Biofabrication of a vascularized islet organ for type 1 diabetes

Accepted Manuscript Biofabrication of a vascularized islet organ for type 1 diabetes A. Citro, P.T. Moser, E. Dugnani, T.K. Rajab, X. Ren, D. Evangelista-Leite, J.M. Charest, A. Peloso, B.K. Podesser, F. Manenti, S. Pellegrini, L. Piemonti, H.C. Ott PII:

S0142-9612(19)30045-6

DOI:

https://doi.org/10.1016/j.biomaterials.2019.01.035

Reference:

JBMT 19070

To appear in:

Biomaterials

Received Date: 26 September 2018 Revised Date:

27 November 2018

Accepted Date: 18 January 2019

Please cite this article as: Citro A, Moser PT, Dugnani E, Rajab TK, Ren X, Evangelista-Leite D, Charest JM, Peloso A, Podesser BK, Manenti F, Pellegrini S, Piemonti L, Ott HC, Biofabrication of a vascularized islet organ for type 1 diabetes, Biomaterials (2019), doi: https://doi.org/10.1016/ j.biomaterials.2019.01.035. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

Title:

Biofabrication of a Vascularized Islet Organ for Type 1 Diabetes

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Authors:

† equally contributed to this work as first author

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†† equally contributed to this work as last author

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A. Citro † 1,2,3, PT. Moser † 2,3,6, E. Dugnani1, TK. Rajab 2,3, X. Ren 2,3, D. EvangelistaLeite 2, JM. Charest 2, A. Peloso 7, BK. Podesser 5,6, F. Manenti1, S. Pellegrini1, L. *†† *†† 2,3,4 Piemonti 1, HC. Ott

Affiliations: 1

San Raffaele Diabetes Research Institute, IRCCS San Raffaele Scientific Institute, Milan, Italy. Center for Regenerative Medicine, Massachusetts General Hospital, Boston, Massachusetts, USA. 3 Harvard Medical School, Boston, Massachusetts, USA. 4 Division of Thoracic Surgery, Department of Surgery, Massachusetts General Hospital, Boston, Massachusetts, USA. 5 Head Center for Biomedical Research, Vienna General Hospital, Vienna, Austria. 6 Vienna Medical School, Vienna, Austria. 7 IRCCS Policlinico San Matteo, Department of Surgery, University of Pavia.

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*Correspondence to: Harald C. Ott, Massachusetts General Hospital, 185 Cambridge Street, CPZN 4700, Boston, MA 02114, [email protected] Lorenzo Piemonti, San Raffaele Diabetes Research Institute, via Olgettina 60, 20132, Milan, Italy, [email protected]

ACCEPTED MANUSCRIPT Abstract:

Islet transplantation is superior to extrinsic insulin supplementation in the treating severe Type 1 diabetes. However, its efficiency and longevity are limited by substantial islet loss post-

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transplantation due to lack of engraftment and vascular supply. To overcome these limitations, we developed a novel approach to bio-fabricate functional, vascularized islet organs (VIOs) ex vivo. We endothelialized acellular lung matrixes to provide a biocompatible multicompartment scaffold

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with an intact hierarchical vascular tree as a backbone for islet engraftment. Over seven days of culture, islets anatomically and functionally integrated into the surrounding bio-engineered

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vasculature, generating a functional perfusable endocrine organ. When exposed to supraphysiologic arterial glucose levels in vivo and ex vivo, mature VIOs responded with a physiologic insulin release from the vein and provided more efficient reduction of hyperglycemia compared to intraportally transplanted fresh islets. In long-term transplants in diabetic mice, subcutaneously implanted VIOs achieved normoglycemia significantly faster and more efficiently compared to

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islets that were transplanted in deviceless fashion. We conclude that ex vivo bio-fabrication of VIOs enables islet engraftment and vascularization before transplantation, and thereby helps to overcome

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limited islet survival and function observed in conventional islet transplantation. Given recent

organs.

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progress in stem cells, this technology may enable assembly of functional personalized endocrine

Keywords: decellularization, extracellular matrix, tissue engineering, lung scaffold, islet transplantation, alternative site, type 1 diabetes.

