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Contents lists available at ScienceDirect

Medical Engineering and Physics

journal homepage: www.elsevier.com/locate/medengphy

Engineering human renal epithelial cells for transplantation in

regenerative medicine
Vita Manzoli a,b, David C. Colter c, Sridevi Dhanaraj d, Alessia Fornoni e,f, Camillo Ricordi a,g,h,i,j,
Antonello Pileggi a,g,h,i, Alice A. Tomei a,g,i,
a
Diabetes Research Institute, University of Miami, 1450 NW 10th Ave, Miami, FL 33136, USA
b
Department of Electronics, Information and Bioengineering, Politecnico di Milano, via Ponzio 34/5, 20133 Milan, Italy
c
Analytical Development, Pharmaceutical Development and Manufacturing Sciences, Janssen R&D, Malvern, PA, USA
d
Biosurgicals R&D, Ethicon Biosurgery, Somerville, NJ, USA
e
Katz Family Division of Nephrology and Hypertension, University of Miami, FL, USA
f
Division of Nephrology, University of Miami, 1475 NW 12th Ave, Miami, FL, 33136, USA
g
Department of Surgery, University of Miami Miller School of Medicine, FL, USA
h
Department of Microbiology and Immunology, University of Miami Miller School of Medicine, FL, USA
i
Department of Biomedical Engineering, University of Miami, 1251 Memorial Dr, Coral Gables, FL, 33146, USA
j
Department of Medicine, University of Miami Miller School of Medicine, FL, USA

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

Article history: Cellular transplantation may treat several human diseases by replacing damaged cells and/or providing a
Received 15 December 2016 local source of trophic factors promoting regeneration. We utilized human renal epithelial cells (hRECs)
Revised 3 March 2017
isolated from cadaveric donors as a cell model. For ecacious implementation of hRECs for treatment of
Accepted 26 March 2017
kidney diseases, we evaluated a novel encapsulation strategy for immunoisolation of hRECs and lentivi-
Available online xxx
ral transduction of the Green Fluorescent Protein (GFP) as model gene for genetic engineering of hRECs
Keywords: to secrete desired trophic factors. In specic, we determined whether encapsulation through conformal
Three-dimensional culture coating and/or GFP transduction of hRECs allowed preservation of cell viability and of their trophic factor
Cell encapsulation secretion. To that end, we optimized cultures of hRECs and showed that aggregation in three-dimensional
Conformal coating spheroids signicantly preserved cell viability, proliferation, and trophic factor secretion. We also showed
Hydrogel that both wild type and GFP-engineered hRECs could be eciently encapsulated within conformal hy-
Gene therapy
drogel coatings through our uid dynamic platform and that this resulted in further improvement of cell
Trophic factors
viability and trophic factors secretion. Our ndings may lay the groundwork for future therapeutics based
on transplantation of genetically engineered human primary cells for treatment of diseases affecting kid-
neys and potentially other tissues.
2017 The Author(s). Published by Elsevier Ltd on behalf of IPEM.
This is an open access article under the CC BY-NC-ND license.
(http://creativecommons.org/licenses/by-nc-nd/4.0/)

1. Introduction

Abbreviations: 2D, Two-dimensional; 3D, Three-dimensional; ALG, alginate; Cellular transplantation holds the potential to treat several
CCE, conformal coating encapsulation; DTT, dithiothreitol; dVS, divinyl sulfone;
EGF, epidermal growth factor; FGFb, basic broblast growth factor; GFP, green
human diseases [110]. Among them, acute kidney injury and
uorescent protein; GM-CSF, granulocyte macrophage colony-stimulating factor; chronic kidney disease are two very prevalent medical condi-
HBSS, Hanks balanced salt solution; HGF, hepatocyte growth factor; hRECs, tions that are associated with raising healthcare costs due to
Human Renal Epithelial Cells; MCP-1, monocyte chemotactic protein-1 (CCL2); high cardiovascular morbidity and mortality [1118]. Strategies to
MMP-2, matrix metalloproteinases-2; MOI, multiplicity of infection; MTT, 3-(4,5-
delay the progression of chronic kidney disease or induce a faster
Dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide; PAI-1, plasminogen activa-
tor inhibitor-1; PBS, phosphate-buffered saline; PEG, polyethylene glycol; pHEMA, recovery from acute kidney injury are highly needed.
Poly(2-hydroxyethyl methacrylate); PPG, polypropylene glycol; RPM, revolutions per
minute; SCF, stem cell factor; TEA, triethanolamine; TGF, transforming growth fac-
tor alpha; TIMP, tissue inhibitor of metalloproteinase; UP-MVG, ultrapure medium

viscosity (> 200 mPas) sodium alginate where minimum 60% of the monomer Corresponding author at: Diabetes Research Institute, University of Miami, 1450
units are guluronate; VCAM-1, vascular cell adhesion molecule-1; VEGF, vascular NW 10th Ave, Miami, FL 33136, USA.
endothelial growth factor. E-mail address: atomei@med.miami.edu (A.A. Tomei).

http://dx.doi.org/10.1016/j.medengphy.2017.03.009
1350-4533/ 2017 The Author(s). Published by Elsevier Ltd on behalf of IPEM. This is an open access article under the CC BY-NC-ND license.
(http://creativecommons.org/licenses/by-nc-nd/4.0/)

