Acta Biomaterialia 44 (2016) 178–187

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Acta Biomaterialia
journal homepage: www.elsevier.com/locate/actabiomat

Full length article

Macroporous biohybrid cryogels for co-housing pancreatic islets with

mesenchymal stromal cells
Danielle J. Borg a,1, Petra B. Welzel b,1, Milauscha Grimmer b, Jens Friedrichs b, Marc Weigelt a,
Carmen Wilhelm a, Marina Prewitz b, Aline Stißel b, Angela Hommel a, Thomas Kurth c,
Uwe Freudenberg b, Ezio Bonifacio a,⇑, Carsten Werner b,⇑
a
Preclinical Approaches to Stem Cell Therapy, DFG-Center for Regenerative Therapies Dresden, Dresden University of Technology, Fetscherstrasse 105, Dresden 01307, Germany
b
Leibniz Institute of Polymer Research Dresden, Max Bergmann Center of Biomaterials Dresden, Hohe Str. 6, 01069 Dresden, Germany
c
Electron Microscopy Facility, DFG-Center for Regenerative Therapies Dresden, Dresden University of Technology, Fetscherstrasse 105, Dresden, 01307, Germany

a r t i c l e i n f o a b s t r a c t

Article history: Intrahepatic transplantation of allogeneic pancreatic islets offers a promising therapy for type 1 diabetes.
Received 11 March 2016 However, long-term insulin independency is often not achieved due to severe islet loss shortly after
Received in revised form 19 July 2016 transplantation. To improve islet survival and function, extrahepatic biomaterial-assisted transplantation
Accepted 5 August 2016
of pancreatic islets to alternative sites has been suggested. Herein, we present macroporous, star-shaped
Available online 6 August 2016
poly(ethylene glycol) (starPEG)-heparin cryogel scaffolds, covalently modified with adhesion peptides,
for the housing of pancreatic islets in three-dimensional (3D) co-culture with adherent mesenchymal
Keywords:
stromal cells (MSC) as accessory cells. The implantable biohybrid scaffolds provide efficient transport
Pancreatic islet
Mesenchymal stromal cell
properties, mechanical protection, and a supportive extracellular environment as a desirable niche for
Cryogel the islets. MSC colonized the cryogel scaffolds and produced extracellular matrix proteins that are impor-
Heparin tant components of the natural islet microenvironment known to facilitate matrix-cell interactions and to
Poly(ethylene glycol) prevent cellular stress. Islets survived the seeding procedure into the cryogel scaffolds and secreted insu-
lin after glucose stimulation in vitro. In a rodent model, intact islets and MSC could be visualized within
the scaffolds seven days after subcutaneous transplantation. Overall, this demonstrates the potential of
customized macroporous starPEG-heparin cryogel scaffolds in combination with MSC to serve as a mul-
tifunctional islet supportive carrier for transplantation applications.

Statement of Significance

Diabetes results in the insufficient production of insulin by the pancreatic b-cells in the islets of
Langerhans. Transplantation of pancreatic islets offers valuable options for treating the disease; however,
many transplanted islets often do not survive the transplantation or die shortly thereafter. Co-
transplanted, supporting cells and biomaterials can be instrumental for improving islet survival, function
and protection from the immune system. In the present study, islet supportive hydrogel sponges were
explored for the co-transplantation of islets and mesenchymal stromal cells. Survival and continued func-
tion of the supported islets were demonstrated in vitro. The in vivo feasibility of the approach was shown
by transplantation in a mouse model.
Ó 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

1. Introduction
⇑ Corresponding authors.
E-mail addresses: danielle.borg@mater.uq.edu.au (D.J. Borg), welzel@ipfdd.de
(P.B. Welzel), grimmer@ipfdd.de (M. Grimmer), friedrichs@ipfdd.de (J. Friedrichs), Pancreatic islet transplantation therapy is a viable option for
marc.weigelt@crt-dresden.de (M. Weigelt), carmenzitawilhelm@web.de type 1 diabetic individuals [1]. However, maintaining long-term
(C. Wilhelm), mc.prewitz@googlemail.com (M. Prewitz), alinestissel@web.de graft efficacy remains challenging since the current intrahepatic
(A. Stißel), angela.hommel@crt-dresden.de (A. Hommel), thomas.kurth@ portal vein transplant site is prone to mechanical stress and
crt-dresden.de (T. Kurth), freudenberg@ipfdd.de (U. Freudenberg), ezio.bonifacio@
crt-dresden.de (E. Bonifacio), werner@ipfdd.de (C. Werner).
inflammation associated with the risk of islet capsule rupture
1
Danielle J. Borg and Petra B. Welzel share first authorship. and prolonged ischemia [2–5]. Furthermore, islet endocrine cells

http://dx.doi.org/10.1016/j.actbio.2016.08.007
1742-7061/Ó 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
 D.J. Borg et al. / Acta Biomaterialia 44 (2016) 178–187 179

