Microtissue 안정화를위한 미세 유체 장치에서 레이어별로 층 콜라겐 증착

Summary

The creation of functional microtissues within microfluidic devices requires the stabilization of cell phenotypes by adapting traditional cell culture techniques to the limited spatial dimensions in microdevices. Modification of collagen allows the layer-by-layer deposition of ultrathin collagen assemblies that can stabilize primary cells, such as hepatocytes, as microfluidic tissue models.

Abstract

Although microfluidics provides exquisite control of the cellular microenvironment, culturing cells within microfluidic devices can be challenging. 3D culture of cells in collagen type I gels helps to stabilize cell morphology and function, which is necessary for creating microfluidic tissue models in microdevices. Translating traditional 3D culture techniques for tissue culture plates to microfluidic devices is often difficult because of the limited channel dimensions. In this method, we describe a technique for modifying native type I collagen to generate polycationic and polyanionic collagen solutions that can be used with layer-by-layer deposition to create ultrathin collagen assemblies on top of cells cultured in microfluidic devices. These thin collagen layers stabilize cell morphology and function, as shown using primary hepatocytes as an example cell, allowing for the long term culture of microtissues in microfluidic devices.

Introduction

Although microfluidics allows for the exquisite control of the cellular microenvironment, culturing cells, especially primary cells, within microfluidic devices can be challenging. Many traditional cell culture techniques have been developed to sustain and stabilize cell function when cultured in tissue culture plates, but translating those techniques to microfluidic devices is often difficult.

One such technique is the culture of cells on or sandwiched between collagen gels as a model of the physiological 3D cell environment.¹ Type I collagen is one of the most frequently used proteins for biomaterials applications because of its ubiquity in extracellular matrix, natural abundance, robust cell attachment sites, and biocompatibility.² Many cells benefit from 3D culture with collagen, including cancer cells^3,45, microvascular endothelial cells⁶, and hepatocytes⁷, among others. While the use of collagen gels is easy in open formats, such as tissue culture plates, the limited channel dimensions and enclosed nature of microfluidic devices makes the use of liquids that gel impractical without blocking the entire channel.

To overcome this problem, we combined the layer-by-layer deposition technique⁸ with chemical modifications of native collagen solutions to create ultrathin collagen assemblies on top of cells cultured in microfluidic devices. These layers can stabilize cell morphology and function similar to collagen gels and can be deposited on cells in microfluidic devices without blocking the channels with polymerized matrix. The goal of this method is to modify native collagen to create polycationic and polyanionic collagen solutions and to stabilize cells in microfluidic culture by depositing thin collagen matrix assemblies onto the cells. This technique has been used to stabilize the morphology and function of primary hepatocytes in microfluidic devices.⁹

Although layer-by-layer deposition has previously been reported with natural and synthetic polyelectrolytes¹⁰ to cover hepatocytes in plate culture^11,12 and as a seeding layer for hepatocytes in microfluidic devices^13,14, this method describes the deposition of a pure collagen layer on top of hepatocytes, mimicking the 3D collagen culture techniques. In this protocol, we use hepatocytes as example cells that can be maintained using 3D collagen layers. The many other types of cells that benefit from 3D culture in collagen may similarly benefit from culture after layer-by-layer deposition of an ultrathin collagen matrix assembly.

Protocol

원주민 수용성 콜라겐 솔루션 1. 준비 준비 또는 내가 1-3 밀리그램 / 등 Piez 등에 의해보고 된 표준 격리 프로토콜을 사용하여 ML. (15)에 쥐의 꼬리에서 콜라겐 산성화, 가용성, 유형의 200 mg의 구입 변성 콜라겐 용액의 목적하는 최종 부피를 기준으로 출발 물질의 양을 규모. 약 수용성 천연 콜라겐의 200 mg의에서, 3 ㎎ / ㎖에서 25 ~ 30 메틸화 ㎖의 석신 콜라겐 솔루션 25 ~…

Representative Results

네이티브 콜라겐 층별 증착에 사용하기위한 폴리 양이온과 폴리 음이온 콜라겐 용액을 만들 메틸화 및 succinylation를 사용하여 수정 될 수있다. Succinylation가 숙시 닐기로 네이티브 콜라겐의 ε 아미노 그룹을 수정하고 메틸화는 메틸기 (도 1a)와 네이티브 콜라겐 카르복실기를 수정한다. 콜라겐 단백질 아미노산 측쇄에 이러한 변형은 용액의 pH에​​ 대한 적정 곡선을 변경. 메틸화는 ?…

Discussion

얇은 순수 콜라겐 어셈블리 변성 콜라겐의 층별 증착을 이용하여 충전 재료 또는 세포 표면 상에 증착 될 수있다. 이 연구의 결과는 메틸화 네이티브 콜라겐 succinylation 세포 (도 2) 상에 얇은 콜라겐 매트릭스 어셈블리를 증착하기 위해 레이어 – 바이 – 레이어 기술로 사용될 수있다 (도 1) 또는 다른 충전 폴리 양이온과 폴리 음이온 콜라겐 용액을 생성 함을 입증 재료 표면…

Materials

collagen type I, rat tail	Life Technologies	A1048301	option for concentrated rat tail collagen
collagen type I, rat tail	Sigma-Aldrich	C3867-1VL	option for concentrated rat tail collagen
collagen type I, rat tail	EMD Millipore	08-115	option for concentrated rat tail collagen
collagen type I, rat tail	R%D Systems	3440-100-01	option for concentrated rat tail collagen
succinic anhydride	Sigma-Aldrich	239690-50G	succinylation reagent
anhydrous methanol	Sigma-Aldrich	322415-100ML	methylation reagent
sodium hydroxide	Sigma-Aldrich	S5881-500G	pH precipitation reagent
hydrochloric acid	Sigma-Aldrich	320331-500ML	pH precipitation reagent
rat collagen type I ELISA	Chondrex	6013	option for detecting collagen content
hydroxyproline assay kit	Sigma-Aldrich	MAK008-1KT	option for detecting collagen content
hydroxyproline assay kit	Quickzyme Biosciences	QZBtotcol1	option for detecting collagen content