Please cite this article as: V. Manzoli et al., Engineering human renal epithelial cells for transplantation in regenerative medicine, Medical
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2 V. Manzoli et al. / Medical Engineering and Physics 000 (2017) 111
Various therapeutic approaches are being explored. These in-
clude transplanting tissue-derived cells either alone or as helper
cells in damaged/dysfunctional tissues. The latters may contribute
to tissue regeneration via local secretion of cytoprotective factors
and/or through the recruitment of cells with regenerative potential
[1114,1924]. The fate of implanted cells depends on their plastic-
ity and on phenotypical changes inuenced by the microenviron-
ment. Culture of most cells in three-dimensional (3D) scaffolds has
shown to better preserve cell phenotype when compared to stan-
dard two-dimensional (2D) culture [22,2533].
Autologous cells are not rejected by the host immune
system and they could integrate into the remodeling tissue
[3439]. Unfortunately, achieving a satisfactory yield when recov-
ering healthy cells from damaged tissue may be a limitation fac-
tor. Autologous somatic cells (i.e., skin cells) that have undergone
in vitroreprogramming are another therapeutic option. These cells
though need expansion and this poses a major challenge. Also,
timely use might not be possible in case of acute organ failure.
Furthermore, if the cause for organ dysfunction is genetically de-
termined, autologous cells require gene therapy prior to utilization.
Allogeneic cells may also represent a viable option for regenera-
tive/tissue repair therapies, as they could be cultured and banked
to be readily available to treat acute medical conditions. The ma-
jor drawback of allogeneic cells is their susceptibility to immune
rejection after implantation. Their use requires concurrent admin-
istration of immunosuppressive drugs that are associated with un-
desirable side effects [40,41]. Moreover, immunosuppression may
also interfere with cell replication and function, potentially alter-
ing the regenerative properties of the implanted cells [42].
As an alternative with the potential to overcome these limi-
tations, immunoisolation through encapsulation of allogeneic cells
and cell clusters may represent an appealing strategy to prevent
immune rejection without the need of life-long immunosuppres-
sion [4346]. Conventional cell microencapsulation is based on the
generation of capsules with a constant diameter that ranges be-
tween 500 and 1500 m. Experimental and computational studies
have shown that 150 m is the maximum distance between cell
and blood supply allowing for proper diffusion of nutrients and
oxygen [4750]. Conventional microcapsules can cause central hy-
poxia of enclosed cell clusters, accumulation of cellular waste and
delayed secretion of trophic factors due to the capsule large size.
Moreover, the high volume of capsule material limits the choice
for transplant sites to the intraperitoneal cavity. To overcome such
limitations, we have recently developed a novel method for con-
formal coating encapsulation (CCE) of cell clusters based on uid
dynamic principles. This process allows shrink-wrapping of cell
clusters with a thin (few tens of microns) layer of biocompatible
polyethylene glycol (PEG) and alginate (ALG) hydrogel without af-
fecting cell viability or functionality of the encapsulated cells [51].
In this study we used previously characterized [52] human re-
nal epithelial cells (hRECs) as a model for showing feasibility of
a cell engineering approach that allows long-term ex vivo culture,
immunoisolation and genetic manipulation of the cells while pre-
serving viability and secretion of trophic factors involved in tissue
regeneration.
2. Materials and methods
2.1. Human renal epithelial cells
Tissue considered unsuitable for transplantation was obtained
through the National Disease Research Interchange (Philadelphia,
PA) following institutional protocol approvals. To remove blood
cells and debris, kidneys were washed in Dulbeccos modied Ea-
gles medium (DMEM, Life Technologies). Tissues were dissected
from the cortex region of the kidneys. The tissues were then me-
Please cite this article as: V. Manzoli et al., Engineering human renal epithelial cells for transplantation in regenerative medicine, Medical
Engineering and Physics (2017), http://dx.doi.org/10.1016/j.medengphy.2017.03.009
[m5G;April 12, 2017;8:30]
chanically dissociated in tissue culture plates and digested in good
manufacturing practice grade enzyme mixtures containing 0.25
units 4-phenylazobenzyloxycarbonyl activity/mL collagenase (NB6,
Serva Electrophoresis GmbH) and 2.5 units/mL dispase II (Roche
Diagnostics Corporation). The enzyme mixture was combined with
renal epithelial growth medium (REGM, Lonza). The conical tubes
containing tissue, medium, and digestion enzymes were incubated
at 37C in an orbital shaker at 225 rpm for 2 h If large pieces of
tissue were still present after the digestion step, they were re-
moved by gravity sedimentation or by slow centrifugation. The
supernatant containing the suspended cells was then transferred
into a new 50 mL tube and centrifuged. Cells were resuspended
in REGM, plated on gelatin-coated tissue culture asks, and cul-
tured at 37C under normal atmospheric conditions for cytological
analyses. Resulting human renal epithelial cells (hRECs), were pas-
saged up to 6 times and demonstrated 88% viability and a normal
46 XY karyotype. Cell characterization has been already published
[52]. All experiments presented here were conducted with hRECs
at passage 6.
2.2. GFP labeling of hRECs
hRECs at passage 4 and 70% conuency were transduced
at multiplicity of infection (MOI) between 1 and 10 with a
pRRLsinPPT-EGFP lentivirus provided by the viral vector core fa-
cility at the University of Miami. Culture media was changed af-
ter 8 h. GFP positivity was conrmed 5 days after transduction by
both uorescence microscopy and ow cytometry with LSRII ma-
chine (BD Biosciences). GFP+ cells were expanded from passage 4
to passage 6. In order to conrm that viral proteins were cleared
from the cell supernatant, two weeks after transduction an HIV-1
p24 antigen ELISA was performed according to manufacturers pro-
tocol (Zeptometrix).
2.3. Generation of three-dimensional hREC spheroids
Several methods for aggregating passage 6 hRECs into 3D
spheroids were evaluated. First, we compared suspension culture
in non-tissue culture-treated, 6-well plastic plates and 35 mm
custom-made silicon-peruorocarbon plates [5355] (to improve
cell oxygenation during culture) with orbital shaking at 125 rev-
olutions per minute (RPM) during incubation at 37C and 5%
CO2 . Three seeding densities were compared: 10,0 0 0, 25,0 0 0,
and 50,0 0 0 cells/cm2 (0.2 106 , 0.5 106 , and 0.9 106 and
cells/well). In an alternative approach, we fabricated biologi-
cally inert plates by coating tissue culture plates with Poly(2-
hydroxyethyl methacrylate) (pHEMA; Sigma Aldrich). A stock so-
lution of 10% was prepared by dissolving 2.4 g of pHEMA in 24 ml
of 95% Ethanol. A 0.5% diluted solution was used for coating tissue
culture treated and non-treated 6-well plates. Plates were dried for
48 h at 37 C, exposed to UV light overnight and stored in sterile
conditions at room temperature for future use. Cells were plated
in tissue culture treated and non-tissue culture treated pHEMA-
coated 6-well plates and incubated at 37 C and 5% CO2 .
2.4. Characterization of hREC spheroid growth
For characterization of spheroid growth, hRECs at passage 6
cells were plated on pHEMA-coated non-tissue culture treated
plate at three cell densities: 0.2 106 , 0.5 106 , and 0.9 106 and
cells/well. Cell aggregates were imaged through phase contrast mi-
croscopy 2, 24 and 48 h after seeding and the aggregate major
axes D1 and D2 (5 elds/well, 3 wells/condition) were measured
for each condition and time point using Zeiss AxioVision 4.8 soft-
ware. Spheroid volume was calculated according to the ellipsoid
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V. Manzoli et al. / Medical Engineering and Physics 000 (2017) 111
volume formula (F1):
4 D1 D2 ( D1 /2 + D2 /2 )
F1 : V =
3 2 2 2
The radius of the sphere with volume equivalent to the ellip-
soid volume was then extrapolated from the spheroid volumes
for each experimental condition and used to quantify spheroid
size. Histograms of spheroid-extrapolated radius for each condition
were generated and tested for Gaussian distribution according to
D Agostino and Pearson omnibus normality test.
2.5. Conformal coating encapsulation (CCE) of spheroids of hRECs
GFP- and GFP+ hREC spheroids underwent CCE 72 h after seed-
ing single cell suspensions as previously described for pancreatic
islets [51]. Briey, 3D spheroids were collected in a 15 ml coni-
cal tube and spun for 3 min at 10 0 0 RPM. The pellet was washed
with Hanks Balanced Salt solution (HBSS, Invitrogen, Grand Is-
land, NY) to remove all traces of culture media. Then a solution
of 5% polyethylene glycol functionalized with divinyl sulfone (PEG-
dVS) (>85% by Michael type addition of dVS (Sigma) to 8-arm
10 kDa PEG (Jenkem Technology)) and dithiothreitol (DTT, Sigma)
at a 1:4 molar ratio and 0.8% w/v of UP-MVG grade alginate (ALG,
ultra-pure medium viscosity sodium alginate with 60% minimum
guluronate, Pronova, FMC BioPolymer AS) at pH 6 was added to
the cell pellet and mixed. The cell spheroids/polymer solution was
then run coaxially to polypropylene glycol (PPG with Mn = 40 0 0,
Sigma) supplemented with 10% Span80 (Sigma) through the mi-
crouidic conformal coating device that we custom made, as de-
scribed [51]. PEG-ALG conformally coated spheroids were collected
in a 50 ml conical tube containing PPG with 10% Span80 and 0.01%
triethanolamine (TEA, Sigma) and the PEG hydrogel coating was
polymerized by TEA-mediated increase in pH. The coated spheroids
were then puried by hexane (Sigma) extraction of PPG and re-
suspended in culture media. GFP- and GFP+ conformally coated
spheroids were imaged in phase contrast microscopy to evaluate
thickness and completeness of CCE. GFP+ spheroids were also im-
aged with a uorescence microscope in order to help distinguish
cell spheroid (GFP+, green) from hydrogel coating (GFP-) and con-
rm coating completeness.
2.6. Cell viability
3D naked and 3D encapsulated hREC clusters (GFP and GFP+)
were cultured up to 14 days and viability and proliferation was
assessed at 1, 7 and 14 days after CCE (number of replicates,
n = 3 for each condition). For viability assessment, cells were col-
lected at each time point and a Live/Dead viability/cytotoxicity kit
(Molecular Probes, Invitrogen) was used following manufacturers
protocol. Hoechst (Molecular Probes, Invitrogen) was used as nu-
clear counterstain. Images were acquired with a Leica SP5 con-
focal microscope. Maximum projections of z-stacks were gener-
ated with Leica Application Suite (LAS) software. For quantitative
assessment of hREC proliferation MTT (3-(4,5-Dimethylthiazol-2-
yl)2,5-diphenyltetrazolium bromide) assay was performed (Cell
Titer 96 non-radioactive cell proliferation assay kit, Promega) ac-
cording to manufacturers protocol. Viability and proliferation data
were normalized to total DNA content. Briey, hREC monolayers
were scraped from the culture dish while 3D aggregates (naked
and encapsulated spheroids) were collected and spun. Cell pellets
were washed with HBSS before being resuspended in Phosphate-
Buffered Saline (PBS, Invitrogen). DNA was extracted with the QI-
Aamp DNA Mini kit (Qiagen, Germantown, MD) according to man-
ufacturers protocol. DNA content was quantied with a NanoDrop.
Please cite this article as: V. Manzoli et al., Engineering human renal epithelial cells for transplantation in regenerative medicine, Medical
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3
2.7. Characterization of trophic factor secretion
2D, 3D naked and encapsulated spheroids of hRECs (both GFP
and GFP+) were cultured for 14 days and trophic factor produc-
tion was assessed at 1, 7 and 14 days after CCE. Twenty-four
hours before assessment, cell culture media was replaced with
2 ml/well basal media. The SearchLight Custom Chemiluminiscent
Arrays (Aushon) was used for quantication of secretion of hu-
man matrix metalloproteinases-2 (MMP-2), human basic brob-
last growth factor (FGF)-b, hepatocyte growth factor (HGF), trans-
forming growth factor (TGF)- , tissue inhibitor of metallopro-
teinase (TIMP)1 and 2 and vascular endothelial growth factor
(VEGF). Luminex ELISA (EMD Millipore) was used for quantication
of secretion of epidermal growth factor (EGF), FGFb, granulocyte
macrophage colony-stimulating factor (GM-CSF), monocyte chemo-
tactic protein-1 (MCP-1), plasminogen activator inhibitor (PAI)1,
vascular cell adhesion molecule (VCAM)1, stem cell factor (SCF).
Secretion data were normalized to DNA content.
3. Statistics
Unless otherwise noted, data are presented as means stan-
dard deviations (SD). Statistical comparisons were based on Stu-
dents t-test or analysis of variance (ANOVA) with Tukey post
hoc test for pairwise comparisons or two-way repeated measures
(mixed model) ANOVA with matched values for each time point
and Bonferroni post-tests to compare replicate means of each
group to each other. A condence level of 95% was considered sig-
nicant. Prism 5.0 for Macintosh software (Graphpad) was used for
analysis.
4. Results
4.1. Optimization and characterization of 3D spheroids of hRECs
Our rst goal was to optimize the protocol for aggregating
hRECs in 3D spheroids with a target average radius between 50
and 75 m (max diameter: 150 m). Suspension culture of hRECs
in non-tissue culture-treated plates in presence of orbital shaking
did not allow successful generation of hREC spheroids. In an alter-
native protocol, we plated hRECs in non-tissue culture-treated and
0.5% pHEMA-coated 6-well plates in absence of any shaking and
observed ecient formation of hREC aggregates.
Then we characterized the kinetic of hREC spheroid formation
after seeding the cells at different densities (0.2 106 , 0.5 106
and 0.9 106 cells/well, respectively) at selected time points
(namely, 2, 24 and 48 h). As expected, hREC spheroid size increased
with culture time (Fig. 1A and B). We found that within 48 h
of culture only seeding densities of 0.5 and 0.9 106 cells/well
gave rise to spheroids with the desired 5075 m radius (diame-
ter: 100150 m), which was larger than the one of spheroids ob-
tained from a 0.2 106 cell/well starting seeding density (Fig. 1B,
p< 0.05). We also found that spheroid growth throughout the cul-
ture well was more uniform (average spheroid radius increasing
proportionally to the culture time) when the cells were seeded at
0.5 106 cells/well than at 0.9 106 . Despite higher seeding den-
sity, the size of spheroids formed by seeding the cells at 0.9 106
cells/well after 24- and 48-h of culture was comparable to the size
of spheroids obtained by seeding the cells at 0.5 106 cells/well
the radius did not increase proportionally with culture time
(Fig. 1B). Spheroid average radius was normally distributed for
0.9 106 cells/well at 2, 24 and 48 h of culture and for 0.5 106
cells/well at 24 and 48 h. Conversely, at initial seeding density of
0.2 106 cells/well, spheroid size never achieved a normal distri-
bution. Therefore, we concluded that the optimal seeding density
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Fig. 1. Optimization of scaffold-less 3D culture of hRECs. cell aggregation into spheroids. (A) Phase contrast microscope images of hRECs seeded at 0.2, 0.5 or 0.9 x 106
cells/well on pHEMA-coated non tissue culture-treated plates and analyzed 2, 24, and 48 h after seeding. Scale bar: 100 m. (B,C) size quantication of spheroids formed
after hRECs culture on pHEMA-coated non tissue culture-treated plates 2, 24, and 48 h after seeding. Radia were extrapolated from a sphere with equivalent volume of the
measured spheroid. Average (B) and histogram (C) of spheroid radius for each seeding density and culture time are shown. 2-hr: 23 10 m for 0.2x106 cells/well, 42
19 m for 0.5x106 cells/well, and 51 19 m for 0.9x106 cells/well; 24-hrs: 35 19 m for 0.2x106 cells/well, 47 23 m for 0.5x106 cells/well, and 50 18 m for 0.9x106
cells/well; 48-hrs: 42 22 m for 0.2x106 cells/well, 63 25 m for 0.5x106 cells/well, and 59 22 m for 0.9x106 cells/well.