frequently undergo anoikis during islet isolation due to detach-
ment from their natural extracellular matrix (ECM) in the pancreas
[6–8]. Thus, transplantation of islets into alternate sites and inhibi-
tion of inflammation and anoikis during and after transplantation
are of particular interest to improve survival and function.
Related efforts mostly focus on the immunoisolation of islets in
diabetic animal models by encapsulation of single insulin-
producing beta cells or whole islets in natural or synthetic bioma-
terials [9]. However, encapsulation of islets for protection against
the immune response was found to hinder their revascularization
after transplantation, resulting in graft hypoxia and attrition
[10,11]. Macroporous cell scaffolds are used to overcome these
limitations by providing mechanical protection, enabling exchange
of oxygen, nutrients, and metabolites, as well as release of insulin
from the pancreatic beta cells [12]. In addition, macroporous mate-
rials facilitate cell infiltration from the surrounding tissue includ-
ing neovascularization [13]. Scaffolds prepared from natural
[14,15], synthetic [12,16–18] and mixtures of natural and synthetic
precursors [19–21], have been successfully applied in housing
islets for periods of up to 60 days ex vivo without affecting cell
survival.
Beyond immunoisolation or scaffolding, biomaterials can sup-
port transplanted islets with the localized provision of bioactive
molecules, such as peptide sequences and ECM proteins, and/or
accessory cells [22–25]. Mesenchymal stromal cells (MSC) define
a promising target, since these accessory cells have been shown
to exert beneficial immunomodulatory, pro-angiogenic, and anti-
apoptotic effects which improved diabetes outcome when co-
transplanted with islets in animal models [26–28]. Moreover,
MSC are known to secrete ECM proteins in response to environ-
mental stimulation [29,30].
Here, we customized a recently introduced macroporous hydro-
gel system based on star-shaped poly(ethylene glycol) (starPEG)
and heparin (starPEG-heparin cryogels) [31–33] with adhesion-
mediating peptide ligands to house islets and MSC together in a
defined, multifunctional microenvironment. The architecture of
this cryogel system with interconnected macropores, allows for
unhindered exchange of nutrients, and affords mechanical protec-
tion, 3D spatial distribution and retention of MSC and islets. MSC
attach to the adhesion peptides bound to the cryogel matrix and
further improve the presented artificial 3D niche by secretion of
ECM proteins, enabling proper cell-matrix interactions (Fig. 1).
Our results show that islets remain as functional units in the cryo-
gel scaffolds verified by the secretion of insulin in response to glu-
cose stimulation in vitro. Subcutaneous transplantation into mice
confirmed the principal feasibility of the approach.
2. Materials and methods
2.1. Preparation and characterization of biofunctional starPEG-
heparin cryogel scaffolds
The fabrication of starPEG-heparin cryogel scaffolds with inter-
connected macropores has been described elsewhere [31,32].
Briefly, network formation via chemical crosslinking (EDC/sulfo-
NHS chemistry) of amino terminated 4-arm poly(ethylene glycol
(starPEG, Mw 10,000 Da, JenKem Technology, USA) and heparin
(Porcine intestinal heparin sodium salt; Mw 14,000 Da; Cal-
biochem, Darmstadt, Germany) was combined with cryogelation
technology [34–37]. Architectural and mechanical scaffold proper-
ties can be modulated by altering the concentrations of either poly-
mer precursors or their molar ratio and also freezing temperature
[31,32]. starPEG-heparin cryogel materials were adjusted to com-
bine high porosity and appropriate pore size with suitable mechan-
ical properties by varying cryogelation processing conditions

Fig. 1. Representative SEM image of the dry cryogel (center) with schematic representation of the underlying starPEG-heparin network (bottom left) and representative
digital image of dry cryogel cylinder and discs (top left). The scaffold was designed to offer mechanical protection and allow 3D distribution and attachment of islets and MSC
accessory cells via bioactive surfaces (RGD peptide) within pores to support continued cell survival. MSC further biofunctionalized the cryogel scaffold by secretion of ECM
proteins. The interconnected pore system allows for the exchange of nutrients, oxygen and waste as well as for the transport of insulin released from the pancreatic beta cells
via convection.
180 D.J. Borg et al. / Acta Biomaterialia 44 (2016) 178–187

