3D High Throughput Assays for Combinatorial Screening of Biomaterials for

Tissue Regeneration

C.P. Bertucci*, S. Ramamoorthy†, P.J. Karande†, D.M. Thompson*

Rensselaer Polytechnic Institute
Department of Biomedical Engineering*
Department of Chemical Engineering†
Troy, NY
Thompd4@rpi.edu

Abstract— Tissue engineered strategies suffer from a lack of
complexity, which manifests itself as unsatisfactory functional
recovery in clinical applications. Better understanding of cell
response to complex combinations of matrix proteins and
growth factors mediating regeneration is necessary for
increased biomaterial performance. A high throughput 3D
multiplex assay has been designed for evaluating cell behavior
in response to differential composite biomaterials. Specifically,
Schwann cell (SC) and neuronal viability, death, and
morphology were measured in response to varying collagen
(COL) hydrogel concentration (1-3 mg/mL) and Matrigel™
(MAT) concentration (0-100% v/v). SCs were sensitive to
differences in MAT and relatively insensitive to COL
differences within this range, whereas neurons were sensitive
to changes in both MAT and COL concentration. This strategy
is capable of rapidly identifying large differences in cell
behavior within combinatorial biomaterials, demonstrating its
utility for narrowing large experimental libraries to optimal
multifunctional composite materials.
Keywords- high throughput, 3D cell culture, combinatorial
screening, neural tissue engineering, extracellular matrix,
biomaterials
I. INTRODUCTION
Tissue repair and regeneration are incredibly complex
biological responses to injury with exact mechanisms of
healing system-dependent. Engineering strategies for
enhancing regeneration requires comprehensive
understanding of the roles of biomolecular factors involved
during normal regenerative processes. These include cell
types, insoluble extracellular matrix (ECM) proteins,
soluble growth factors (GFs), and the complex signaling
patterns involved therein. Furthermore, understanding how
the inclusion of exogenous factors modify regenerative
mechanisms is necessary for advancing tissue engineered
regeneration. Current approaches for tissue engineering
regeneration focus on simplifying biological systems to the
introduction of 1-2 cell types into a scaffold composed of 1-
2 biological factor. While these studies may prove
successful and yield important information in vitro, they fail
to offer the same regenerative benefits compared to
autologous grafts in vivo due to a lack of complexity,
whether it is architectural and/or compositional, required to
provide a growth-supportive environment.
This project aims to design high throughput assays for
identifying cell metrics important during regeneration in 3D
cultures. Screened parameters include cell metabolism,
death, morphology, and migration. A high throughput
approach is necessary due to the prohibitively large
experimental space derived from the combinations of
potential substrates, ECM factors, and GFs. These assays
are designed for flexibility amongst biological systems but
are being applied towards peripheral nerve injuries. As a
proof-of-concept, COL hydrogels were modified by addition
of increasing amounts of MAT and SC and neuronal
responses were measured. COL is the most abundant matrix
protein and present in peripheral nerve tissue as a structural
component [1], whereas MAT is derived from basement
membrane containing proteins important in Schwann cell
and neuronal adhesion and signaling [2].
II. METHODS
Polydimethylsiloxane (PDMS) sheets were perforated and
adhered to glass coverslips to serve as wells. COL-MAT
libraries were mixed on ice varying COL (1, 2, 3 mg/mL)
and MAT (0, 20, 35, 50, 100% v/v) and cells were
subsequently encapsulated for either 1 or 3 days. Schwann
cells and neuronal primary cells were obtained from
Sprague-Dawley rat pup (P3) sciatic nerves and dissociated
dorsal root ganglia, respectively.
Cell metabolism was measured via manufacturer’s
protocol for Alamar blue, with a 24hr incubation period
being the exception for enhanced sensitivity in 3D cultures.
Similarly, cell death was measured via manufacturer’s
protocol for LDH assay, with an 8hr incubation period.
Cell morphology was measured via fixation,
permeabilization, and staining of cytoskeleton. Schwann cell
and neuronal morphologies were assessed through actin and
III-tubulin staining, respectively. Images were rapidly
acquired via fluorescent flatbed scanner (Typhoon Trio+,
GE). Higher resolution images used to validate data from the
fluorescent scanner were acquired through confocal
microscopy (LSM 510 Meta, Zeiss). Image processing and
analysis were performed through ImageJ (NIH). Two factor
ANOVA followed by post-hoc Tukey-Kramer testing was
used for statistical analysis (Excel).