Please cite this article as: V. Manzoli et al., Engineering human renal epithelial cells for transplantation in regenerative medicine, Medical
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V. Manzoli et al. / Medical Engineering and Physics 000 (2017) 111 5

Fig. 2. Encapsulation and viability/proliferation of hREC 3D spheroids. (A, B) Images of 3D naked (A) and 3D encapsulated (B, through conformal coating with PEG ALG: 3D
CCE) spheroids 72 h after seeding. Scale bar: 100 m. C: Confocal imaging of 2D hRECs vs. 3D naked vs. 3D encapsulated (CCE) spheroids at day 1, 7 and 14 h after encap-
sulation stained with a viability (live, green) and an apoptotic (dead, red) marker. Hoechst (blue) was used as nuclear counterstain. Scale bar: 100 m. (D, E) Quantication
of proliferation of hRECs cultured in 2D, 3D naked, or 3D encapsulated (CCE) spheroids as a function of culture time (day 1, 7 and 14 h after encapsulation) (D) and as
a function of culture method (2D hRECs vs. 3D naked vs. 3D CCE) for each time point as assessed by MTT assay and normalized to total DNA. (For interpretation of the
references to color in this gure legend, the reader is referred to the web version of this article.)

to generate spheroids with average radius in the 5075 m ranges
within 48 h of culture time was 0.5 106 cells/well (Fig. 1C).
4.2. Conformal coating encapsulation of 3D hREC spheroids
hREC spheroids underwent CCE through our microuidic device
with PEG-ALG hydrogels. Through phase contrast microscopy,
we conrmed that spheroids were completely coated with a
very thin layer (a few tens of microns) of PEG-ALG hydrogel
and irregularities of spheroid shapes (Fig. 2A) did not affect
coating completeness (Fig. 2B). Viability of encapsulated hREC
3D spheroids cultured for 1, 7 and 14 days after encapsulation
was comparable to 3D naked spheroids and to cells cultured
in 2D monolayers (Fig. 2C). No central necrosis was present in
encapsulated cells, even in the largest aggregates (radius up to
100 m, Fig. 2C), suggesting that polymer coating did not impair
diffusion of oxygen and nutrients through the capsule.
Proliferation of hRECs cultured in a 2D monolayer (2D hREC)
gradually decreased during culture up to 14 days (Fig. 2D). Cul-
ture of hRECs in 3D naked spheroids preserved the cell prolifera-
tive capacity during culture up to 14 days; at day 7 and 14 naked
3D cultures showed higher proliferation capacity of hRECs than 2D
(Figs. 2D and E). Though at day 1 after CCE proliferation of 3D en-
capsulated hRECs was lower than 2D and 3D naked hREC cells, at
day 7 and day 14 this was completely reversed and encapsulated
cells showed higher proliferation than both 2D and 3D naked cells
(Fig. 2E). These data suggest that: (i) culture of hRECs in 3D pre-
vents the decrease in cell proliferation that is associated with long-