[31,32]. For the present study a molar ratio of starPEG to heparin of
c = 3 and a total precursor concentration of 4.3% (w/w) was used,
resulting in a material with appropriate architecture and stiffness
for housing of islets and MSC and subcutaneous transplantation.
Gels were fluorescently labeled by mixing heparin with 1% (w/w)
of Alexa FluorÒ 488- or Alexa FluorÒ 405-labeled heparin (prepared
from Alexa FluorÒ 488 or Alexa FluorÒ 405, respectively, Gibco,
UK). Specifically, starPEG and heparin were each dissolved in
one-third of the total volume of deionized, decarbonized water
(Milli-Q) on ice. After treating the solutions in an ultrasonic bath
filled with ice-water for approximately 3 5 min, they were kept
on ice. Applying the same procedure, EDC and sulfo-NHS (molar
ratio of EDC/sulfo-NHS of 2:1) were each dissolved in one-sixth
of the total volume of ice-cold Milli-Q. Based on the amount of
NH2 groups of starPEG, a 2-fold molar excess of EDC was used.
The EDC and sulfo-NHS solutions were mixed with the heparin
solution, and incubated for 10 min on ice to activate the heparin
carboxylic groups. Finally, the starPEG solution was added to the
activated heparin and mixed for 15 s on a vortexer (Minishaker
MS2, IKA, Germany). The resulting reaction mixtures were added
to the cavities of 96-well plates (350 ll per well); frozen at
20 °C overnight, and lyophilized for 24 h. The produced cryogel
cylinders were cut into discs, punched to the desired diameter
using a punching tool (Hoffmann GmbH Qualitätswerkzeuge, Ger-
many), and were finally swollen and repeatedly washed in phos-
phate buffered saline (PBS, pH 7.4). Determination of swelling
properties, and 3D architecture of the cryogel is reported else-
where [31,32]. Pore sizes were determined from cross-sectional
confocal images of Alexa FluorÒ 405-fluorescently labeled PBS
swollen cryogels (Fig. 2A; methods described in the Supplementary
Information) using Image J software for image processing. The
mean pore size and the pore size distribution were obtained by
quantifying the dimensions of more than 1,200 pores using discs
from 3 different cylinders. In addition, cryogel scaffolds were infil-
trated with poly(L-lysine) coated, Alexa FluorÒ 633-conjugated
pancreatic islet-sized glass beads (£ 125 and 250 lm, MO-SCI Spe-
cialty Products, Rolla, MO, provided by Dr. Sophie Pautot, Center
for Regenerative Therapies Dresden, Germany) to test their appli-
cability for housing islets. Uniaxial compression tests were utilized
to achieve the global (bulk) mechanical properties of the material.
Moreover, the local mechanical properties (stiffness of the cryogel
struts) were characterized by an AFM nanoindentation approach
on thin cryogel sections (see Supplementary Information).
Cryogel scaffolds used for in vitro and in vivo studies were 2 mm
in height and 8 mm in diameter in the PBS swollen state. To
improve cell adhesion, the starPEG-heparin cryogel scaffolds were
biofunctionalized with a RGD (cyclo(Arg-Gly-Asp-D-Tyr-Lys)) con-
taining peptide sequence (Peptides International, USA) as
described [32]. The PBS swollen scaffolds were first sterilized in
Proclin/0.02% PBS (Supelco, USA) overnight and washed three
times with fresh PBS. After another washing step (three times)
with 67 mM phosphate buffer (pH 5) at 4 °C, carboxylic acid groups
of heparin were activated with EDC/sulfo-NHS solution (50 mM
EDC, 25 mM sulfo-NHS in 67 mM phosphate buffer (pH 5)) for
1 h. Subsequently, the scaffolds were washed three times in borate
buffer (100 mM, pH 8, 4 °C) to remove unbound EDC/sulfo-NHS
and then incubated in 500 lL cyclo(Arg-Gly-Asp-D-Tyr-Lys)-
solution (160 lg/mL; dissolved in borate buffer) for 3 h at room
temperature. Finally, all scaffolds were washed in PBS three times,
and maintained in cell culture medium until use.

