微生理細胞培養模型，即器官芯片，作為模擬活體器官基本單位的一個新平臺正迅速崛起。構建這些微生理細胞培養模型通常需要由多層細胞培養腔組成的可灌注微流控系統，有利于不同細胞的共培養從而構建多層組織結構。具有納米或微米空隙的半透膜是這種微型裝置的關鍵組分，它通常被夾在兩相鄰細胞培養腔中間用作細胞培養基底，以模擬體內基底膜并創建細胞生長和分化的微環境。然而，現有的薄膜主要是由合成高分子材料組成，并不能很好地模擬細胞與天然細胞外基質間的相互作用。同時，高分子薄膜也不能模擬自然基質的纖維結構和物理性能。

近期，美國賓夕法尼亞大學的Dongeun Huh教授團隊通過自然蒸發天然細胞外基質（ECM）制備得到可用于微型裝置的細胞培養半透膜。得到的ECM薄膜具有纖維結構、透光性、可滲透性且機械性能穩定，該研究為克服傳統半透膜基底局限性提供了新的思路。

該研究中，ECM基薄膜從制備到用于微流控細胞培養的流程如圖1所示。首先，ECM水凝膠溶液均勻分散于PDMS平板上并在37℃孵化1小時使其成膠，并在室溫下蒸發過夜，之后用去離子水再水化，4小時后吸去水分即得到ECM基薄膜，將薄膜從PDMS上剝下并剪成需要的尺寸用于微流控裝置（圖1A-E）。為構建多層微流控裝置，該團隊選擇由上中下三層組成的兩通道微裝置（圖1F-H）。將細胞培養于上述的微流控裝置內，在對裝置進行預處理后，將細胞懸液灌注于上層培養腔，2小時后移除未貼壁細胞，將裝置與微型注射泵相連使細胞在穩定的培養基液體流中培養（圖 1I-J）。

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.

在該研究中，研究人員制備了三種不同組分的ECM薄膜，I型膠原（COL）、I型膠原和基質膠（COL-MAT）、I型膠原和海藻酸鹽（COL-ALG），其性能也有差異。ECM薄膜的肉眼觀察和表面微觀結構圖片如圖2所示。

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.

在微流控細胞培養中，半透膜插入物主要作為分隔相鄰細胞培養腔的物理屏障，同時膜上的空隙允許液體和可溶因子在培養腔間的運輸。這種設計通常應用于兩種不同細胞的共培養。作為初步研究，研究人員將人肺上皮細胞培養于裝置中，用來重建空氣-肺界面（圖3A）。基于此，研究人員進一步構建共培養模型并模擬兩不同組織結構間的生物界面。首先，研究人員制備了模擬包括人支氣管上皮細胞（BESA-2b）和原代人肺成纖維細胞（NHLFs）的“上皮—基質”界面結構。同樣地，研究人員制備了“上皮—內皮”和“血管—基質”界面結構。

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.

本研究由美國賓夕法尼亞大學的Dongeun Huh教授團隊完成，于2017年8月發表于Lab on A Chip。

論文信息：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.

論文鏈接：

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

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