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term culture of hRECs ex vivo in 2D monolayers; and (ii) CCE of 3D
hRECs further improves cell proliferation during long-term culture.
4.3. Trophic factor secretion of 2D, 3D naked and 3D encapsulated
hRECs
Next, we quantied the secretion of trophic factors that are
important for cell growth and survival, of factors that have
anti-apoptotic and pro-angiogenic potential and of factors with
immunomodulation properties. Secretion from hRECs cultured in
a 2D monolayer was compared to 3D naked and 3D encapsulated
spheroids for up to 14 days (Fig. 3). hRECs cultured in 2D secrete
negligible (<2 (pg/ml)/ng of DNA) amounts of FGFb, HGF, TGF, EGF,
GM-CSF, and SCF at all time points analyzed (Fig. 3AF). Secretion
of TIMP-1, TIMP-2 and PAI-1 decreases with culture time, while
secretion of MMP-2, VCAM-1, MCP-1, and VEGF are not statistically
different at all time points (Fig. 3GM).
hRECs cultured in 3D also secrete negligible amounts of FGFb,
HGF, TGF , EGF, GM-CSF, and SCF at all time points (Fig. 3A
F). Secretion of TIMP-1 and TIMP-2 decreases with culture time
while secretion of PAI-1, MMP-2, VCAM-1, MCP-1, and VEGF
are not different at all time points (Fig. 3GM). When com-
pared to culture of hRECs in 2D, culture in 3D spheroids in-
creases secretion of SCF at day 1, 7 and 14 (Fig. 3F), of TIMP-
1 at day 1 and day 7 (Fig. 3G), of VCAM-1 at day 7 (Fig. 3K),
of MCP-1 at day 7 and 14 (Fig. 3L), and decreases secretion of PAI-1
at day 1 (Fig. 3I).
CCE hRECs cultured in 3D also secrete negligible amounts of
HGF, GM-CSF and SCF at all time points (Fig. 3B, E and F). Secretion
of FGFb and TGF is very low (<2 (pg/ml)/ng of DNA) at day 1 and
14 and it peaks [<10 (pg/ml)/ng of DNA] at day 7 after encapsula-
tion (Fig. 3A and C). Secretion of EGF, TIMP-1, TIMP-2, PAI-1, MMP-
2, VCAM-1, MCP-1, and VEGF peaks at day 7 before decreasing to
baseline levels, except for TIMP-1 and MCP-1 that remain higher
than baseline (day 1) (Fig. 3D, GM). When compared to culture
of hRECs in 2D and 3D naked spheroids, culture of CCE 3D clus-
ters increases secretion of FGFb, EGF, GM-CSF, VCAM-1, and VEGF
at day 7 (Fig. 3A, D, E, K, and M), of TGF , SCF, TIMP-2, MMP-2,
and MCP-1, at day 7 and 14 (Fig. 3C, F, H, J and L), and of TIMP-
1 at day 1, 7 and 14 (Fig. 3G). Similarly to the 3D naked, culture
in 3D capsules reduces day 1 secretion of PAI-1, while secretion at
day 7 peaks compared to both 2D and 3D naked, before returning
to baseline values at day 14 (Fig. 3I).
4.4. Gene transduction and encapsulation of hRECs
GFP was chosen as model transgene for assessing the effect of
gene transduction of hRECs on cell growth in 3D spheroids, en-
capsulation eciency, viability and trophic factor secretion dur-
ing long-term culture in vitro. No difference in size of GFP+ cells
compared to GFP- spheroid was found at all time points analyzed:
2, 24, and 48 h (Fig. 4A and B, p>0.05). Thickness and complete-
ness of CCE of GFP+ spheroids were equivalent to what we ob-
served for GFP- cells as we assessed by analysis of phase contrast
(Fig. 4C) and uorescence (Fig. 4E) microscopy images.
Viability of GFP+ hRECs cultured in 2D, 3D naked and 3D en-
capsulated spheroids at 1, 7 and 14 days after CCE was comparable
and as high as we observed for GFP- cells, including absence of
central necrosis (Fig. 4F). Proliferation of GFP+ cultured in 2D was
similar to GFP- cells (Fig. 4G). Proliferation of hRECs cultured in
3D spheroids was comparable to 2D cells at day 1 and day 7 but
it was higher at day 14 as quantied by MTT assay normalized to
total DNA (Fig. 4H). Proliferation of GFP+ 3D encapsulated hRECs
was lower than 2D cells at day 1, but comparable to 2D and 3D
naked cells at day 7 and to 2D cells at day 14 (Fig. 4H).
Secretion of trophic factors was moderately affected by GFP
gene transduction (Fig. 5A). GFP+ hRECs cultured in 2D, 3D and
3D conformal capsules secrete negligible [<2 (pg/ml)/ng of DNA]
amounts of FGFb, HGF, TGF , GM-CSF, and SCF at all time points
analyzed (Fig. 5BF) similarly to GFP- cells (Fig. 3).
When compared to culture of GFP+ hRECs in 2D, culture in
3D spheroids increases secretion of FGFb, GM-CSF, SCF, TIMP-1 and
VCAM-1 at day 1, 7 and 14 (Fig. 5B, E, F, H and I, p< 0.05), of TIMP-
2 and VEGF at day 14 (Fig. 5J and K, p< 0.05), of EGF at day 1 and
14 (Fig. S2K, p< 0.05) and decreases secretion of PAI-1 at day 1, 7
and 14 (Fig. 5M, p< 0.05).
Culture in 3D conformal capsules increases secretion of FGFb at
day 1 compared to 2D and 3D naked and at day 7 and 14 when
compared to 3D naked (Fig. 5B, p<0.05). Secretion of TGF is in-
creased at day 1, 7 and 14 when compared to 2D and 3D naked
(Fig. 5D, p<0.05). Secretion of GM-CSF is comparable to 2D and
lower than 3D naked at day 1 and day 7, whereas it is higher than
2D but lower than 3D naked at day 14 (Fig. 5E, p<0.05). Secretion
of SCF is higher than 2D but lower than 3D naked at day 1, 7, and
14 (Fig. 5F, p<0.05). Secretion of MMP-2 is increased at day 1 when
compared to 2D and 3D naked, while it is higher than 3D naked at
day 7 (Fig. 5G, p<0.05). Secretion of TIMP-1 is increased at day 1
when compared to 2D but it is lower than 3D naked, while it is
higher than 2D and comparable to 3D naked at day 7 and day 14
(Fig. 5H, p<0.05). Secretion of VCAM-1 is comparable to 2D but
lower than 3D naked at day 1 and 7 and higher than 2D but lower
than 3D naked at day 14 (Fig. 5I, p<0.05). Secretion of VEGF is in-
creased at day 1 when compared to 2D and 3D naked, while it
is comparable to 2D and lower than 3D naked at day 14 (Fig. 5K,
p < 0.05). Secretion of EGF is higher than 2D and 3D naked at day
1, higher than 2D but comparable to 3D naked at day 7 and higher
than 2D but lower than 3D naked at day 14 (Fig. 5L, p < 0.05). Se-
cretion of PAI-1 is decreased at day 1, 7 and 14 when compared to
2D but higher than 3D naked (Fig. 5M, p < 0.05).
5. Discussion
The end goal of regenerative medicine is treatment of dis-
eases either by cell replacement of injured tissue and/or by pro-
moting host tissue regeneration through the local release of re-
generative factors. In this in vitro study, we aimed at assessing
the effects of engineering cells through our microuidic confor-
mal coating platform, which could enable transplantation without
immunosuppression and through genetic engineering, which could
enable local delivery of regenerative factors at the cell transplant
site.
As cell model we chose hRECs, which might be transplanted
in the subrenal capsule for treatment of kidney diseases. The
rationale for transplanting hRECs aggregated in 3D spheroids is
based on previous studies demonstrating that 3D culture could al-
low hRECs to maintain a more in vivo-like morphology and func-
tion than cells cultured in conventional 2D conditions [5658]. This
will likely maximize their regenerative potential after transplanta-
tion. Standard 3D cell culture requires suspension of hRECs in an
extracellular matrix scaffold. We decided to develop and optimize
a new protocol for culture of hRECs in scaffold-less 3D conditions
through spheroid formation. The absence of a supporting scaffold
will minimize the size of cell aggregates (spheroids) to an average
diameter of 100150 m. This may allow local injection of aggre-
gates and more even distribution, simplifying the transplantation
procedure. It also allows encapsulation of single aggregates, min-
imizing the size of immunoisolating devices and allowing trans-
plantation in sites like the subrenal capsule. Absence of supporting
scaffolds might as well allow easier regulatory procedures for clin-
ical translation.