Fig. 2. Properties of the PBS swollen cryogel scaffolds. (A) Representative scanning confocal image of an Alexa FluorÒ 405-labeled cryogel section (blue). Scale bar = 500 lm
(B) Representative uniaxial compression stress strain curve. (C) Pore size distribution (2426 pore dimensions were measured from n = 3 scaffolds). Refer to Supplementary
Information for more details. (D) Representative digital image of a PBS swollen cryogel disc. (For interpretation of the references to colour in this figure legend, the reader is
referred to the web version of this article.)
 D.J. Borg et al. / Acta Biomaterialia 44 (2016) 178–187
2.2. Experimental animals
C57BL/6 J and B6.Cg-Tg(Ins1-EGFP)1Hara/J mice were pur-
chased from Jackson Laboratories (Jackson Laboratories, USA) and
maintained and sacrificed for MSC or islet isolation, respectively,
or transplanted and sacrificed 7 days after transplantation in accor-
dance with the institutional animal ethics guidelines (24D-
9168.11-1/2012-50).
2.3. MSC isolation and expansion
MSC were isolated from bone marrow aspirates from the femur
and tibia of 9–10 week old C57BL/6 J donor mice. Passage 0 MSC
were cultured for 3–4 weeks in MSC medium (IMDM medium sup-
plemented with 10% fetal calf serum (Gibco, USA), 10% equine
serum (Hyclone Laboratories, USA), 1% L-glutamine (Lonza,
Switzerland), 1% antibiotic-antimycotic (Gibco, USA)) at 37 °C/5%
CO2 with a medium change after 24 h of culture and every 3–
4 days thereafter. MSC were subcultured using 0.25% trypsin/1 mM
EDTA (Gibco, USA) when 60–80% confluent until passage 10, at a
cell density of 2,500 cells/cm2. MSC were used for experiments at
passage 8–10. MSC were characterized by flow cytometry as previ-
ously reported [27]. MSC were able to differentiate in vitro into
chondrocytes, osteoblasts and adipocytes as determined by aggre-
can, osteopontin and BODIPY staining, respectively [27].
2.4. Islet isolation
Islets were isolated from the pancreas of 8–9 week old B6.Cg-Tg
(Ins1-EGFP)1Hara/J or 9–10 week old C57BL/6 J donor mice by col-
lagenase digestion (0.7 mg/mL; Sigma-Aldrich, USA) and purified
using a discontinuous Ficoll density gradient, as described previ-
ously [27]. Islets were washed with islet medium: RPMI-1640
medium supplemented with 1% (v/v) L-glutamine (Sigma-Aldrich,
USA), 1% penicillin-streptomycin (Sigma-Aldrich, USA), 5.5 mM
glucose (Sigma-Aldrich, USA) and 5% fetal calf serum (Gibco,
USA). Isolated, free-floating islets were rested 18–24 h prior to
seeding into the cryogel scaffolds at 37 °C/5% CO2.
2.5. Seeding of MSC and islets into cryogel scaffolds
Islets were seeded into cryogels as follows. The cryogel scaffolds
were placed on sterile filter paper (to remove culture medium).
One hundred islets in 250 lL of islet medium were seeded on top
of the scaffold that was then placed in a 24-well plate to allow
the islets to recover at 37 °C/5% CO2, for 30 min, prior to adding
islet medium.
MSC were seeded into cryogel scaffolds in a two-step process.
The cryogels were placed on sterile filter paper and 250,000 MSC
in 500 lL MSC medium were seeded on top of the scaffold. Cryo-
gels were then placed in a 24-well plate to allow MSC to attach
at 37 °C/5% CO2, for 1 h. After this time, a second suspension of
250,000 MSC in 500 lL medium was added to the top of the cryo-
gel. Newly seeded MSC were allowed to attach at 37 °C/5% CO2, for
1 h, before filling the well with MSC medium.
Cryogels containing MSC and islets were prepared as follows.
First, MSC were seeded into the scaffolds as described above. Cryo-
gels were cultured for 3 days with daily medium changes. There-
after, islets were seeded as described above and were analyzed
or transplanted 2 h after islet seeding.
2.6. Analysis of MSC and islet distribution within cell-laden scaffolds
in vitro
For scanning electron microscopy (SEM), scaffolds were first
fixed with 4% formalin and subsequently post-fixed in modified
181
Karnovsky (2% paraformaldehyde/2% glutaraldehyde in 50 mM
HEPES) followed by 1% osmium tetroxide in PBS, dehydrated in
a graded series of ethanol, critically point dried using a CPD
030 (Leica Microsystems, Austria), and coated with gold using a
sputter coater device (SCD 050; Leica Microsystems, Austria).
Samples were analyzed with a TM 1000 tabletop SEM (Hitachi
High-Technologies Europe GmbH, Germany) or a JSM 7500F cold
field emission SEM (Jeol GmbH, Germany). To determine whether
MSC and islets penetrated the whole scaffolds, cryogels were
fixed as described for SEM analysis and were dehydrated in etha-
nol, infiltrated and embedded in the glycol methacrylate resin
TechnovitÒ 7100 (Heraeus-Kulzer, Germany). Sections (2 lm)
were stained with 1% toluidine blue/0.5% borax and multiple
images were captured using a Biozero 8000 light microscope
and tile images were stitched into larger mosaics using the image
merge function of the associated BZ Analyzer software (Keyence,
Germany).
2.7. Characterization of extracellular matrix deposition within cell-
laden cryogels in vitro
Seeded scaffolds were washed in PBS, fixed in cold 4% forma-
lin, submerged in cold 30% sucrose overnight, and frozen in liquid
nitrogen. Cryogels were sectioned (5 lm; Cryostat) from the top
seeding surface until cells were observed under a light micro-
scope. Serial cryosections (5 lm) throughout the cryogels were
stained with anti-laminin IgG (Sigma-Aldrich, USA) and anti-
fibronectin IgG (Rockland Immunochemicals Inc, USA) in
PBS/0.1% Tween for 1 h at room temperature and thereafter
labeled with respective secondary antibodies (Alexa FluorÒ 546;
Invitrogen, USA) for 1 h at room temperature. Cells were stained
for actin using Phalloidin-Alexa FluorÒ 633 (Invitrogen, USA) for
30 min at room temperature. Images were acquired using a SP5
laser scanning confocal microscope (Leica Microsystems,
Germany).
2.8. Analysis of MSC and islet viability within cell-laden scaffolds
in vitro
Cryogel scaffolds were washed with PBS and submerged in
1 lM of Calcein-AM and EthD-1, provided in the LIVE/DEADÒ Via-
bility/Cytotoxicity Kit (Invitrogen, USA) and incubated in the dark,
for 30 min at 37 °C/5% CO2. Cryogels were washed in PBS and its
top region and bottom site imaged within 30 min 1 h after stain-
ing using a Leica SP5 multi-photon laser scanning confocal micro-
scope (Leica Microsystems, Germany). For the top region (93–
195 lm from the top seeding surface), z stacks (approximately
every 10 lm) were deconvoluted using Imaris software and were
reported as maximum projections or 3D projections (see Supple-
mentary Information).
2.9. In vitro islet function: glucose-stimulated insulin secretion
The glucose stimulated insulin secretion assay involved the
static resting of islets ± MSC inside cryogels, or freely sus-
pended, in resting buffer containing a glucose concentration of
2.8 mM for 2 h and subsequent stimulation of islets in a buffer
containing a high glucose concentration (25 mM) as previously
described [38]. Secreted insulin was detected from supernatants
using a commercial mouse insulin ELISA kit (Crystal Chem Inc.,
USA) according to the manufacturers’ instructions. A stimulation
index was calculated as the ratio of the insulin released during
high glucose to the insulin release during low glucose
treatment.
182 D.J. Borg et al. / Acta Biomaterialia 44 (2016) 178–187

