| Biological sciences & biotechnology |
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| Construction of Caco-2 cell hollow fiber bioreactor for the study on functional substances |
| XU Dongdong1, CHU Qiang1, YANG Yunyun1, LÜ Xiangmin1, ZHANG Yanzhen2, ZHENG Xiaodong1* |
| (1. College of Biosystems Engineering and Food Science, Zhejiang University, Hangzhou 310058, China; 2. The Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310009, China) |
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Abstract Human colon carcinoma cell line Caco-2 has been extensively used over the last thirty years as a model of the intestinal barrier. The parental cell line undergoes a process of spontaneous differentiation that leads to the formation of a monolayer of cells, expressing several morphological and functional characteristics of the mature enterocyte. Hollow fiber membrane bioreactors can offer an in vivo like microenvironment for adherent cell types, such as Caco-2 cells, and enable a high quantity of the desired cellular product with less population variation and favorable operation costs.
The objective of this study is to construct a novel Caco-2 cell hollow fiber membrane bioreactor, to explore the feasibility for further application by morphological observation and functional test of Caco-2 cells.
In this experiment, the Caco-2 cells were cultured in polyethersulfone (PES) and polyvinylidenefluoride (PVDF) hollow fiber membranes as well as the classic Transwell culture plate. The morphological features of the cells were observed by optical microscope and scanning electron microscope (SEM), and the cell growth characters were examined by 3-（4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT). Meanwhile, the integrity of the cell monolayer was verified by fluorescence microscopy. The polarization and compactification of the monolayer was determined by brush border enzymatic activity, including the activities of alkaline phosphatase and γ glutamyltransferase. In this way, the proliferation and differentiation of Caco-2 cells were compared and evaluated in threedimensional hollow fiber bioreactor and two-dimensional models.
Subsequently, time-dependent morphological observation of cell layers indicated that the Caco-2 cells cultured on hollow fiber, spread well and showed clear outline, and gradually formed confluent monolayer within 10 days. The brush border enzymes were well differentiated with higher expressed function within 8-14 days compared to the traditional model, and all of them tended to be consistent in the late cultivation.
〖JP2〗In conclusion, both the PES and PVDF hollow fiber membranes can effectively accelerate the proliferation and differentiation of Caco-2 cells during the 21 day culture period, resulting in the construction of a highly stable three-dimensional cell model. Eventually, the threedimensional Caco-2 cell hollow fiber membrane bioreactors of PES and PVDF are successfully constructed, and the model has advantages over the traditional Transwell monolayer model.
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Published: 20 July 2016
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可用于功能性成分研究的Caco-2细胞中空纤维反应器的构建
为了构建一种新型的Caco-2细胞中空纤维膜反应器，探究该模型应用于研究功能性成分吸收转运的可行性，利用体外细胞培养技术，将Caco-2细胞接种于聚醚砜（polyethersulfone, PES）和聚偏氟乙烯（polyvinylidenefluoride, PVDF）中空纤维膜上，与经典的Transwell单层培养模型进行比较，探讨不同材料对细胞模型的影响；同时，利用光学显微镜和扫描电镜观察细胞形态学特点，通过细胞功能表征方法，包括用3-(4，5-二甲基噻唑-2)-2，5-二苯基四氮唑溴盐比色法绘制细胞生长曲线，荧光免疫染色观察验证细胞单层的完整性和通透性，测定细胞的碱性磷酸酶和谷氨酰转肽酶活性来验证细胞的刷状缘酶系活性，从而对Caco2细胞在3维中空纤维反应器与2维模型中的增殖分化状况进行对比和评价。细胞形态学观察结果表明：Caco-2细胞于接种10 d后铺展良好，轮廓清晰，逐渐融合形成细胞单层和完整的紧密连接；刷状缘酶系活性检测表明：Caco-2细胞于接种后8~14 d内完成功能分化并且活性水平高于在Transwell模型上培养的细胞。因此，聚醚砜和聚偏氟乙烯中空纤维膜能有效促进细胞增殖，缩短细胞分化周期，从而形成高效稳定的3维细胞模型；同时，中空纤维膜Caco-2细胞3维反应器已经具备甚至高于传统的Transwell单层模型所具有的功能。
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