Please cite this article as: V. Manzoli et al., Engineering human renal epithelial cells for transplantation in regenerative medicine, Medical
Engineering and Physics (2017), http://dx.doi.org/10.1016/j.medengphy.2017.03.009
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V. Manzoli et al. / Medical Engineering and Physics 000 (2017) 111 7

Fig. 3. Trophic factor secretion of hRECs cultured in 2D, 3D naked and 3D encapsulated as a function of culture time and culture condition. Multiplex ELISA on supernatant
from in vitro culture of 2D hREC passage 6, 3D hREC naked and 3D hREC encapsulated (through conformal coating: 3D CCE) spheroids as a function of culture time: day 1,
7, and 14 after encapsulation (left) and as a function of culture conditions (right). Trophic factor concentrations in the supernatant (pg/ml) have been normalized to total
DNA (ng). SearchLight Custom Chemiluminiscent array was used for quantication of secretion of human FGF-b (A), HGF (B), TGF (C), TIMP-1 (G), TIMP-2 (H), MMP-2 (J),
VEGF (M). Luminex ELISA was used for quantication of secretion of EGF (D), GM-CSF (E), SCF (F), PAI-1 (I), VCAM-1 (K), MCP-1 (L). = p < 0.01; = p < 0.05. n= 3. Two-way
repeated measures (mixed model) ANOVA with matched values for each time point and Bonferroni post-tests to compare replicate means of each group to each other groups
was used for statistical analysis.

Please cite this article as: V. Manzoli et al., Engineering human renal epithelial cells for transplantation in regenerative medicine, Medical
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8 V. Manzoli et al. / Medical Engineering and Physics 000 (2017) 111

Fig. 4. Gene transduction and encapsulation of hRECs. (A) Phase contrast microscope images of hRECs transduced with GFP (GFP+) vs. control non transduced cells (GFP-),
seeded at 0.5 106 cells/well on pHEMA-coated non tissue culture-treated plates and analyzed 2, 24, and 48 h after seeding. Scale bar: 100 m. (B) spheroid size quanti-
cation after culture of GFP+ vs. GFP- hRECs on pHEMA-coated non tissue culture-treated plates as a function of culture time (2, 24, and 48 h after seeding). Radia were
extrapolated from a sphere with volume equivalent to the measured spheroid. (C, D) Images of GFP+ 3D naked (C) and 3D encapsulated (D, through conformal coating
with PEG ALG: 3D CCE) spheroids 72 h after seeding. Scale bar: 100 m. (E) Fluorescence microscope image of GFP+ hREC spheroid (uorescent green) encapsulated with
PEG-ALG (CCE, phase contrast). Scale bar: 50 m (F) Confocal imaging of GFP+ 2D hRECs vs. 3D naked vs. 3D encapsulated (CCE) spheroids at day 1, 7 and 14 h after encap-
sulation stained with a viability (live, green) and an apoptotic (dead, red) marker. Hoechst (blue) was used as nuclear counterstain. Scale bar: 100 m. (G) Quantication of
proliferation of GFP+ vs. GFP- hRECs cultured in 2D for each time point (1, 7, 14 days culture time) as assessed by MTT assay normalized to total DNA. (H) Quantication
of proliferation of hRECs as a function of culture method (2D hRECs vs. 3D naked vs. 3D encapsulated spheroids: CCE) for each time point (1, 7, 14 days culture time) as
assessed by MTT assay normalized to total DNA. (For interpretation of the references to color in this gure legend, the reader is referred to the web version of this article.)

Since traditional methods were not successful for generating 3D
spheroids, we exploited culture on dishes with surface-anchored
polymer layers of pHEMA, which completely blocks cell adhe-
sion and encourages cells to aggregate and proliferate within 3D
clusters [59]. Optimization was focused on generating spheroids
of hRECs with a 100150 m diameter. We aimed at maximizing
cell-cell interactions by increasing size of aggregates, without
imposing diffusion limitations that may lead to hypoxia of core
cells and spheroid central necrosis (diameter < 150 m and radius
< 75 m) [4750]. We show that culture of hRECs in 3D spheroids
counteracts the loss in viability and proliferation that we observed
during long-term culture of hRECs in 2D standard culture con-

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Fig. 5. Trophic factor secretion of GFP hRECs at 1, 7, and 14 days after culture in 2D, 3D naked and 3D encapsulated (3D CCE) as a function of culture condition. Multi-
plex ELISA on supernatants from in vitro culture of passage 6 GFP+ 2D hRECs normalized to GFP- cells at day 1 after culture (A) and of 2D hRECs, 3D hREC naked and
3D hREC encapsulated (CCE) spheroids at day 1, 7, and 14 after encapsulation as a function of culture condition (2D vs. 3D naked vs. 3D encapsulated). MMP-2 (1.58 0.05
times, p < 0.05), TIMP-1 (0.49 0.03 times, p < 0.05), TIMP-2 (0.42 0.13 times, p < 0.05), VEGF (0.59 0.12 times, p < 0.05), PAI-1 (0.56 0.04 times, p < 0.05), VCAM-
1 (0.23 0.02 times, p < 0.05), EGF (0.36 0.03 times, p < 0.05), GM-CSF (0.094 0.005 times, p < 0.05), SCF (0.623 0.038 times, p < 0.05), HGF (0.564 0.156 times,
p < 0.05), and TGF (0.208 0.026 times, p < 0.05). (BM). Trophic factor concentrations in the supernatant (pg/ml) was normalized to total DNA (ng) (BM). SearchLight
Custom Chemiluminiscent arrays was used for quantication of secretion of human FGF-b (B), HGF (C), TGF (D), MMP-2 (G), TIMP-1 (H), TIMP-2 (J), VEGF (K). Luminex
ELISA was used for quantication of secretion of PAI-1 (M), VCAM-1 (I), EGF (L), GM-CSF (E), SCF (F). = p < 0.01; = p < 0.05. n= 3. Two-way repeated measures (mixed
model) ANOVA with matched values for each time point and Bonferroni post-tests to compare replicate means of each group to each other groups was used for statistical
analysis.