2.10. Co-transplantation of cell-laden cryogel scaffolds into a rodent
model
Nine week old anesthetized (100 mg/kg Ketamine and 10 mg/kg
Xylazine) C57BL6/J mice were transplanted subcutaneously with
Alexa FluorÒ 405-labeled cryogels containing no cells (n = 3), 100
green fluorescent protein (GFP) positive islets (n = 3) or 100 GFP+
islets plus 500,000 MSC (passage 8–10) labeled with a cell tracker
dye (CM-DiI; Invitrogen, USA) (n = 3). Mice were monitored daily
and 7 days post-transplant, mice were sacrificed and cryogel
implants were removed. Scaffolds were washed, fixed, frozen and
sectioned as described above. Fluorescent images were acquired
randomly throughout the cryogel on an upright Leica TCS SP5 con-
focal system (Leica Microsystems, Germany).
2.11. Statistical analysis
Statistical analyses were performed using GraphPad Prism soft-
ware (La Jolla, USA). Data with a Gaussian normal distribution are
reported as mean values with standard deviation. P values were
determined by the unpaired Student’s t-test or one-way ANOVA.
Comparison of insulin secretion under multiple conditions was
performed using the general linear model. A two-tailed p value
of <0.05 was considered significant. Changes in weight post-
transplant were analyzed using linear regression and slope com-
parison. Overall slopes were pooled if no significant differences
were observed.
demonstrated by scanning electron microscopy (SEM, Fig. 1). The
scaffolds prepared from a reaction mixture with a molar ratio of star-
PEG to heparin of c = 3 and a total precursor concentration of 4.3%
(w/w) using a freezing temperature of 20 °C, exhibited a high total
porosity of approximately 98%, determined from mass and volume
swelling data [31]. The mean pore size in the PBS swollen state, mea-
sured from cross-sectional confocal images of fluorescently labeled
cryogels (Fig. 2A), was 228 ± 130 lm (pore size distribution is shown
in Fig. 2C). Uptake of fluorescently labeled model glass beads with
diameters (125 and 215 lm) comparable to murine islets (50–
250 lm) was demonstrated (Fig. S1), suggesting that the scaffolds
should allow for infiltration and housing of whole islets.
A global (bulk) Young’s modulus of 0.6 ± 0.1 kPa was calculated
from the linear elastic part of stress-strain curves that were
obtained by uniaxial compression of the whole PBS swollen cryogel
scaffolds (Fig. 2B). The local Young’s modulus of the pore walls
(struts) was determined by an AFM-based nanoindentation
approach [31,34] and was found to be 68 ± 38 kPa (a representative
force-distance curve is given in Fig. S2).
As previously demonstrated, the starPEG-heparin hydrogel plat-
form can be readily functionalized with a variety of adhesion-
mediating peptides and glycosaminoglycan-binding growth factors
[34,35]. Herein, the scaffolds (Fig. 2D) were additionally modified
by conjugating a RGD (Arg-Gly-Asp)-containing peptide sequence
(Fig. 1) via carbodiimide chemistry to remaining carboxylic acid
functionalities of the heparin component of the gels for anchoring
MSC to the macropore walls prior to islet loading.

3. Results 3.2. Co-housing of islets and MSC within cryogel scaffolds in vitro

3.1. Engineering and characterization of macroporous starPEG-
heparin cryogel scaffolds
Macroporous starPEG-heparin cryogel scaffolds were prepared
using chemical crosslinking (EDC/sulfo-NHS chemistry) of amino
terminated starPEG and heparin in aqueous solution at sub-zero
temperatures as previously described [31,32,34,35]. Lyophilization
of the incompletely frozen gel (Fig. 1) resulted in sponge-like
materials with a system of interconnected macropores as
The potential of the cryogel scaffolds to co-house islets and MSC
accessory cells was tested in a series of in vitro experiments. Mouse
bone marrow-derived MSC were seeded on RGD-functionalized,
starPEG-heparin cryogel scaffolds to investigate cell attachment
and survival. Subsequently, the infiltration of mouse pancreatic
islets into empty scaffolds and scaffolds pre-cultivated with MSC
for 3 days was studied.
When seeded into the cryogel scaffolds, MSC maintained a
fibroblast-like morphology and remained viable after 3 days of

Fig. 3. Islets and MSC survive in cryogels in vitro. Live (green; Calcein AM+) and dead (red; Ethidium homodimer-1(EthD-1)+) staining reveals minimal cell death after (A)
3 days of MSC culture, (D) 2 h of islet culture or (G) 2 h of islet and 3 day cultured-MSC co-culture in cryogels (blue); maximum intensity confocal images (deconvoluted
maximum projections of Z stacks ranging from 93 lm to 195 lm from the top seeding surface of the cryogel); (scale bar = 100 lm; n = 3). Representative scanning electron
images on the periphery of the cryogels (n = 3) show flat multilayered MSC (B; scale bar = 10 lm) with several protrusions and deposition of extracellular matrix (C; scale
bar = 10 lm), whole islets (E; scale bar = 10 lm) with intact microvilli and cilia indicated by the black arrowheads (F; scale bar = 10 lm) and islets surrounded by MSC (H;
scale bar = 10 lm) with intact microvilli after seeding (I; scale bar = 10 lm). A representative scanning electron image of a cryogel seeded with islets and MSC demonstrates
the presence of islets (asterisks) which are surrounded by MSC (dotted line), within the pores of the cryogel (J, scale bar = 100 lm). (For interpretation of the references to
colour in this figure legend, the reader is referred to the web version of this article.)
 D.J. Borg et al. / Acta Biomaterialia 44 (2016) 178–187 183

