Fabrication of size-controllable human mesenchymal stromal cell spheroids from micro-scaled cell sheets
Fabrication of size-controllable human mesenchymal stromal cell spheroids from micro-scaled cell sheets
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DOI：
10.1088/1758-5090/ab21f6
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发表时间：
2019-07-01
期刊：
影响因子：
9
通讯作者：
Shin, Heungsoo
中科院分区：
文献类型：
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作者：
Byun, Hayeon;Lee, Yu Bin;Shin, Heungsoo
近年来，基质细胞球体在组织再生中的应用研究十分活跃。在这项研究中，我们报道了一种利用温敏水凝胶从微尺度细胞片(MU CS)中制备尺寸可控的不同大小的基质细胞球体的方法，并研究了它们对基质细胞功能的影响。从人鼻甲组织、骨髓和脂肪组织中分离出的间充质基质细胞选择性地附着在水凝胶表面的每个微图案(宽度分别为100和400微米的正方形)上，形成MU CS。球体的直径由图案的大小(对于100和400亩m微图案，直径分别为45+/-5和129+/-4微米)和播种密度(对于5.0，10.0和15.0×10(4)细胞厘米(-2)，分别为129+/-4,149±/-6和163+/-6微米)调节。此外，无论基质细胞来源如何，均能成功制备出球体，球体的直径也受细胞培养皿上细胞铺展面积的影响。茎标记在球体中高度表达，与球体的大小无关。E-钙粘蛋白表达的增加和N-钙粘蛋白基因表达的降低表明有不同大小的球体稳定形成。缺氧诱导因子的基因表达水平和血管内皮生长因子的分泌水平与球体的直径成正比(单层分别为13.2+/-1.4、32 5+/-83.4和534.3+/-121.5 pg ng(-1)DNA，以及10 0和40 0µm微纹球体)。即使在注射、冷冻和悬浮培养7d后，球体的大小仍保持不变，存活率接近90%。总之，这种制造尺寸可控的球体的新技术可以广泛应用于再生医学中需要尺寸可控的各种应用。
Recently, stromal cell spheroids have been actively studied for use in tissue regeneration. In this study, we report a method for the fabrication of size-controllable stromal cell spheroids in different sizes from micro-scaled cell sheets (mu CS) using thermosensitive hydrogels and investigated their effects on stromal cell function. Mesenchymal stromal cells isolated from different tissues such as human turbinate tissue, bone marrow, and adipose tissue were adhered selectively to each micro-pattern (squares with widths of 100 and 400 mu m) on the surface of the hydrogel and formed mu CS. The diameters of the spheroids were modulated by the size of the patterns (45 +/- 5 and 129 +/- 4 mu m in diameter for the 100 and 400 mu m micro-patterns, respectively) and the seeding density (129 +/- 4, 149 +/- 6, and 163 +/- 6 mu m for 5.0, 10.0, and 15.0 x 10(4) cells cm(-2), respectively, on 400 mu m micropattern). In addition, the spheroids were successfully fabricated regardless of stromal cell origin, and the diameter of the spheroids was also affected by cell spreading area on a cell culture dish. Stemness markers were highly expressed in the spheroids regardless of the spheroid size. Furthermore, an increase in E-cadherin and decrease in N-cadherin gene expression showed the stable formation of spheroids of different sizes. Gene expression levels of hypoxia inducible factors and secretion of vascular endothelial growth factor were increased (13.2 +/- 1.4, 325 +/- 83.4 and 534.3 +/- 121.5 pg ng(-1) DNA in a monolayer, and 100 and 400 mu m micro-patterned spheroids, respectively) proportional to the diameters of the spheroids. The size of spheroids were maintained even after injection, cryopreservation and 7 d of suspension culture with high viability (similar to 90%). In conclusion, this novel technique to fabricate spheroids with controlled size could be widely applied in various applications that require a controlled size in regenerative medicine.