ditions and allows preserving hRECs viability and proliferative
capacity during culture up to 14 days, conrming our hypothesis
and consistently with previous ndings.
We also evaluated the effects of immunoisolation of hRECs
through CCE using PEG-ALG hydrogels on cell viability, prolifer-
ation and trophic factor secretion. The rationale for immunoiso-
lation of hRECs is twofold. First, encapsulated cell clusters will
maintain their 3D integrity even after transplantation. Therefore,
cells in a persistent 3D conguration may receive extracellular
signals that could render them more ecient in secreting trophic
factors that have a benecial effect on tissue regeneration, includ-
ing cell recruitment, survival, proliferation and tissue remodeling.
Second, the polymeric hydrogel coating would provide shielding
from immune cells and prolong survival of implanted cells even
in fully major histocompatibility complex-mismatched recipients.
We chose CCE because this microuidic method of encapsulation
that we designed minimizes capsule thickness [51]. This in turn
minimizes diffusion barriers for oxygen, nutrients and trophic
factors and the total volume of non-biological capsule material.
Furthermore, smaller capsules will minimize the size of the cell
graft allowing transplantation in a variety of sites, including the
subrenal capsule. We found that encapsulation does not impair
hREC function during long-term culture. Conceivably, CCE will
likely not impact the in vivo regenerative potential of hRECs after
transplantation in sites of injury. More importantly, we found that
combining CCE and 3D culture of hRECs improves cell prolifera-
tion, likely by enhancing cell-cell interactions. This phenomenon is
consistent with the improved survival and performance reported
after encapsulation also for other type of cell clusters [60].
For evaluation of the effects of encapsulation and genetic en-
gineering of hRECs, we focused on trophic factors that are im-
portant for kidney repair. Among those, MMP-2, TIMP-1 and
TIMP-2 are important during acute kidney injury, glomeruloscle-
rosis/tubulointerstitial brosis, chronic allograft nephropathy, dia-
betic nephropathy [61], polycystic kidney disease, and renal cell
carcinoma [62]. 3D culture of hRECs increases secretion of TIMP-
1 when compared to 2D, and encapsulation of 3D cells overall
increases secretion of TIMP-1, TIMP-2 and MMP-2 with peaks at
day 7 after encapsulation. These results suggest that 3D culture
and CCE may have a more profound effect on tissue remodeling
after transplantation and this could be exploited therapeutically.
Secretion of VEGF, which is important for revascularization after
transplant, is constant over culture time and comparable between
all culture conditions analyzed, suggesting similar pro-angiogenic
potential. PAI-1 is a determinant of glomerular brin deposition
and renal injury [63]. Therefore, the decrease in PAI-1 secretion

