Stepwise construction of dynamic microscale concentration gradients around hydrogel-encapsulated cells in a microfluidic perfusion culture device.

Shinya Yamahira,Toshiyuki Kanamori,Eri Nakatani,Masato Tamura,Taku Satoh,Shinji Sugiura,Yuta Kusama,Toshiyuki Takagi,Kimio Sumaru,Fumiki Yanagawa

doi:10.1098/rsos.200027

Abstract

Inside living organisms, concentration gradients dynamically change over time as biological processes progress. Therefore, methods to construct dynamic microscale concentration gradients in a spatially controlled manner are needed to provide more realistic research environments. Here, we report a novel method for the construction of dynamic microscale concentration gradients in a stepwise manner around cells in micropatterned hydrogel. In our method, cells are encapsulated in a photodegradable hydrogel formed inside a microfluidic perfusion culture device, and perfusion microchannels are then fabricated in the hydrogel by micropatterned photodegradation. The cells in the micropatterned hydrogel can then be cultured by perfusing culture medium through the fabricated microchannels. By using this method, we demonstrate the simultaneous construction of two dynamic concentration gradients, which allowed us to expose the cells encapsulated in the hydrogel to a dynamic microenvironment.

Highlights

Many biological processes rely on concentration gradients that dynamically change over time as the processes progress for processes including embryo development [1], immune responses [2], wound healing [3] and angiogenesis [4]
To produce the photodegradable hydrogel, we used dibenzocyclooctyl-terminated photocleavable tetra-arm-polyethylene glycol (DBCO-PC-4armPEG), which is a click cross-linkable and photocleavable PEG that we developed in our previous study [29], and azideterminated tetra-arm-PEG
After formation of the hydrogel in the gel chamber of the device, the outlet channel was filled with DMEM (10% FBS), and the inlet channel was filled with 0.5 μM fluorescein isothiocyanate (FITC)– dextran (MW: 40 k; Sigma-Aldrich, MO) in DMEM (10% FBS) to allow us to visualize the microchannels

Summary

Introduction

Many biological processes rely on concentration gradients that dynamically change over time as the processes progress for processes including embryo development [1], immune responses [2], wound healing [3] and angiogenesis [4]. Methods for creating concentration gradients of ions [7], small molecules [8], peptides [9], proteins [10], physiological parameters of the extracellular matrix [11,12] and cells [13] have been developed to construct research environments that mimic those found in vivo To generate these gradients, diffusion [14], heat [15], gravity [16], electric force [17], light [18] and precise fluid control using microfluidic devices [12,19,20,21,22] have been used. A versatile method capable of changing the spatial pattern of concentration gradients potentially creates a new opportunity to recapitulate dynamic biological events

Methods

Results

Discussion

Conclusion