in vitro culture as demonstrated by the large proportion of Calcein
AM+ cells (Fig. 3A, S3A and S4A). SEM revealed multiple layers of
MSC exhibiting a flattened morphology within the macropores
(Fig. 3B).
Deposition of extracellular matrix in the MSC-seeded cryogel
scaffolds was observed by SEM (Fig. 3C) and the matrix structures
were shown to contain laminin and fibronectin as visualized by
immunochemistry staining (Fig. 5A and 5B).
SEM and scanning confocal images revealed that intact islets
survived the seeding procedure, entered the macropores, and spa-
tially distributed within the sponge-like cryogel material (Fig. 3,
S3B and S4B). The cryogel-supported islets exhibited minimal
rounding of individual endocrine cells (Fig. 3E) with villi and
microvilli present, supporting contact between neighbouring
endocrine cells (Fig. 3F) and remained viable with little cell death
(Fig. 3D, G, S3B and S4B).
When seeded in scaffolds pre-cultivated with MSC, islets were
found within the macropores adjacent to the adherent MSC
(Fig. 3H and J), with villi and microvilli present on the endocrine
cells of the islet (Fig. 3I) as demonstrated by SEM images. Scanning
confocal images of the stained cell-laden scaffolds (LIVE/DEADÒ
Viability/Cytotoxicity kit) illustrate that most islets and MSC sur-
vived and maintained their spheroid or fibroblast-like morphology,
respectively, within the macropores of the cryogel (Fig. 3G, S3C and
S4C). The results suggest that the cryogel scaffolds may keep the
accessory MSC in close proximity to transplanted islets.
To better observe how far MSC and islets could penetrate into
the cryogel, co-seeded cryogels were embedded in TechnovitÒ gly-
col methacrylate resin and thin sections (2 lm) were stained with
toluidine blue. MSC attached homogenously throughout the entire
thickness of the 2 mm thick cryogel scaffold (Fig. 4A). Even after
seeding of only 100 islets onto the rather large scaffold (2 mm

Fig. 4. Islets and MSC distribute throughout the scaffold. Representative light microscopy images of islet/MSC co-cultures in starPEG-heparin cryogels scaffolds (purple)
stained with toluidine blue after embedding in resin and sectioning (2 lm, n = 1 cryogel). (A) MSC (grey; spindle) are attached throughout an entire cross-section after 3 days
of culture within the scaffold. Asterisk represents the top of the cryogel. Scale bar = 50 lm. In (B) and (C) islets are visible (grey-blue; indicated by asterisk) in a depth of
550 lm (B) and 625 lm (C) from the top seeding surface. Scale bar = 20 lm. (For interpretation of the references to colour in this figure legend, the reader is referred to the
web version of this article.)

