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首页> 外文期刊>Biotechnology Progress >Flow Cytometric Cell Cycle Analysis of Muscle Precursor Cells Cultured Within 3D Scaffolds in a Perfusion Bioreactor
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Flow Cytometric Cell Cycle Analysis of Muscle Precursor Cells Cultured Within 3D Scaffolds in a Perfusion Bioreactor

机译：在灌注生物反应器中的3D支架内培养的肌肉前体细胞的流式细胞仪细胞周期分析

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It has been widely demonstrated that perfusion bioreactors improve in vitro three-dimensional(3D)cultures in terms of high cell density and uniformity of cell distribution;however,the studies reported in literature were primarily based on qualitative analysis(histology,immunofluorescent staining)or on quantitative data averaged on the whole population(DNA assay,PCR).Studies on the behavior,in terms of cell cycle,of a cell population growing in 3D scaffolds in static or dynamic conditions are still absent.In this work,a perfusion bioreactor suitable to culture C2C_(12)muscle precursor cells within 3D porous collagen scaffolds was designed and developed and a method based on flow cytometric analyses for analyzing the cell cycle in the cell population was established.Cells were extracted by enzymatic digestion of the collagen scaffolds after 4,7,and 10 days of culture,and flow cytometric live/dead and cell cycle analyses were performed with Propidium Iodide.A live/ dead assay was used for validating the method for cell extraction and staining.Moreover,to investigate spatial heterogeneity of the cell population under perfusion conditions,two stacked scaffolds in the 3D domain,of which only the upstream layer was seeded,were analyzed separately.All results were compared with those obtained from static 3D cultures.The live/dead assay revealed the presence of less than 20% of dead cells,which did not affect the cell cycle analysis.Cell cycle analyses highlighted the increment of cell fractions in proliferating phases(S/G2/M)owing to medium perfusion in long-term cultures.After 7-10 days,the percentage of proliferating cells was 8-12% for dynamic cultures and 3-5% for the static controls.A higher fraction of proliferating cells was detected in the downstream scaffold.From a general perspective,this method provided data with a small standard deviation and detected the differences between static and dynamic cultures and between upper and lower scaffolds.Our methodology can be extended to other cell types to investigate the influence of 3D culture conditions on the expression of other relevant cell markers.
机译：已有广泛研究表明，灌注生物反应器在高细胞密度和细胞分布均匀性方面改善了体外三维（3D）培养;然而，文献报道的研究主要基于定性分析（组织学，免疫荧光染色）或在整个种群中平均获得的定量数据（DNA分析，PCR）。关于在静态或动态条件下在3D支架中生长的细胞群体的行为，在细胞周期方面的研究仍然缺乏。在这项工作中，灌注生物反应器设计并开发了适合在3D多孔胶原支架中培养C2C_（12）肌肉前体细胞的方法，建立了一种基于流式细胞仪分析细胞群细胞周期的方法，酶消化后提取细胞培养4,7，和10天，并使用碘化丙啶进行流式细胞术活/死和细胞周期分析。此外，为了研究细胞在灌注条件下的空间异质性，分别在3D结构域中堆叠了两个堆叠的支架，其中仅接种了上游层，将它们分别进行了分析。活/死分析显示死细胞少于20％，这并不影响细胞周期分析。细胞周期分析突出显示了增殖期（S / G2 / M）中细胞分数的增加由于在长期培养中进行了培养基灌注.7-10天后，动态培养的增殖细胞百分比为8-12％，静态对照的增殖细胞百分比为3-5％。从一般的角度来看，该方法提供的数据具有较小的标准偏差，并且可以检测静态和动态培养之间以及上，下支架之间的差异。扩展到其他细胞类型，以研究3D培养条件对其他相关细胞标志物表达的影响。

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