Download Engineering the Cellular Microenvironment Molly S

Engineering the Cellular Microenvironment
Molly S. Shoichet, Shawn C. Owen, Stephanie A. Fisher, Roger Y. Tam
Department of Chemical Engineering & Applied Chemistry, Institute of Biomaterials & Biomedical
Engineering, Department of Chemistry, Donnelly Center, University of Toronto, Toronto ON Canada
Introduction: The cellular microenvironment is defined
by chemical, physical and mechanical properties of the
extracellular matrix in addition to other cells [1]. The
fields of tissue engineering and regenerative medicine
have been focused on understanding this environment with
the goal of creating biomimetic matrices with which to
grow cells and guide their migration in vivo for tissue
regeneration and in vitro to study disease progression.
This has inspired the use of hydrogels as the extracellular
matrix to mimic soft tissues, and their modification with
biomolecules to guide cell adhesion and growth. We have
been particularly interested in transparent hydrogels, such
as agarose and hyaluronan, that allow 3D chemical
patterning with multiphoton lasers and control of
mechanical properties with chemical crosslinking.
Crosslinked hyaluronan hydrogels have been particularly
compelling to study cancer biology because of the natural
abundance of hyaluronan in cancer tissues. The overall
hypothesis of this research is that hyaluronan hydrogels
can be defined to mimic the microenvironment of breast
cancer, thereby providing a system that better represents
the in vivo paradigm than current 2D cell culture studies,
enabling greater insight into disease progression. The
overall objectives are to: (1) synthesize a crosslinked
hyaluronan hydrogel with controlled mechanical, physical
and chemical properties; and (2) test the hydrogel for
cellular response in term of adhesion and migration.
peptide crosslinked hydrogel, cell invasion was promoted.
Both naturally invading (MDA-MB-231 cells) and noninvading (MCF-7) cells were compared for cell penetration
into these hydrogels, with the goal of controlling invasion
and understanding mechanism. Moreover, cryogelation
was used to control the porosity of the crosslinked
hydrogels, which also influenced cell penetration into the
gels. By careful control of the processing conditions,
cryogels were synthesized that were sufficiently
transparent to allow chemical patterning with multiphoton
lasers. The multiphoton laser technology enables precise
control of the chemical cues present at distinct volumes.
As shown below with fluorescently-tagged epidermal
growth factor, concentration gradients of growth factors
can be achieved with this technology.
Materials and Methods: Hyaluronan is purchased from
Lifecore and sterile-filtered prior to use. It is chemically
modified to introduce furan functional groups, as
previously described [2]. Peptides were custom
synthesized on a peptide synthesizer. Dimaleimidefunctionalized poly(ethylene glycol) and peptides were
used to crosslink the hyaluronan hydrogels [3]. Peptide
modification was characterized by amino acid analysis.
Young’s modulus was measured by compression testing
with a micromechanical tester.
Discussion and Conclusions: A series of hyaluronancrosslinked hydrogels were synthesized with distinct
control of mechanical, chemical and topographical
properties, enabling a thorough investigation of cellular
interactions to be probed. The crosslinked hydrogels
enable cell-cell and cell-matrix interactions to be
controlled with the goal of creating a biomimetic niche for
further evaluation. The cell morphology and penetration
into the gels were influenced by mechanical and chemical
properties, resulting in greater insight into their influence
in disease progression.
Results:
Crosslinked hyaluronan hydrogels were
synthesized with a series of Young moduli, from
1.46±0.24 to 16.12±0.94 kPa, with either the concentration
of the hydrogel or the degree of furan substitution used to
control mechanical properties.
By introducing cell
adhesive peptides, the chemical properties were tuned
allowing cell adhesion to be independently controlled from
the mechanical properties. In contrast, using a peptide
crosslinker, mechanical and chemical properties could be
simultaneously controlled, thereby demonstrating the
versatility of this platform technology. With a degradable
Acknowledgments: We are grateful to NSERC and
CIHR for partial funding of this research.
References:
[1] Shoichet, M.S. Macromolecules, 43, 581, 2010
[2] Nimmo C, Owen S, Shoichet MS. Biomacromolecules
12, 824, 2011.
[3] Owen S, Fisher S, Tam R. Langmuir doi:
10.1021/la305000w, 2013.