Fig. 5. Representative images taken throughout Alexa FluorÒ 405-labeled cryogels (blue) seeded with MSC secreting (A) laminin (green) or (B) fibronectin (green) after three
days of in vitro culture. The actin cytoskeleton of the MSC is labeled in red (scale bar = 50 lm; n = 3; sections: 5 lm). (For interpretation of the references to colour in this
figure legend, the reader is referred to the web version of this article.)
184 D.J. Borg et al. / Acta Biomaterialia 44 (2016) 178–187
thickness, 8 mm diameter), we were able to detect islets at a depth
of 550 and 625 lm from the top seeding surface of the cryogel
(Fig. 4B and C, respectively), which is 3- to 4-fold of an islet average
size or one-third to one-fourth of the total cryogel thickness.
3.3. Islet function within cryogel scaffolds in vitro
Insulin secretion was assessed by glucose stimulation of the
islets 2 h after cryogel housing (Fig. 6A). Islets maintained basal
insulin secretion in low glucose either freely suspended
(32.6 ± 26.6 ng/mL) or housed in cryogels (59.2 ± 42.2 ng/mL,
p = 0.25). Islets were able to secrete insulin in stimulating condi-
tions in cryogels, similar to that of freely suspended islets
(84.2 ± 42 ng/mL; suspension vs 158.7 ± 105.3 ng/mL; cryogel).
Co-culture of MSC did not impede basal (69.9 ± 64.9 ng/mL) or
stimulated (155.4 ± 73.8 ng/mL) insulin secretion in suspension,
and insulin secretion was similar when islet and MSC were co-
housed in cryogels (basal, 42.7 ± 29.5 ng/mL; stimulated,
168.8 ± 27 ng/mL). Islets alone or islets plus MSC cultured within
cryogels revealed a similar insulin secretion as islets in suspension
(resting; p = 0.81, stimulation; p = 0.34; general linear regression).
No differences were observed in the stimulation index between
groups (p = 0.185; one-way ANOVA, Fig. 6B). Thus, the viability
and functionality of islets loaded within the cryogel scaffold was
similar to controls, which were cultured in suspension.
3.4. Graft assessment of islet and MSC laden cryogel scaffolds after
syngeneic transplantation in rodents
In order to test the transplantation of islets in starPEG-heparin
cryogel scaffolds and the co-transplantation of MSC and islets,
mice were transplanted subcutaneously with empty, islet (GFP+
islets) laden or islet plus MSC (CM-DiI labeled) laden cryogels.
No adverse behavioral effects were observed in the recipients
post-transplant, and mice maintained their weight 7 days post-
transplant with no differences in weight change over time between
groups (pooled slope = 0.14, p = 0.85; linear regression, Fig. 7B). A
thin fibrous capsule surrounding the cryogel was observed in all
cryogels removed. Sequential sectioning and imaging of the cryo-
gels by means of fluorescence microscopy revealed the presence
of intact islets and MSC in the scaffolds after 7 days in vivo
throughout the scaffold (Fig. 7A) as well as the presence of host
F4/80+ macrophages (Fig. S5).
Fig. 6. In vitro insulin secretion after glucose stimulation (A) Islets with or without MSC in suspension (n = 5–6) or cryogels (n = 6) were rested in Krebs buffer containing
2.8 mM glucose (open symbols) and stimulated in Krebs buffer containing 25 mM glucose (closed symbols) for 2 h. Mean insulin concentrations ± SD are shown. (B)
Stimulation index calculated from insulin secreted under resting and stimulating conditions in (A). Scatter dot plots with the mean ± SD are shown.
4. Discussion
Transplantation of pancreatic islets for type 1 diabetic individu-
als can delay or stabilize diabetic complications by providing
appropriate glycemic control [1]. However, the poor long-term
effectiveness of transplanted islets directs current research
towards engineering superior transplant microenvironments
[10,12] in order to overcome central problems like the lack of vas-
culature and ECM attachment/integrin ligation [25].
Here, we applied a recently developed macroporous, starPEG-
heparin cryogel material [31–33] and MSC as a synergistic strategy
to design multifunctional 3D islet-supportive carriers. Since pan-
creatic islets of a given species are not uniform in size, the broad
pore size distribution (Fig. 2C) of the cryogel scaffolds was consid-
ered advantageous for an optimal uptake. Figs. 2–4 as well as
Figs. S1, S3 and S4 clearly demonstrate that the size of the macro-
pores is large enough and the interconnectivity is sufficient to
allow for 3D distribution and housing of living MSC and whole
islets within the cryogel scaffolds. Although fewer islets were
observed on the bottom site of the cryogel (Fig. S3B and S3C), a
number of islets were detected throughout the cryogel
(Fig. 4B and C). The difficulty in islet location may be attributed
to the small number of seeded islets (100), relative to the large
cryogel scaffold (diameter 8 mm, thickness 2 mm).
Due to their high porosity (approximately 98%) and their thin
but stiff struts (Fig. S2), the cryogel scaffolds are soft but robust
upon compression (Fig. 2B), and can withstand large deformation
(approximately 90%) without losing integrity [31,32]. The local
stiffness of the cryogel pore walls (struts) was found to be orders
of magnitudes higher in comparison to the low global (bulk) stiff-
ness of the whole cryogel scaffold. This enables both mechanical
protection of the MSC and islets in the macropores, and at the same
time matches the tissue stiffness at potential extra-hepatic trans-
plantation sites. The global Young’s modulus of the cryogel scaf-
folds (0.6 kPa) is comparable to the Young’s modulus of muscle
[39] and normal adipose tissue [40], and thus should be advanta-
geous for subcutaneous implantation applications. Adaptation to
the tissue stiffness of other transplantation sites is similarly possi-
ble by choosing different experimental conditions during scaffold
preparation. This may further allow integration into alternate pan-
creatic transplantation sites such as brachioradialis muscle of the
forearm [41], or the omentum pouch [42]. Despite its low bulk
stiffness, the scaffold provides a stiff, local microenvironment that
 D.J. Borg et al. / Acta Biomaterialia 44 (2016) 178–187 185

Fig. 7. Performance of Alexa FluorÒ 405 cryogels as islet carriers 1 week post-subcutaneous transplantation in C57BL6/J mouse hosts. A cryogel (n = 1/mouse) contained
either 100 GFP+ islets alone or together with 500,000 CM-DiI MSC (n = 3 mice/group). Empty Alexa FlourÒ 405-conjugated cryogels were also transplanted (n = 3).
Representative fluorescence microscopy images of cryogel slices (5 lm) were acquired with a laser scanning microscope throughout the cryogel. (A) GFP+ islets (green) and
CM-DiI labeled MSC (red) located within the cryogel 7 days post-transplant (blue). Empty cryogels (blue) seeded without cells are shown in the right panel. Scale bar = 50 lm.
(B) Mean weight of mice from the time of transplant to sacrifice. Error bars are shown as SEM. (For interpretation of the references to colour in this figure legend, the reader is
referred to the web version of this article.)