ACCEPTED MANUSCRIPT Introduction

To meet the metabolic demands of its tissues and organs, the human body maintains a tightly controlled blood glucose level 1. Pancreatic islet cells regulate this level through insulin and

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glucagon secretion. Autoimmune destruction of endocrine function results in Type 1 diabetes (T1D)2. Intrahepatic islet cell therapy is a promising treatment option for severe T1D and represents an extremely valuable success of cellular therapy 3. However, in order to deliver isolated islets to

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the portal vasculature, fresh islets have to be mechanically and enzymatically dissociated from their native tissue specific extra cellular matrix (ECM) and their vascular environment4,5. After

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transplantation, this process contributes to increase islet vulnerability and inevitably lead to islet loss of 50 to 75%6,7. Indeed, substantial loss of β cell mass and insulin content can be observed within three days after transplantation also due to the avascular phase described as the absence of integration between hepatic vascular bed and islet capillary network8,9. Intensive research efforts

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have been dedicated to identifying innovative strategies for improving islet transplantation outcome10,11. Scaffold generation plays an increasingly important role for beta cell replacement12; in particular, whole organ decellularization, more than the ECM manipulation13,14, supports the native

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tissue framework for endocrine regeneration perspective12,15. Recent evidence shows that decellularized organs other than pancreas16-18 can be repurposed to pancreatic endocrine function,

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i.e. spleen19, pericardium20 and kidney21. However, immediate vascularization and islet cell engraftment have not yet been accomplished both after standard intrahepatic transplantation of islets 22-24

and even in previously described decellularized organs19-21. To date, most approaches focus on

improvements of vascularization and tissue integration exclusively in vivo

11,25,26

. To overcome

these limitations, we hypothesized that by eliminating the avascular phase and facilitating ex vivo islet cell engraftment in a decellularized and re-vascularized organ, we could improve endocrine function after transplantation. We aimed to design and construct the first biofabricated vascularized islet organ (VIO), based on decellularized rat lung lobe, endothelial cells and pancreatic islets,

ACCEPTED MANUSCRIPT which provides an appropriate microenvironment for intra-islet vascular perfusion and islet engraftment prior to transplantation.

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Material and Methods

Animals

Experiments involving rats were performed under protocols approved and monitored by the

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Massachusetts General Hospital Institutional Animal Care and Use Committee and performed in compliance with the Animal Welfare Act. Experiments involving mice were performed under

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protocols approved and monitored by Animal Care and Use Committee of San Raffaele Scientific Institute. Male Lewis rats (225-250g; Charles River Laboratories) and C57BL/6 (25-26g; Charles River Laboratories, Calco, Italy) served as islet donors. Male Lewis rats served as scaffold donors. Male RNU rats (250–300 g; Charles River Laboratories) and NSG mice (24-26 g; Charles River

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Laboratories, calco, Italy) were used as recipients.

Preparation of acellular lung scaffolds

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. In brief,

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Cadaveric rat lungs were isolated and perfusion decellularized as previously described

cadaveric lungs were explanted from male Lewis rats (225-250 g, Charles River Laboratories) after

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systemic heparinization. The pulmonary artery (PA) was cannulated with an 18G cannula (McMaster-Carr), the pulmonary veins (PV) were cannulated through the left atrial appendage using a miniball cannula with tip basket (1.9 mm ID) (Harvard Apparatus), and the aorta was ligated. The trachea was cannulated with a 16G cannula (McMaster-Carr) and the right main bronchus was ligated. Subsequently, the right upper, middle and lower lobes were tied off and removed. Decellularization was performed by perfusing the PA (constant pressure, 40 mm Hg) sequentially with heparinized (10 units/ml) phosphate-buffered saline (PBS, 10 min), 0.1% sodium dodecyl sulfate (SDS) in deionized water (2 h), deionized water (15 min) and 1% Triton X-100 in

ACCEPTED MANUSCRIPT deionized water (10 min). The resulting scaffolds were washed with PBS supplemented with 1% antibiotics and antimycotics for 72 h to remove residual detergent and cellular debris. All reagents were sourced from Sigma-Aldrich. Scaffolds showing evidence of infection, vascular air trapping or

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vascular leakage were excluded from the study.

