Wawrzyk Dawid, Goncerz Paweł, Rajfur Zenon
Institute of Physics, Faculty of Physics, Astronomy and Applied Computer Science, Jagiellonian University, 30-348 Kraków, Poland.
Undergraduate Program in Biophysics, Faculty of Physics, Astronomy and Applied Computer Science, Jagiellonian University, 30-348 Kraków, Poland.
bioRxiv. 2025 Jun 17:2025.06.12.659070. doi: 10.1101/2025.06.12.659070.
Recently, cellular heterogeneity has gained attention in several scientific studies. It's a phenomenon of the occurrence of differences in cellular parameters, for example, morphology or migration parameters of cells from a single cell line. It has been studied primarily in cancer cells; however, normal cells exhibit analogous behavior. This study investigated morphological and migratory parameters heterogeneity in mouse embryonic fibroblast (MEF 3T3) cells cultured on polyacrylamide (PA) substrates with elasticity of 20 and 40 kPa, which were functionalized with fibronectin solutions in 1, 5, 10 and 20 μg/ml concentrations. Two-hour time lapse microscopy made it possible to quantify the cells spread area and velocity. Based on these parameters and observations of cellular migration strategies, it was possible to classify normal cells into four subpopulations - mesenchymal, amoeboid, slow amoeboid and polygonal/bigonal. Cells grown on softer substrates (20 kPa) showed a greater sensitivity to fibronectin than those grown on stiffer substrates. Cells area was maximal, with the lowest velocity for cells grown on PA substrates functionalized with a 10 μg/ml solution, making their parameters closest to those grown on glass. Cells underwent spontaneous transitions between subpopulations, but no direct transition between mesenchymal and amoeboid or slow amoeboid has been observed. Overall, substrate elasticity and concentration of fibronectin in the functionalizing solution impact the morphology and migration parameters of cells. These findings demonstrate the occurrence of heterogeneity phenomenon in normal mouse fibroblasts. This study also shows the impact of the concentration of fibronectin in the functionalization solution on cells' behavior.
最近，细胞异质性在多项科学研究中受到关注。它是一种细胞参数出现差异的现象，例如，来自单一细胞系的细胞的形态或迁移参数。它主要在癌细胞中得到研究；然而，正常细胞也表现出类似行为。本研究调查了在弹性分别为20 kPa和40 kPa的聚丙烯酰胺（PA）基质上培养的小鼠胚胎成纤维细胞（MEF 3T3）的形态和迁移参数异质性，这些基质用浓度为1、5、10和20 μg/ml的纤连蛋白溶液进行了功能化处理。两小时的延时显微镜观察使得量化细胞铺展面积和速度成为可能。基于这些参数以及对细胞迁移策略的观察，有可能将正常细胞分为四个亚群——间充质样、阿米巴样、慢阿米巴样和多边形/双边形。在较软基质（20 kPa）上生长的细胞比在较硬基质上生长的细胞对纤连蛋白更敏感。对于用10 μg/ml溶液功能化处理的PA基质上生长的细胞，其面积最大，速度最低，使其参数最接近在玻璃上生长的细胞。细胞在亚群之间发生自发转变，但未观察到间充质样和阿米巴样或慢阿米巴样之间的直接转变。总体而言，基质弹性和功能化溶液中纤连蛋白的浓度会影响细胞的形态和迁移参数。这些发现证明了正常小鼠成纤维细胞中存在异质性现象。本研究还显示了功能化溶液中纤连蛋白浓度对细胞行为的影响。