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Engineering and Physics (2017), http://dx.doi.org/10.1016/j.medengphy.2017.03.009
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10 V. Manzoli et al. / Medical Engineering and Physics 000 (2017) 111
that we observed for 2D cells during culture up to 14 days and
the reduced expression in 3D and 3D encapsulated cells at 14 days
may be benecial for preventing brosis after transplantation of
engineered hRECs. VCAM-1 is upregulated during kidney inam-
mation [64] and may be deleterious after transplant because it
may worsen the inammatory response. It is secreted in compa-
rable low amounts in 2D, 3D naked and 3D encapsulated cells. SCF
has been shown to protect against apoptosis after renal injury [65].
We observed that SCF is secreted in negligible amounts by hRECs,
with higher values for 3D and 3D encapsulated cells suggesting
that the therapeutic potential of SCF can be exploited by 3D cul-
ture and encapsulation. We also found that MCP-1, which plays a
role in the pathogenesis and progression of renal failure [66] is
expressed in lower amounts by 2D hRECs. Furthermore, we found
that GM-CSF, which has been found to be upregulated in renal in-
terstitial brosis, is expressed in negligible amount by 2D hRECs
and it decreases during culture [67]. It was shown that intersti-
tial FGFb protein and mRNA expression correlate with scarring
[68]. Therefore, our ndings that FGFb is expressed in negligible
amounts in 2D and 3D cells, and 3D encapsulated cells further sup-
port a benecial effect of our engineered cells for renal tissue engi-
neering. Similarly, expression of negligible amounts of HGF that we
found for all conditions analyzed may be benecial because HGF
disrupts the polarity of renal epithelial cells in vitro [69]. Our re-
sults that show negligible secretion of TGF and EGF for 2D and
3D naked hRECs are in agreement with previous ndings [70].
Overall, we observed that encapsulated 3D cells display a peak
of expression of trophic factors at day 7, which returns to baseline
by 14 days. This may be exploited for cell-based therapy in renal
tissue engineering by timing their transplant accordingly.
Finally, we have shown that hRECs can be eciently transduced
with a gene of interest and encapsulated through conformal coat-
ing after gene transduction without affecting cell viability and pro-
liferation. We chose GFP as gene of interest and lentiviruses as
gene transduction vectors for their proven eciency in transduc-
ing most primary cells. We found that secretion of trophic factors
from GFP+ cells is moderately reduced when compared to GFP-
for most factors analyzed and the effect of 3D culture and encap-
sulation on trophic factor secretion is partially different than what
we observed for GFP- cells. This suggests that gene transduction
may affect the secretory pattern of hRECs and may be designed
and exploited for local delivery of therapeutic agents after trans-
plantation.
Collectively, our data support the feasibility of engineering
hRECs for regenerative and reparative medicine. We successfully
created 3D clusters from hREC with standardized size, encapsu-
lated them through the CCE protocol that we previously designed
and genetically modied them. The entire process did not affect,
and in certain cases even ameliorated, the proliferative potential
and the secretory properties of hRECs. This approach may there-
fore represent one step forward in the translation of the use of
hREC transplantation for the treatment of growing epidemic of kid-
ney diseases. Providing isolated cells and cell clusters with their
own extracellular matrix (i.e. renal ECM to renal epithelial cells)
has demonstrated to be a powerful approach to improve viability
and functionality of both renal cells [71,72] and other cells, includ-
ing pancreatic islets [73,74]. Therefore, our approach for engineer-
ing renal epithelial cells can be further rened by incorporating
renal ECM to conformal capsules.
Disclosure
A.A.T. is co-inventor of intellectual property used in the study
and may gain royalties from future commercialization of the
technology. C.R. is Advisory Board Member and stakeholder, A.P.,
A.A.T. and V.M. are stock holders of Converge Biotech Inc. that
Please cite this article as: V. Manzoli et al., Engineering human renal epithelial cells for transplantation in regenerative medicine, Medical
Engineering and Physics (2017), http://dx.doi.org/10.1016/j.medengphy.2017.03.009
[m5G;April 12, 2017;8:30]
is the exclusive licensee of the intellectual property used for cell
encapsulation.
Disclaimer
This article was prepared while Dr. Pileggi was employed at
the University of Miami. He is currently employed at Center for
Scientic Review of the National Institutes of Health. The opinions
expressed in this article are the authors own and do not neces-
sarily reect the views of the National Institutes of Health, the
Department of Health and Human Services, or the United States
government.
Conict of interest
No conict of interest.
Acknowledgments
The authors are grateful to the members of the Diabetes Re-
search Institute and University of Miami Sylvester Comprehensive
Cancer Center Imaging and Flow Cytometry Cores, to the Univer-
sity of Miami Viral Vector Core, to Karen Newman for help with
some technical aspects of the project and to Dr. Christopher A.
Fraker for discussion and support. Funding was provided by ATRM,
the Juvenile Diabetes Research Foundation and the Leona M. and
Harry B. Helmsley charitable trust (Grant 17-2010-5) and in part
through philanthropic funds from the Diabetes Research Institute
Foundation. AF was supported by R01DK104753 and R01DK090316.
References
[1] Consolo F, Bariani C, Mantalaris A, Montevecchi F, Redaelli A, Morbiducci U.
Computational modeling for the optimization of a cardiogenic 3D biopro-
cess of encapsulated embryonic stem cells. Biomech Model Mechanobiol
2012;11:26177.
[2] Minguez-Castellanos A, Escamilla-Sevilla F, Hotton GR, Toledo-Aral JJ, Ortega
Moreno A, Mendez-Ferrer S, et al. Carotid body autotransplantation in Parkin-
son disease: a clinical and positron emission tomography study. J Neurol Neu-
rosurg Psychiatry 2007;78:82531.
[3] Chang PL, Van Raamsdonk JM, Hortelano G, Barsoum SC, MacDonald NC, Stock-
ley TL. The in vivo delivery of heterologous proteins by microencapsulated re-
combinant cells. Trends Biotechnol 1999;17:7883.
[4] Daoud J, Petropavlovskaia M, Rosenberg L, Tabrizian M. The effect of extracel-
lular matrix components on the preservation of human islet function in vitro.
Biomaterials 2010;31:167682.
[5] Meier RP, Montanari E, Morel P, Pimenta J, Schuurman HJ, Wandrey C,
et al. Microencapsulation of Hepatocytes and mesenchymal stem cells for ther-
apeutic applications. Methods Mol Biol 2017;1506:25971.
[6] Koh RH, Jin Y, Kang BJ, Hwang NS. Chondrogenically primed tonsil-derived
mesenchymal stem cells encapsulated in riboavin-induced photocrosslinking
collagen-hyaluronic acid hydrogel for meniscus tissue repairs. Acta Biomater
2017.
[7] Gomes ED, Mendes SS, Leite-Almeida H, Gimble JM, Tam RY, Shoichet MS,
et al. Combination of a peptide-modied gellan gum hydrogel with
cell therapy in a lumbar spinal cord injury animal model. Biomaterials
2016;105:3851.
[8] Zhao S, Zhang L, Han J, Chu J, Wang H, Chen X, et al. Conformal nanoencap-
sulation of allogeneic t cells mitigates graft-versus-host disease and retains
graft-versus-leukemia activity. ACS nano 2016;10:6189200.
[9] Li H, Ham TR, Neill N, Farrag M, Mohrman AE, Koenig AM, et al. A hydrogel
bridge incorporating immobilized growth factors and neural stem/progenitor
cells to treat spinal cord injury. Adv Healthcare Mater 2016;5:80212.
[10] Vegas AJ, Veiseh O, Gurtler M, Millman JR, Pagliuca FW, Bader AR,
et al. Long-term glycemic control using polymer-encapsulated human stem
cell-derived beta cells in immune-competent mice. Nat Med 2016;22:30611.
[11] Rinsch C, Dupraz P, Schneider BL, Deglon N, Maxwell PH, Ratcliffe PJ, et al. De-
livery of erythropoietin by encapsulated myoblasts in a genetic model of se-
vere anemia. Kidney Int 2002;62:1395401.
[12] McQuilling JP, Arenas-Herrera J, Childers C, Pareta RA, Khanna O, Jiang B,
et al. New alginate microcapsule system for angiogenic protein delivery and
immunoisolation of islets for transplantation in the rat omentum pouch.
Transplant Proc 2011;43:32624.
[13] Kobayashi T, Aomatsu Y, Iwata H, Kin T, Kanehiro H, Hisanga M, et al. Sur-
vival of microencapsulated islets at 400 days posttransplantation in the omen-
tal pouch of NOD mice. Cell Transplant 2006;15:35965.
JID: JJBE
ARTICLE IN PRESS
V. Manzoli et al. / Medical Engineering and Physics 000 (2017) 111
[14] Scharp DW, Marchetti P. Encapsulated islets for diabetes therapy: history,
current progress, and critical issues requiring solution. Adv Drug Deliv Rev
2014;67-68:3573.
[15] Omotoso BA, Turgut F, Abdel-Rahman EM, Xin W, Ma JZ, Scully KW, et al. Dial-
ysis requirement and long-term major adverse cardiovascular events in pa-
tients with chronic kidney disease and superimposed acute kidney injury.
Nephron 2017.
[16] Barasch J, Zager R, Bonventre JV. Acute kidney injury: a problem of denition.
Lancet 2017;389:77981.
[17] Bhatt GC, Das RR. Early versus late initiation of renal replacement therapy in
patients with acute kidney injury-a systematic review & meta-analysis of ran-
domized controlled trials. BMC Nephrol 2017;18:78.
[18] Lo C, Teede H, Fulcher G, Gallagher M, Kerr PG, Ranasinha S, et al. Gaps and
barriers in health-care provision for co-morbid diabetes and chronic kidney
disease: a cross-sectional study. BMC Nephrol 2017;18:80.
[19] Li Z, Araoka T, Wu J, Liao HK, Li M, Lazo M, et al. 3D culture supports
long-term expansion of mouse and human nephrogenic progenitors. Cell stem
cell 2016;19:51629.
[20] Takasato M, Er PX, Chiu HS, Little MH. Generation of kidney organoids from
human pluripotent stem cells. Nat Protoc 2016;11:168192.
