微生理細(xì)胞培養(yǎng)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(gòu)建這些微生理細(xì)胞培養(yǎng)模型通常需要由多層細(xì)胞培養(yǎng)腔組成的可灌注微流控系統(tǒng)，有利于不同細(xì)胞的共培養(yǎng)從而構(gòu)建多層組織結(jié)構(gòu)。具有納米或微米空隙的半透膜是這種微型裝置的關(guān)鍵組分，它通常被夾在兩相鄰細(xì)胞培養(yǎng)腔中間用作細(xì)胞培養(yǎng)基底，以模擬體內(nèi)基底膜并創(chuàng)建細(xì)胞生長和分化的微環(huán)境。然而，現(xiàn)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(xì)胞與天然細(xì)胞外基質(zhì)間的相互作用。同時，高分子薄膜也不能模擬自然基質(zhì)的纖維結(jié)構(gòu)和物理性能。

近期，美國賓夕法尼亞大學(xué)的Dongeun Huh教授團(tuán)隊(duì)通過自然蒸發(fā)天然細(xì)胞外基質(zhì)（ECM）制備得到可用于微型裝置的細(xì)胞培養(yǎng)半透膜。得到的ECM薄膜具有纖維結(jié)構(gòu)、透光性、可滲透性且機(jī)械性能穩(wěn)定，該研究為克服傳統(tǒng)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(xì)胞培養(yǎng)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(fā)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(gòu)建多層微流控裝置，該團(tuán)隊(duì)選擇由上中下三層組成的兩通道微裝置（圖1F-H）。將細(xì)胞培養(yǎng)于上述的微流控裝置內(nèi)，在對裝置進(jìn)行預(yù)處理后，將細(xì)胞懸液灌注于上層培養(yǎng)腔，2小時后移除未貼壁細(xì)胞，將裝置與微型注射泵相連使細(xì)胞在穩(wěn)定的培養(yǎng)基液體流中培養(yǎng)（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(zhì)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(jié)構(gòu)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(xì)胞培養(yǎng)中，半透膜插入物主要作為分隔相鄰細(xì)胞培養(yǎng)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(yǎng)腔間的運(yùn)輸。這種設(shè)計通常應(yīng)用于兩種不同細(xì)胞的共培養(yǎng)。作為初步研究，研究人員將人肺上皮細(xì)胞培養(yǎng)于裝置中，用來重建空氣-肺界面（圖3A）。基于此，研究人員進(jìn)一步構(gòu)建共培養(yǎng)模型并模擬兩不同組織結(jié)構(gòu)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(xì)胞（BESA-2b）和原代人肺成纖維細(xì)胞（NHLFs）的“上皮—基質(zhì)”界面結(jié)構(gòu)。同樣地，研究人員制備了“上皮—內(nèi)皮”和“血管—基質(zhì)”界面結(jié)構(gòu)。

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(xué)的Dongeun Huh教授團(tuán)隊(duì)完成，于2017年8月發(fā)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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