Pezzanite Lynn, Chow Lyndah, Griffenhagen Gregg, Dow Steven, Goodrich Laurie
Department of Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO, United States.
Department of Microbiology, Immunology and Pathology, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO, United States.
Front Vet Sci. 2021 Apr 30;8:634064. doi: 10.3389/fvets.2021.634064. eCollection 2021.
Culture and expansion of equine mesenchymal stromal cells (MSCs) are routinely performed using fetal bovine serum (FBS) as a source of growth factors, nutrients, and extracellular matrix proteins. However, the desire to minimize introduction of xenogeneic bovine proteins or pathogens and to standardize cellular products intended for clinical application has driven evaluation of alternatives to FBS. Replacement of FBS in culture for several days before administration has been proposed to reduce antigenicity and potentially prolong survival after injection. However, the functional consequences of MSC culture in different serum types have not been fully evaluated. The objective of this study was to compare the immunomodulatory and antibacterial properties of MSCs cultured in three serum sources: FBS or autologous or allogeneic equine serum. We hypothesized that continuous culture in FBS would generate MSCs with improved functionality compared to equine serum and that there would not be important differences between MSCs cultured in autologous vs. allogeneic equine serum. To address these questions, MSCs from three healthy donor horses were expanded in medium with FBS and then switched to culture in FBS or autologous or allogeneic equine serum for 72 h. The impact of this 72-h culture period in different sera on cell viability, cell doubling time, cell morphology, bactericidal capability, chondrogenic differentiation, and production of cytokines and antimicrobial peptides was assessed. Altering serum source did not affect cell viability or morphology. However, cells cultured in FBS had shorter cell doubling times and secreted more interleukin 4 (IL-4), IL-5, IL-17, RANTES, granulocyte-macrophage colony-stimulating factor, fibroblast growth factor 2, eotaxin, and antimicrobial peptide cathelicidin/LL-37 than cells cultured in either source of equine serum. Cells cultured in FBS also exhibited greater spontaneous bactericidal activity. Notably, significant differences in any of these parameters were not observed when autologous vs. allogeneic equine serum was used for cell culture. Chondrogenic differentiation was not different between different serum sources. These results indicate that MSC culture in FBS will generate more functional cells based on a number of parameters and that the theoretical risks of FBS use in MSC culture should be weighed against the loss of MSC function likely to be incurred from culture in equine serum.
马间充质基质细胞（MSCs）的培养和扩增通常使用胎牛血清（FBS）作为生长因子、营养物质和细胞外基质蛋白的来源。然而，尽量减少异种牛蛋白或病原体的引入以及规范用于临床应用的细胞产品的需求促使人们对FBS的替代物进行评估。有人提议在给药前几天在培养中替换FBS，以降低抗原性并可能延长注射后的存活时间。然而，不同血清类型中MSCs培养的功能后果尚未得到充分评估。本研究的目的是比较在三种血清来源中培养的MSCs的免疫调节和抗菌特性：FBS或自体或同种异体马血清。我们假设，与马血清相比，在FBS中连续培养会产生功能更好的MSCs，并且在自体与同种异体马血清中培养的MSCs之间不会有重要差异。为了解决这些问题，从三匹健康供体马中获取的MSCs在含有FBS的培养基中扩增，然后切换到在FBS或自体或同种异体马血清中培养72小时。评估了这72小时在不同血清中培养对细胞活力、细胞倍增时间、细胞形态、杀菌能力、软骨生成分化以及细胞因子和抗菌肽产生的影响。改变血清来源不影响细胞活力或形态。然而，与在任何一种马血清来源中培养的细胞相比，在FBS中培养的细胞具有更短的细胞倍增时间，并且分泌更多的白细胞介素4（IL-4）、IL-5、IL-17、调节激活正常T细胞表达和分泌因子（RANTES）、粒细胞-巨噬细胞集落刺激因子、成纤维细胞生长因子2、嗜酸性粒细胞趋化因子和抗菌肽cathelicidin/LL-37。在FBS中培养的细胞也表现出更强的自发杀菌活性。值得注意的是，当使用自体与同种异体马血清进行细胞培养时，在这些参数中的任何一个方面都未观察到显著差异。不同血清来源之间的软骨生成分化没有差异。这些结果表明，基于多个参数，在FBS中培养MSCs会产生功能更强的细胞，并且在MSCs培养中使用FBS的理论风险应与在马血清中培养可能导致的MSCs功能丧失相权衡。
