Saini Amarjit, Rullman Eric, Lilja Mats, Mandić Mirko, Melin Michael, Olsson Karl, Gustafsson Thomas
Division of Clinical Physiology, Department of Laboratory Medicine, Karolinska Institutet, Karolinska University Hospital Huddinge, Stockholm, Sweden.
Cardiovascular Theme, Karolinska Institutet, Karolinska University Hospital Huddinge, Stockholm, Sweden.
PLoS One. 2018 Feb 5;13(2):e0192384. doi: 10.1371/journal.pone.0192384. eCollection 2018.
For successful growth and maintenance of primary myogenic cells in vitro, culture medium and addition of sera are the most important factors. At present it is not established as to what extent sera of different origin and composition, supplemented in media or serum-free media conditions influence myoblast function and responses to different stimuli. By assessing markers of proliferation, differentiation/fusion, quiescence, apoptosis and protein synthesis the aim of the current study was to elucidate how primary human myoblasts and myotubes are modulated by different commonly used serum using FCS (foetal calf serum), (CS-FCS charcoal-stripped FCS, a manufacturing process to remove hormones and growth factors from sera), HS (horse serum) as well as in serum free conditions (DMEM). To characterise the biological impact of the different serum, myoblasts were stimulated with Insulin (100 nM) and Vitamin D (100 nM; 1α,25(OH)2D3, 1α,25-Dihydroxycholecalciferol, Calcitriol), two factors with characterised effects on promoting fusion and protein synthesis or quiescence, respectively in human myoblasts/myotubes. We demonstrate that sera of different origin/formulation differentially affect myoblast proliferation and myotube protein synthesis. Importantly, we showed that quantifying the extent to which Insulin effects myoblasts in vitro is highly dependent upon serum addition and which type is present in the media. Upregulation of mRNA markers for myogenic fusion, Myogenin, with Insulin stimulation, relative to DMEM, appeared dampened at varying degrees with serum addition and effects on p70S6K phosphorylation as a marker of protein synthesis could not be identified unless serum was removed from media. We propose that these asymmetric molecular and biochemical responses in human myoblasts reflect the variable composition of mitogenic and anabolic factors in each of the sera. The results have implications for both the reproducibility and interpretation of results from experimental models in myoblast cells/myotubes.
对于原代成肌细胞在体外的成功生长和维持，培养基以及血清的添加是最重要的因素。目前，尚不清楚在培养基或无血清培养基条件下添加的不同来源和成分的血清在多大程度上会影响成肌细胞的功能以及对不同刺激的反应。通过评估增殖、分化/融合、静止、凋亡和蛋白质合成的标志物，本研究的目的是阐明使用胎牛血清（FCS）、活性炭处理的胎牛血清（CS-FCS，一种从血清中去除激素和生长因子的制造工艺）、马血清（HS）以及在无血清条件下（DMEM），不同常用血清如何调节原代人成肌细胞和肌管。为了表征不同血清的生物学影响，用胰岛素（100 nM）和维生素D（100 nM；1α,25(OH)2D3，1α,25-二羟基胆钙化醇，骨化三醇）刺激成肌细胞，这两种因子分别对促进人成肌细胞/肌管中的融合和蛋白质合成或静止具有特定作用。我们证明，不同来源/配方的血清对成肌细胞增殖和肌管蛋白质合成有不同影响。重要的是，我们表明，量化胰岛素在体外对成肌细胞的作用程度高度依赖于血清的添加以及培养基中存在的血清类型。相对于DMEM，胰岛素刺激下成肌融合的mRNA标志物肌细胞生成素的上调，在添加血清时会不同程度地受到抑制，并且除非从培养基中去除血清，否则无法确定作为蛋白质合成标志物的p70S6K磷酸化的影响。我们认为，人成肌细胞中的这些不对称分子和生化反应反映了每种血清中有丝分裂和合成代谢因子的可变组成。这些结果对成肌细胞/肌管实验模型结果的可重复性和解释都有影响。
