Functionalization, preparation and use of cell-laden gelatin
methacryloyl-based hydrogels as modular tissue culture
platforms
Author
Loessner, Daniela, Meinert, Christoph, Kaemmerer, Elke, Martine, Laure C, Yue, Kan, Levett,
Peter A, Klein, Travis J, Melchels, Ferry PW, Khademhosseini, Ali, Hutmacher, Dietmar W
Published
2016
Journal Title
Nature Protocols
Version
Accepted Manuscript (AM)
DOI
https://doi.org/10.1038/nprot.2016.037
Copyright Statement
© 2016 Nature Publishing Group. This is the author-manuscript version of this paper.
Reproduced in accordance with the copyright policy of the publisher. Please refer to the journal
website for access to the definitive, published version.
Downloaded from
http://hdl.handle.net/10072/343838
Griffith Research Online
https://research-repository.griffith.edu.au
1
DOI: nprot.2016.037
Categories:
Cell culture,
Ontology:
Biological sciences / Biological techniques / Cytological techniques / Cell culture
Physical sciences/Materials science/Biomaterials/Bioinspired materials
Biological sciences / Biotechnology / Biomimetics
Keywords: GelMA, gelatin-methacryloyl hydrogel, 3D cell culture model, tissue culture, cell
culture, GelMA polymer, 3D cell culture, biomaterial, methacryloyl, methacrylamide
Related MS: doi:10.1016/j.actbio.2014.02.035
Tweet #NewNProt: GelMA-based hydrogels for use in 3D cell culture models
Editorial summary: This protocol describes how to make semi-synthetic gelatin-methacryloyl
(GelMA)-based hydrogels for use in 3D cell culture models for cancer and stem cell research, and
tissue engineering
Functionalization, preparation, and use of cell-laden gelatin-methacryloyl-based hydrogels as
modular tissue culture platforms
Daniela Loessner
1#*
, Christoph Meinert
1#
, Elke Kaemmerer
1
, Laure C Martine
1
, Kan Yue
2,3
, Peter A
Levett
1
, Travis J Klein
1
, Ferry PW Melchels
1,4,5
, Ali Khademhosseini
2,3,6,7,8*
, Dietmar W
Hutmacher
1,9,10,11*
1
Queensland University of Technology (QUT), Brisbane 4059, Australia;
2
Biomaterials Innovation
Research Center, Division of Biomedical Engineering, Department of Medicine, Brigham and
Women’s Hospital, Harvard Medical School, Boston 02139, MA, USA;
3
Harvard-Massachusetts
Institute of Technology, Division of Health Sciences and Technology, Massachusetts Institute of
Technology, Cambridge 02139, MA, USA;
4
Department of Orthopaedics, University Medical
Center Utrecht, Utrecht, The Netherlands;
5
Institute of Biological Chemistry, Biophysics and
Bioengineering, School of Engineering and Physical Sciences, Heriot-Watt University, Edinburgh
EH14 4AS, United Kingdom;
6
Wyss Institute for Biologically Inspired Engineering, Harvard
University, Boston 02115, MA, USA;
7
Department of Bioindustrial Technologies, College of
Animal Bioscience and Technology, Konkuk University, Hwayang-dong, Gwangjin-gu, Seoul 143-
701, Republic of Korea;
8
Department of Physics, King Abdulaziz University, Jeddah 21569, Saudi
Arabia;
9
Australian Prostate Cancer Research Centre-Queensland, Translational Research Institute,
Queensland University of Technology, Brisbane 4059, Australia;
10
George W Woodruff School of
2
Mechanical Engineering, Georgia Institute of Technology, Atlanta 30332, GA, USA;
11
Institute for
Advanced Study, Technische Universität München, Munich 85748, Germany.
# These authors contributed equally to this work.
*Corresponding authors:
Dietmar W Hutmacher, PhD, MBA Ali Khademhosseini, PhD
Institute of Health and Biomedical Innovation Biomaterials Innovation Research Center
Queensland University of Technology Harvard Medical School
60 Musk Avenue, Kelvin Grove PRB-252, 65 Landsdowne Street
Queensland 4059, Australia Cambridge 02139, MA, USA
E-mail: dietmar.hutmacher@qut.edu.au E-mail: alik@rics.bwh.harvard.edu
Phone: +61 7 3138 6077 617 768 8395
Fax: +61 7 3138 6030 617 768 8477
Daniela Loessner, PhD
Institute of Health and Biomedical Innovation
Queensland University of Technology
60 Musk Avenue, Kelvin Grove
Queensland 4059, Australia
E-mail: daniela.lossner@qut.edu.au
Phone: +61 7 3138 6441
Fax: +61 7 3138 6030
E-MAIL ADDRESSES OF CONTRIBUTING AUTHORS
Daniela Loessner, E-mail: daniela.lossner@qut.edu.au
Christoph Meinert, E-mail: christoph.meinert@qut.edu.au
Elke Kaemmerer, E-mail: e.kaemmerer@uq.edu.au
Laure C Martine, E-mail: laure.thibaudeau@qut.edu.au
Kan Yue, E-mail: kanyue@mit.edu
Peter A Levett, E-mail: peter.levett@outlook.com
Travis J Klein, E-mail: t2.klein@qut.edu.au
Ferry PW Melchels, E-mail: f.melchels@hw.ac.uk
Ali Khademhosseini, E-mail: alik@rics.bwh.harvard.edu
Dietmar W Hutmacher, E-mail: dietmar.hutmacher@qut.edu.au
3
ABSTRACT
Progress in advancing a system-level understanding of the complexity of human tissue development
and regeneration is hampered by a lack of biological model systems that recapitulate key aspects of
these processes in a physiological context. Hence, growing demand by cell biologists for organ-
specific extracellular mimics has led to the development of a plethora of three-dimensional (3D)
cell culture assays based on natural and synthetic matrices. We developed a physiological
microenvironment of semi-synthetic origin, called gelatin-methacryloyl (GelMA)-based hydrogels,
which combine the biocompatibility of natural matrices with the reproducibility, stability, and
modularity of synthetic biomaterials. We describe here a step-by-step protocol for the preparation
of the GelMA polymer, which takes 1–2 weeks to complete, and can be used to prepare hydrogel-
based 3D cell culture models for cancer and stem cell research, and tissue engineering. We also
describe quality control and validation procedures, including how to assess the degree of GelMA
functionalization, plus mechanical and diffusive properties, to ensure reproducibility in
experimental and animal studies.