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978-1-4799-8360-5/15/$31.00 ©2015 IEEE.
 III. RESULTS
A. Schwann Cell Response to COL-MAT Library
SCs were encapsulated in conditions as described in
methods (Figure 1a). There was no significant differences in
cell viability, indicative of even seeding across groups.
Additionally, there was low cell death (<10%) following
seeding (data not shown). Morphology data was calculated
as actin area/image and normalized to initial seeding density
and negative control COL-only (0% MAT) samples. By day
1, at every COL concentration, significant SC spreading
occurred in 20, 35, and 50% MAT, whereas there was no
significant differences between these MAT values. Within
20, 35, and 50% MAT there was increased SC spreading in 2
mg/mL COL over 1 mg/mL COL. Confocal microscopy
image analysis confirms that 20, 35, and 50% MAT induces
greatest SC spreading (data not shown).
B. Neuronal Response to COL-MAT Library
Neurons were encapsulated in conditions identical to SCs
(Figure 1b). No significant differences in cell viability or cell
death (<15%) were observed between all groups (data not
shown). Morphology data was calculated as III-tubulin
area/image and normalized to initial seeding density and
negative control COL-only samples. Neurite outgrowth
significantly increased at 20 and 35% MAT from COL-only,
which further increased in 50% MAT at 1 mg/mL COL and
MAT-only (100% MAT). Within COL-only and 50% MAT,
1 mg/mL COL supported greatest neurite outgrowth. Within
20 and 35% MAT 1 and 2 mg/mL supported greatest neurite
outgrowth. Confocal microscopy image analysis confirms
that 20, 35, and 50% MAT induces greatest neurite
outgrowth (data not shown).
IV. DISCUSSION
COL and MAT contain essential biomolecular
components in peripheral nerve tissue. COL-MAT mixtures
best resemble the SC-neuronal in vivo microenvironment
through basement membrane, namely laminin, addition into
culture that SCs and neurons adhere and extend processes on
[2]. Maximal SC spreading occurred in COL-MAT mixtures,
Figure 1: Schwann cell and neuronal morphology COL-MAT screens: (a) SC normalized actin area two-factor screen evaluates COL and MAT as
base substrates for SC spreading at day 1. Spreading is maximal in COL-MAT mixtures and not pure substrates. (n=3-5; * p<0.05) (b) Neuronal
normalized III-tubulin two-factor screen evaluates COL and MAT as base substrates for neurite outgrowth at day 3. Neurite outgrowth is maximal
in 1 mg/mL COL and 50% MAT. (n=3-5; *,†,^ p<0.05) (Gray scale: 1 mg/mL COL; Blue scale: 2 mg/mL COL; Red scale: 3 mg/mL COL)
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with little to no spreading occurring in COL- and MAT-only.
COL-only does not support SC spreading due to a lack of
laminin, and MAT-only substrates may be too soft to support
3D SC spreading. SCs display greater sensitivity to changes
in MAT content as opposed to COL concentration whereas
neurons display sensitivity to both COL and MAT
concentrations. Neurons are known to prefer softer substrates
which this data supports. Importantly, the flatbed scanner is
capable of rapidly identifying differences in cell morphology
supporting its use in higher throughput studies.
V. FUTURE WORK
Novel SC- and neuronal-specific composite biomaterials
will be identified by rationally screening complex mixtures
of ECM factors and GFs for enhanced morphology, viability,
outgrowth, and migration. Our lab has previously developed
a method for screening migration in combinatorial materials
that will be incorporated [3]. Tissue-specific composite
biomaterials will then be identified through coculture
screening of previously identified cell-specific composite
biomaterials. Ultimately, this strategy serves as a general
guide that identifies a number of novel combinations that
provide blueprints for 1) 3D models for deeper
understanding of cell-cell signaling and 2) biomaterials that
can potentially enhance tissue regeneration.
ACKNOWLEDGMENT
This project is funded by NSF.1067208 and CB is an NIH
pre-doctoral fellow.
REFERENCES
[1] R Deumens et al., “Repairing injured peripheral nerves: Bridging the
gap,” Prog. in Neurobio, vol. 92, 2010, pp. 245-276, doi:
10.1016/j.pneurobio.2010.10.002
[2] C. Ide, “Peripheral nerve regeneration,” Neuroscience Research, vol.
25, 1996, pp. 101-121
[3] C.M. Dumont, P.J. Karande, D.M. Thompson, “Rapid assessment of
migration and proliferation, a novel 3D high-throughput platform for
rational and combinatorial screening of tissue-specific biomaterials,”
Tissue Engineering: Part C, vol. 20, 2014, pp. 620-629, doi:
10.1089/ten/tec.2013.0362