间充质基质细胞（MSC）群体的潜力。然而，迄今为止，在转化应用背景下对人类细胞进行微图案化是不可能的，因为仅反刍动物培养基补充剂（例如胎牛血清（FBS））被确定可用于微图案（MP）。因此，目前没有符合药品生产质量管理规范（GMP）的MP可用培养基。本研究测试了一种无动物源的人血浆和血小板裂解物（hP + PL）培养基补充剂，以确定其与MP的兼容性。未过滤的hP + PL培养基导致大量蛋白质沉积，形成一层“地毯状”层，使MP失效。过滤（3次/5次）消除了这种影响。重要的是，使用液滴数字PCR进行的定量比较显示，所有培养基类型中的人MSC均表现出相似的特征，即肌源性钙调蛋白1/转胶蛋白2（TAGLN2）表达强，成骨碱性磷酸酶/ runt相关转录因子2标记物表达弱，而成脂（脂蛋白脂肪酶/过氧化物酶体增殖物激活受体γ）和软骨生成（II型胶原/聚集蛋白聚糖）表达则弱得多，且特征以肌源性标记物为主导。在这些相似的特征中，与含FBS的培养基相比，所有hP + PL培养基对肌源性标记物TAGLN2的诱导作用更强。总体而言，这表明FBS可以被hP + PL替代而不改变分化特征。然而，评估单个MSC对具有明确类别的各种MP类型的反应发现，未过滤的hP + PL培养基无法使用。重要的是，FBS和3次过滤的hP + PL培养基在每个分化类别中具有可比性。总之，本研究推荐3次过滤的hP + PL作为一种无动物源且可能符合GMP的替代品，以替代FBS作为培养基补充剂，用于基础研究和转化研究中的细胞群体微图案化，这将确保用于治疗的MSC微图案化一致且可靠。