INTRODUCTION
In multicellular organisms, cells are embedded in a pericellular and/or an extracellular matrix
(ECM). Structurally, the ECM of native tissues is subdivided into two general types: filamentous
protein networks, as found in connective tissues
1
, and thin layers with sheet-like organization,
which are found in basement membranes
2
. It is now thought that the ECM represents more than just
a structural architecture that provides adhesion sites for cell surface receptors
3
. ECM homeostasis is
a critical factor in preserving normal tissue function and tissue-specific mechanical and biochemical
properties
4
. The interaction between cells and the surrounding ECM regulates a variety of
physiological cellular processes, including motility, migration, invasion, and proliferation
5,6
. On the
other hand, the crosstalk of cells with the local microenvironment promotes the development and
progression of various diseases, including cancer
7-9
.
The physico-chemical properties of the cellular microenvironment directly influence the state of
differentiation of various cell types. For example, with regard to chondrocytes, the properties of the
native or tissue-engineered ECM play a key role in the successful development of a functional
cartilage matrix
10-12
. These effects of the extracellular microenvironment on cellular behavior are
well-known; thus, experimental organ-specific model systems need to mimic physiological
4
conditions in humans, and their development has, therefore, become a major focus of biomedical
research
13-15
.
Two-dimensional (2D) cell culture systems, which are based on cells propagated as monolayers and
are routinely utilized in research, differ greatly from the native microenvironment and often fail to
adequately model normal tissue and disease processes
16
. To address this fundamental drawback of
2D cell culture systems, tissue-engineered in vitro platforms have become the preferred model
system for experimental organ-specific studies
17
. These platforms can rely on different
technologies, so that they consist of a three-dimensional (3D) modular culture system, rather than a
stand-alone model. These modular systems can, therefore, enable researchers to adequately
engineer complex tissue-specific niches. Several 3D culture approaches based on natural, synthetic
and semi-synthetic biomaterials are available to serve as physiologically relevant mimics of the
ECM for cell biology, tissue engineering, and regenerative medicine applications
18
. Most of these
3D cell culture systems consist of hydrogels, which are highly hydrated matrices of crosslinked
polymer chains that mimic the 3D networks observed in native connective tissues
19,20
.
Current 3D cell culture systems
A wide variety of natural, synthetic, and semi-synthetic hydrogels have been successfully used as
3D cell culture systems that mimic the extracellular microenvironment during disease development
and progression
21,22
or that provide a cell delivery vehicle for animal experiments
23
. Biomaterials
utilized for these purposes include natural hydrogel-forming proteins, like collagen type I
24
, and
more complex mixtures based on reconstructed basement membrane proteins, such as Matrigel
25
, as
well as synthetic polymers, like polyethylene glycol
13,23
. Although collagen type I gels effectively
recapitulate the properties of connective tissues and Matrigel enables researchers to conduct cell
growth and differentiation studies
17
, such hydrogels derived from natural proteins often lack
consistent properties and contain impurities resulting in high batch-to-batch variations
16
. Hence,
researchers in the field of materials science have developed an array of synthetic hydrogels that are
widely explored as 3D ECM mimics
26
. Although these synthetic polymers have the advantage of
reproducible, well-defined, and tunable physico-chemical properties, they lack the cell-binding and
protease cleavage motifs that are naturally found in ECM. This limitation can be overcome by
adding cell-responsive sites, such as integrin cell-binding motifs, to synthetic polymers, thus
engineering biomimetic materials
27
.
Interestingly, other 3D cell culture approaches utilize scaffolds instead of hydrogels. For example,
the work by the Cukierman group introduced cell-derived matrices as 3D culture and assay system
that have the characteristics of basement membrane-like substrates in order to analyze cell
morphology and behavior
28
. To create a specific type of basement membrane, the Lengyel team