[21] Nieskens TT, Wilmer MJ. Kidney-on-a-chip technology for renal proximal
tubule tissue reconstruction. Eur J Pharmacol 2016;790:4656.
[22] Xu Y, Shi T, Xu A, Zhang L. 3D spheroid culture enhances survival and ther-
apeutic capacities of MSCs injected into ischemic kidney. J Cell Mol Med
2016;20:120313.
[23] Batchelder CA, Martinez ML, Tarantal AF. Natural scaffolds for renal differenti-
ation of human embryonic stem cells for kidney tissue engineering. PLoS One
2015;10:e0143849.
[24] Moon KH, Ko IK, Yoo JJ, Atala A. Kidney diseases and tissue engineering. Meth-
ods 2016;99:11219.
[25] Kragl M, Lammert E. Basement membrane in pancreatic islet function. Adv Exp
Med Biol 2010;654:21734.
[26] Papas KK, Pisania A, Wu H, Weir GC, Colton CK. A stirred microchamber for
oxygen consumption rate measurements with pancreatic islets. Biotechnol Bio-
eng 2007;98:107182.
[27] Pugliese A. Advances in the etiology and mechanisms of type 1 diabetes. Disc
Med 2014;18:14150.
[28] Forlenza G.P., Rewers M.. The epidemic of type 1 diabetes: what is it telling
us? Current opinion in endocrinology, diabetes, and obesity. 2011;18:24851.
[29] Sanchez-Romero N, Schophuizen CM, Gimenez I, Masereeuw R. In vitro sys-
tems to study nephropharmacology: 2D versus 3D models. Eur J Pharmacol
2016;790:3645.
[30] Jakob PH, Kehrer J, Flood P, Wiegel C, Haselmann U, Meissner M, et al. A 3-D
cell culture system to study epithelia functions using microcarriers. Cytotech-
nology 2016;68:181325.
[31] Shen C, Zhang G, Wang Q, Meng Q. Fabrication of collagen gel hollow bers
by covalent cross-linking for construction of bioengineering renal tubules. ACS
Appl Mater Interf 2015;7:1978997.
[32] Imai M, Furusawa K, Mizutani T, Kawabata K, Haga H. Three-dimensional mor-
phogenesis of MDCK cells induced by cellular contractile forces on a viscous
substrate. Sci Rep 2015;5:14208.
[33] Huang HC, Chang YJ, Chen WC, Harn HI, Tang MJ, Wu CC. Enhancement of
renal epithelial cell functions through microuidic-based coculture with adi-
pose-derived stem cells. Tissue Eng Part A 2013;19:202434.
[34] Maahs DM, West NA, Lawrence JM, Mayer-Davis EJ. Epidemiology of type 1
diabetes. Endocrinol Metab Clin North Am 2010;39:48197.
[35] van Belle TL, Coppieters KT, von Herrath MG. Type 1 diabetes: etiology, im-
munology, and therapeutic strategies. Physiol Rev 2011;91:79118.
[36] Wilm B, Tamburrini R, Orlando G, Murray P. Autologous cells for kidney bio-
engineering. Curr Transplant Rep 2016;3:20720.
[37] George SK, Abolbashari M, Jackson JD, Aboushwareb T, Atala A, Yoo JJ. Potential
use of autologous renal cells from diseased kidneys for the treatment of renal
failure. PLoS One 2016;11:e0164997.
[38] Liu T, Zhang Y, Shen Z, Zou X, Chen X, Chen L, et al. Immunomodulatory effects
of OX40Ig gene-modied adipose tissue-derived mesenchymal stem cells on
rat kidney transplantation. Int J Mol Med 2017;39:14452.
[39] Ezzelarab MB, Raich-Regue D, Lu L, Zahorchak AF, Perez-Gutierrez A, Humar A,
et al. Renal allograft survival in nonhuman primates infused with donor anti-
gen-pulsed autologous regulatory dendritic cells. Am J Transpl 2016.
[40] Bruni A, Gala-Lopez B, Pepper AR, Abualhassan NS, Shapiro AJ. Islet cell trans-
plantation for the treatment of type 1 diabetes: recent advances and future
challenges. Diabetes, Metab Syndr Obes Targets Therapy 2014;7:21123.
[41] Lim GJ, Zare S, Van Dyke M, Atala A. Cell microencapsulation. Adv Exp Med
Biol 2010;670:12636.
[42] Schweicher J, Nyitray C, Desai TA. Membranes to achieve immunoprotection of
transplanted islets. Front Biosci (Landmark Ed) 2014;19:4976.
[43] Verhoef JJ, Carpenter JF, Anchordoquy TJ, Schellekens H. Potential induction of
anti-PEG antibodies and complement activation toward PEGylated therapeu-
tics. Drug Discov Today 2014.
[44] Ratliff BB, Ghaly T, Brudnicki P, Yasuda K, Rajdev M, Bank M, et al. Endothe-
lial progenitors encapsulated in bioarticial niches are insulated from sys-
temic cytotoxicity and are angiogenesis competent. Am J Physiol Renal Physiol
2010;299:F17886.
[45] Balyura M, Gelfgat E, Ehrhart-Bornstein M, Ludwig B, Gendler Z, Barkai U,
et al. Transplantation of bovine adrenocortical cells encapsulated in alginate.
Proc Natl Acad Sci USA 2015;112:252732.
Please cite this article as: V. Manzoli et al., Engineering human renal epithelial cells for transplantation in regenerative medicine, Medical
Engineering and Physics (2017), http://dx.doi.org/10.1016/j.medengphy.2017.03.009
[m5G;April 12, 2017;8:30]
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[46] Hashemi M, Kalalinia F. Application of encapsulation technology in stem cell
therapy. Life Sci 2015;143:13946.
[47] Buchwald P. A local glucose-and oxygen concentration-based insulin secretion
model for pancreatic islets. Theor Biol Med Model 2011;8:20.
[48] Williams SJ, Huang HH, Kover K, Moore W, Berkland C, Singh M, et al. Reduc-
tion of diffusion barriers in isolated rat islets improves survival, but not insulin
secretion or transplantation outcome. Organogenesis 2010;6:11524.
[49] Rabinovitch A, Fuller L, Mintz D, Severyn W, Noel J, Flaa C, et al. Responses of
canine lymphocytes to allogeneic and autologous Islets of Langerhans in mixed
cell cultures. J Clin Invest 1981;67:150716.
[50] Swift SM, Rose S, London NJ, James RF. Development and optimization of the
human allogeneic mixed lymphocyte islet (MLIC) and acinar (MLAC) coculture
system. Transpl Immunol 1996;4:16976.
[51] Tomei AA, Manzoli V, Fraker CA, Giraldo J, Velluto D, Najjar M, et al. Device de-
sign and materials optimization of conformal coating for islets of Langerhans.
Proc Natl Acad Sci USA 2014.
[52] Garcia AJ. PEG-maleimide hydrogels for protein and cell delivery in regenera-
tive medicine. Ann Biomed Eng 2014;42:31222.
[53] Fraker CA, Alvarez S, Papadopoulos P, Giraldo J, Gu W, Ricordi C, et al. En-
hanced oxygenation promotes beta-cell differentiation in vitro. Stem Cells
2007;25:315564.
[54] Pelosi A, Sheriff J, Stevanella M, Fiore GB, Bluestein D, Redaelli A. Compu-
tational evaluation of the thrombogenic potential of a hollow-ber oxygena-
tor with integrated heat exchanger during extracorporeal circulation. Biomech
Model Mechanobiol 2014;13:34961.
[55] Cechin S, Alvarez-Cubela S, Giraldo JA, Molano RD, Villate S, Ricordi C, et al. In-
uence of in vitro and in vivo oxygen modulation on beta cell differen-
tiation from human embryonic stem cells. Stem Cells Trans Med
2014;3:27789.
[56] Gildea JJ, McGrath HE, Van Sciver RE, Wang DB, Felder RA. Isolation, growth,
and characterization of human renal epithelial cells using traditional and 3D
methods. Methods Mol Biol 2013;945:32945.
[57] Grith LG, Swartz MA. Capturing complex 3D tissue physiology in vitro. Nat
Rev Mol Cell Biol 2006;7:21124.
[58] Mettler E, Trenkler A, Feilen PJ, Wiegand F, Fottner C, Ehrhart F, et al. Magnetic
separation of encapsulated islet cells labeled with superparamagnetic iron ox-
ide nano particles. Xenotransplantation 2013;20:21926.
[59] Korpos E, Kadri N, Kappelhoff R, Wegner J, Overall CM, Weber E, et al. The
peri-islet basement membrane, a barrier to inltrating leukocytes in type 1
diabetes in mouse and human. Diabetes 2013;62:53142.
[60] Kobayashi T, Arefanian H, Harb G, Tredget EB, Rajotte RV, Korbutt GS,
et al. Prolonged survival of microencapsulated neonatal porcine islet
xenografts in immune-competent mice without antirejection therapy. Cell
Transplant 2008;17:124356.
[61] Tzakis AG, Ricordi C, Alejandro R, Zeng Y, Fung JJ, Todo S, et al. Pancreatic
islet transplantation after upper abdominal exenteration and liver replace-
ment. Lancet 1990;336:4025.
[62] Dolgin E. Encapsulate this. Nat Med 2014;20:911.
[63] Pedraza E, Coronel MM, Fraker CA, Ricordi C, Stabler CL. Preventing hypoxi-
a-induced cell death in beta cells and islets via hydrolytically activated, oxy-
gen-generating biomaterials. Proc Natl Acad Sci USA 2012;109:424550.
[64] Fraker CA, Mendez AJ, Stabler CL. Complementary methods for the determi-
nation of dissolved oxygen content in peruorocarbon emulsions and other
solutions. J Phys Chem B 2011;115:1054752.
[65] Calaore R, Basta G. Clinical application of microencapsulated islets: actual
prospectives on progress and challenges. Adv Drug Deliv Rev 2013.
[66] Viedt C., Orth S.R.. Monocyte chemoattractant protein-1 (MCP-1) in the kid-
ney: does it more than simply attract monocytes? Nephrol Dial Transplant.
2002;17:20437.
[67] Frank J, Engler-Blum G, Rodemann HP, Muller GA. Human renal tubular cells
as a cytokine source: PDGF-B, GM-CSF and IL-6 mRNA expression in vitro. Exp
Nephrol 1993;1:2635.
[68] Gattas-Asfura KM, Stabler CL. Bioorthogonal layer-by-layer encapsulation
of pancreatic islets via hyperbranched polymers. ACS Appl Mater Interf
2013;5:996474.
[69] Rokstad AM, Lacik I, de Vos P, Strand BL. Advances in biocompatibility and
physico-chemical characterization of microspheres for cell encapsulation. Adv
Drug Deliv Rev 2013.
[70] Darrabie MD, Kendall WF Jr, Opara EC. Characteristics of
Poly-l-Ornithine-coated alginate microcapsules. Biomaterials
2005;26:684652.
[71] Petrosyan A, Zanusso I, Lavarreda-Pearce M, Leslie S, Sedrakyan S, De Fil-
ippo RE, et al. Decellularized renal matrix and regenerative medicine of the
kidney: a different point of view. Tissue Eng Part B, Rev 2016;22:18392.
[72] Katari R, Peloso A, Zambon JP, Soker S, Stratta RJ, Atala A, et al. Renal bioengi-
neering with scaffolds generated from human kidneys. Nephron Exper Nephrol
2014;126:119.
[73] Chaimov D, Baruch L, Krishtul S, Meivar-Levy I, Ferber S, Machluf M. Innova-
tive encapsulation platform based on pancreatic extracellular matrix achieve
substantial insulin delivery. J Control Release 2016. doi:10.1016/j.jconrel.2016.
07.045.
[74] Peloso A, Urbani L, Cravedi P, Katari R, Maghsoudlou P, Fallas ME, et al. The
Human Pancreas as a Source of Protolerogenic Extracellular Matrix Scaffold for
a New-generation Bioarticial Endocrine Pancreas. Ann Surg 2016;264:16979.