Methods in Molecular Biology (2022) 2436: 241–256

DOI 10.1007/7651_2021_442

© Springer Science+Business Media, LLC 2021

Published online: 02 November 2021

Applying Stirred Perfusion to 3D Tissue Equivalents

to Mimic the Dynamic In Vivo Microenvironment

Henry W. Hoyle, Claire L. Mobbs, and Stefan A. Przyborski

Abstract

Complex three-dimensional (3D) tissue equivalents have been widely developed with applications with a

multitude of organs and tissues. While these systems lead to significant improvements over conventional

two-dimensional culture, the static conditions of the surrounding medium still present a limitation to the

physiological relevance of these models. Medium perfusion and convective mixing can be introduced to

these models through a variety of techniques using equipment such as pumps and rockers. These systems

can easily become very complex or suffer from limited control over the fluid flow properties. We have

developed a bioreactor enabling controlled perfusion of 3D tissue equivalents utilizing a magnetic stirrer–-

based system, allowing for scalability and ease of use. This system has demonstrated potential applications in

a range of tissues such as the liver, intestine, and skin, with many other potential applications yet to be

tested. Our solution provides users with a low cost and easy to use alternative to complex bioreactor systems

while still providing high levels of control over fluid flow and structural properties of the tissue constructs.

Key words Bioreactor, Perfusion, Organotypic culture, Alvetex^®, Tissue engineering, Three-dimen-

sional cell culture

1

Introduction

Cells and tissues in vivo exist in a highly complex and dynamic

microenvironment. This has a wide range of factors influencing

their structure and function. These include but are not limited to:

high levels of cell–cell and cell–matrix contact, facilitating commu-

nication and the response of tissues to stimuli; the presence of an

extracellular matrix (ECM) composed of a variety of different ECM

proteins and supporting three-dimensional (3D) growth of cells;

fluid flow from the bloodstream and other fluids present in tissues,

providing low levels of shear stress and allowing adequate turnover

of nutrients and cellular waste. These factors are all important for

the healthy function of tissues, however only a subset of these are

usually present in tissue models in vitro [1, 2].

A variety of methods have been developed to overcome this

limitation and therefore maximize the physiological relevance of

in vitro tissue equivalents. One of the major areas of study for this

purpose is the use of 3D growth substrates. These can range from

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