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天然细胞外基质制备的半透膜插入物用于微流控细胞培养

微生理细胞培养模型,即器官芯片,作为模拟活体器官基本单位的一个新平台正迅速崛起。构建这些微生理细胞培养模型通常需要由多层细胞培养腔组成的可灌注微流控系统,有利于不同细胞的共培养从而构建多层组织结构。具有纳米或微米空隙的半透膜是这种微型装置的关键组分,它通常被夹在两相邻细胞培养腔中间用作细胞培养基底,以模拟体内基底膜并创建细胞生长和分化的微环境。然而,现有的薄膜主要是由合成高分子材料组成,并不能很好地模拟细胞与天然细胞外基质间的相互作用。同时,高分子薄膜也不能模拟自然基质的纤维结构和物理性能。

近期,美国宾夕法尼亚大学的Dongeun Huh教授团队通过自然蒸发天然细胞外基质(ECM)制备得到可用于微型装置的细胞培养半透膜。得到的ECM薄膜具有纤维结构、透光性、可渗透性且机械性能稳定,该研究为克服传统半透膜基底局限性提供了新的思路。

该研究中,ECM基薄膜从制备到用于微流控细胞培养的流程如图1所示。首先,ECM水凝胶溶液均匀分散于PDMS平板上并在37℃孵化1小时使其成胶,并在室温下蒸发过夜,之后用去离子水再水化,4小时后吸去水分即得到ECM基薄膜,将薄膜从PDMS上剥下并剪成需要的尺寸用于微流控装置(图1A-E)。为构建多层微流控装置,该团队选择由上中下三层组成的两通道微装置(图1F-H)。将细胞培养于上述的微流控装置内,在对装置进行预处理后,将细胞悬液灌注于上层培养腔,2小时后移除未贴壁细胞,将装置与微型注射相连使细胞在稳定的培养基液体流中培养(图 1I-J)。

ECM基薄膜从制备到用于微流控细胞培养的流程

Fig. 1 Fabrication of ECM-derived membrane inserts for microfluidic cell culture. A. ECM hydrogel cast on a PDMS surface.B. Dehydration of ECM hydrogel to yield a dried ECM film. C. Rehydration of the dried ECM film to remove salts and other impurities followed by transglutaminase cross-linking (for fabrication of COL + MAT). D. Dehydration of the purified and cross-linked ECM film to yield ECM membranes used as microfluidic cell culture inserts. E. Peeling of the ECM membrane from the underlying PDMS surface using forceps, followed by manual trimming with scissors if necessary. F. Microfluidic channel slabs fabricated by soft lithography are stamped with uncured PDMS to facilitate bonding of ECM membrane inserts over microfluidic channels. G. An ECM membrane is placed over the lower channel using forceps. H. The upper channel slab stamped with uncured PDMS is bonded to the lower channel slab to create an enclosed three-layer channel system. The cross-sectional view of the fully assembled device is shown in I and J. I. Cells are seeded on the ECM-derived membrane inserts in microfluidic devices.J. During perfusion culture, the seeded cells proliferate on the membrane surface to form stable, confluent monolayers in microdevices.

在该研究中,研究人员制备了三种不同组分的ECM薄膜,I型胶原(COL)、I型胶原和基质胶(COL-MAT)、I型胶原和海藻酸盐(COL-ALG),其性能也有差异。ECM薄膜的肉眼观察和表面微观结构图片如图2所示。

ECM薄膜的肉眼观察和表面微观结构图片

Fig. 2 Gross appearance, surface ultrastructure, and composition of ECM-derived membranes. A. Digital photo of a COL–MAT membrane held by forceps demonstrating mechanical integrity and transparency. B. Scanning electron microscopy (SEM) visualization of collagen type I (COL) membrane surface ultrastructure, scale bar = 10 μm. Inset: Characteristic banding pattern visible in larger fibrils. C. SEM visualizationof collagen type I and Matrigel composite (COL–MAT) membrane surface ultrastructure, scale bar = 10 μm.

在微流控细胞培养中,半透膜插入物主要作为分隔相邻细胞培养腔的物理屏障,同时膜上的空隙允许液体和可溶因子在培养腔间的运输。这种设计通常应用于两种不同细胞的共培养。作为初步研究,研究人员将人肺上皮细胞培养于装置中,用来重建空气-肺界面(图3A)。基于此,研究人员进一步构建共培养模型并模拟两不同组织结构间的生物界面。首先,研究人员制备了模拟包括人支气管上皮细胞(BESA-2b)和原代人肺成纤维细胞(NHLFs)的“上皮—基质”界面结构。同样地,研究人员制备了“上皮—内皮”和“血管—基质”界面结构。

究人员将人肺上皮细胞培养于装置中,用来重建空气-肺界面

Fig. 3 Tunable biophysical properties of engineered ECM-derived membranes. A. Plot of membrane absorbance from 350–700 nm. The ECM derived membranes exhibit superior optical transparency compared to traditional transparent cell culture inserts such as Transwell polyester membranes. B. Digital photograph of COL–MAT membrane demonstrating its optical clarity. This membrane was trimmed to the approximate size used for device bonding and held over printed text using forceps. C. Plot of relative membrane permeability representing measurements of 20 kDa FITC-dextran transport across COL, COL–MAT, COL–ALG, and PE membrane inserts over a period of 6 hours under continuous parallel flow perfusion at a flow rate of 100 μl h-1. ** and ns represent P < 0.01 and not significant, respectively. D. SEM visualization of collagen type I–alginate (COL–ALG) membrane surface ultrastructure demonstrating larger pores and fenestrations (arrows) created by using alginate as a water-soluble sacrificial material, scale bar = 2 μm. E. Atomic force microscopy (AFM) nanoindentation measurement of the elastic modulus for hydrated COL, 80 : 20 COL–MAT, 50 : 50 COL–MAT, 20 : 80 COL–MAT, and Transwell PE membranes. * represents P < 0.05.

本研究由美国宾夕法尼亚大学的Dongeun Huh教授团队完成,于20178月发表于Lab on A Chip

 

论文信息:Mark J. Mondrinos, Yoon-Suk Yi, Nan-Kun Wu, Xueting Ding and Dongeun Huh*. Native extracellular matrix-derived semipermeable, optically transparent, and inexpensive membrane inserts for microfluidic cell culture. Lab on A Chip 2017,17:3146-3158.

 

论文链接:

http://pubs.rsc.org/-/content/articlelanding/2017/lc/c7lc00317j#!divAbstract

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