shields the cells in the macropores from mechanical stress, as elas-
tic energy is adsorbed by the cryogel struts [43].
The heparin component of the underlying hydrogel matrix
offers the possibility to introduce adhesion-mediating peptide
ligands (here: RGD). The architecture and bioactive motifs of the
cryogel system were found to support adhesion of MSC within
the pores of the cryogel. The flattened cell morphology suggests
tight interaction with the RGD-modified gel matrix of the cryogel
struts whilst maintaining cell-cell contact with other MSC
(Fig. 3B). The scaffolds were fully compatible to both cell types as
proven by live/dead staining (Fig. 3, S3 and S4).
MSC survived the seeding procedure (Fig. 3A, S3A and S4A) and
maintained their ability to secrete ECM proteins (Fig. 5), which are
important cues for survival, function and proliferation of pancre-
atic islets [44–46]. The significance of islet-ECM interactions for
recapitulating the native environment of functional pancreatic
islets in order to further minimize anoikis has been highlighted
in literature. Within their native environment, functional pancre-
atic islets are supported by a basement membrane rich in ECM pro-
teins, which is destroyed during islet isolation by collagenase
treatment [7,8]. Thus, there have been many attempts to coat or
functionalize 2D and 3D biomaterials for islet cell culture with var-
ious ECM proteins, such as laminin 5 and collagen type IV [6,47]. In
our approach, we used pre-seeded scaffold-bound MSC for secre-
tion and deposition of ECM proteins in the cryogel scaffolds for
three days prior to islet seeding. In addition, the MSC, previously
shown to be beneficial for islet priming as well as during trans-
plantation [26–28], remained in the cryogel scaffold after islet
loading. Additional in vitro time-course studies following the pro-
gression of ECM protein deposition by MSC may optimize this
cell-based modification of the cryogel scaffold.
Our in vitro experiments demonstrated islet anchorage near
MSC, suggesting a favorable local niche at the struts for islet
attachment. SEM images revealed that endocrine cells of the islets
maintained cell-to-cell contact and maintained the presence of
microvilli. Microvilli are found on native islets [48] and the loss
of microvilli in a beta cell line has been reported as an early feature
of apoptosis [49]. Thus, starPEG-heparin cryogel scaffolds support
pancreatic islets by mechanical, architectural and biomolecular
cues and enable the localized interaction of islets with accessory
cells and cell-secreted ECM proteins (Fig. 1). Further, islets
remained responsive to glucose suggesting their function remained
short-term in vitro (Fig. 6). The co-housing of MSC with islets how-
ever did not significantly enhance insulin secretion in contrast to
previous encapsulation studies [22]. Future in vitro studies would
benefit from glucose challenges over long-term culture, to contin-
ually monitor islet function within the scaffold, and to determine
additional support provided by the artificial 3D microenvironment
and/or the MSC, which may have been masked due to the short-
term culture period in the present study.
186 D.J. Borg et al. / Acta Biomaterialia 44 (2016) 178–187
Subcutaneous transplantation of MSC/islet-seeded cryogel scaf-
folds confirmed the principal feasibility of the approach in a mouse
model. Our provided data indicate that implantation of the cell-
seeded cryogels did not interfere with the localization of islet
and MSC. GFP+ islets and cell membrane-labeled MSC could be
visualized within the cryogel 7 days post-transplantation
(Fig. 7A). The cryogel material did induce the production of a
fibrous capsule around the implant and macrophage infiltration
(Fig. S5). Similar host responses were reported in pancreatic islet
transplantation experiments when using PLGA scaffolds [50],
alginate-encapsulated islets [51] and islet containing devices
[52]. Nevertheless, mice maintained their weight throughout the
transplantation period (Fig. 7B). Despite host responses, short-
term islet engraftment was demonstrated after 7 days post-
transplantation. In this pilot study however, we were unable to
confirm islet function, namely insulin secretion in vivo, by
immunofluorescence, due to technical limitations caused by fixa-
tion. Thus, to assist in the translation of this approach in clinical
islet transplantation, long-term experiments in a type 1 diabetes
animal model under autogenic and allogenic settings to assess islet
function and survival, are still required. It would also be useful to
further examine the beneficial aspects of MSC as accessory cells
within the cryogel such as subsequent immunomodulatory or neo-
vascularization effects, which was not the subject of this pilot
study. Indeed, ongoing research aims at further adjusting the
cryogel-based islet carriers with respect to their revascularization
potential utilizing the heparin component of the underlying biohy-
brid material for additional administration of pro-angiogenic
growth factors [33,53].
5. Conclusion
An islet-supportive carrier was designed by combining the
advantageous properties of macroporous starPEG-heparin cryogels
with benefits of bone marrow-derived MSC accessory cells. The
interconnectivity and size distribution of the macropores enabled
MSC and whole islets to enter the cryogel scaffolds with their stiff
pore walls that shield the cells from mechanical stress. RGD-
modification mediated adhesion of pre-seeded MSC which
secreted extracellular matrix proteins to provide further attach-
ment support for the pancreatic islets in the cryogel and to prevent
their anoikis. The survival and continued function of cryogel-
housed islets was demonstrated short-term in vitro with no
adverse effect on insulin secretion, and short-term implantation
in vivo in a mouse model confirmed the feasibility of the approach.
Thus, starPEG-heparin cryogel scaffolds tuneable in architecture,
mechanical characteristics and biomolecular functionalization,
with the ability to load accessory cells, are highly promising as
supportive carriers for pancreatic islets in the context of transplan-
tation in various alternate sites.
Acknowledgements
Financial support was provided by the German Research Foun-
dation (Deutsche Forschungsgemeinschaft) through Grant num-
bers: SFB-TR 67, WE 2539-7 and FOR/EXC999, by the Leibniz
Association (SAW-2011-IPF-2 68) and by the European Union
through the Integrated Project ANGIOSCAFF (Seventh Framework
Program). Moreover, this work was supported by the DFG-Center
for Regenerative Therapies Dresden, Cluster of Excellence (FZT
111) and the Federal Ministry of Education and Research, Germany
(01GN0945). D. J. Borg was enrolled in the Regenerative Medicine
Ph.D. Program of the Dresden International Graduate School for
Biomedicine & Bioengineering (DIGS-BB) as part of the Technical
University of Dresden. D. J. Borg’s current affiliation is the
Glycation and Diabetes and Inflammatory Diseases and Therapeu-
tics Group, Mater Research Institute- University of Queensland,
Translational Research Institute, Brisbane, Australia.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.actbio.2016.08.
007.
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