Preparation of acellular pancreas scaffolds

Cadaveric pancreata were explanted from male Lewis rats (225-250 g, Charles River Laboratories)

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after systemic heparinization. The pancreatic duct (PD) was cannulated with a 25G cannula (McMaster-Carr). The pancreas was dissected out from all adjacent tissues. Decellularization was

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performed by perfusion of the PD (constant pressure, 40 mm Hg) sequentially with heparinized (10 units/ml) phosphate-buffered saline (PBS, 10 min), 0.1% SDS in deionized water (5 h), deionized water (15 min) and 1% Triton X-100 in deionized water (15 min). The resulting scaffolds were

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washed with PBS supplemented with 1% antibiotic and antimycotic solution for 72 hours.

Liquid chromatography - tandem mass spectrometry proteomic matrix analysis Samples of left lung (n=3) and pancreatic (n=3) decellularized scaffolds were prepared for mass

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spectrometry as previously described.28,29 Briefly, samples were lyophilized and then homogenized in modified RIPA buffer (PBS, pH 7.4, SDS 0.1%, sodium deoxycholate 0.25%) with Halt Protease

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Inhibitor cocktail (Thermo Fisher) at a 1:100 dilution. The RIPA buffer was used to remove excess fat from the samples as extracellular matrix proteins have been reported to be mostly insoluble in RIPA.29 After centrifugation, the RIPA fraction was discarded, and the pellet was incubated under constant agitation in urea lysis buffer (PBS, pH 7.4, 5.0 M urea, 2.0 M thiourea, 50mM DTT, 0.1% SDS) for 3h at room temperature, followed by acetone precipitation. As described elsewhere

28,29

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samples were alkylated, digested with trypsin and injected onto a microcapillary C18 column. Subsequently, samples were analyzed by microcapillary liquid chromatography tandem mass spectrometry (LC-MS/MS) with linear ion-trap MS technology and reverse-phase gradient.

ACCEPTED MANUSCRIPT Resulting MS/MS spectra were searched using SEQUEST (The Scripps Research Institute La Jolla, CA) and linked to Swiss-Prot (UniProt consortium, United Kingdom, Switzerland, and Washington DC) protein database for identification. The spectral counts were used as a semi-quantitative

non-zero in any of the replicates (n=3) we ran for each organ.

Islet isolation and cell culture

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measurement of proteins in the samples. A protein was considered present if the hit counts were

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Cadaveric pancreatic islets were freshly isolated from systemically heparinized Lewis rats and

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C57BL/6 using collagenase digestion.30 Briefly, 2 mL of cold Hanks buffer/collagenase type V solution (1 mg/mL; Sigma-Aldrich) was infused via the PD in situ. The pancreas was then carefully dissected out from the surrounding tissue and digested at 37°C for 12 minutes. Islets were purified on a discontinuous Ficoll gradient (Sigma-Aldrich). All islets (250 islets/mL) were suspension cultured (37°C, 5% CO2) in conventional islet culture medium (ICM) (RPMI 1640, BioWhittaker

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11.1 mM glucose) supplemented with l-glutamine (Sigma-Aldrich), penicillin-streptomycin (P/S; 1000U/mL to 10mg/mL; Sigma-Aldrich), and 10% fetal bovine serum (FBS, HyClone) over night

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before use for experiments. Islet purity was consistently over 90%. C57BL/6 donor islets were counted and batch matched transplanted into t DL, SC or infuse in VIOs

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at the equivalent islet dose of 500 ± 10% IEq with purity of 95 ± 5% per diabetic recipient. Human umbilical vein endothelial cells (HUVECs, Lonza) were plated on 0.1% gelatin-coated flasks (Sigma-Aldrich) and cultured in complete EGM-2 (Lonza). Medium was changed every other day and passages 5-8 were used for seeding acellular scaffolds.

Study of islet delivery into decellularized left lung lobes. Islet cell seeding experiments into decellularized rat left lung lobes were performed in a custommade bioreactor allowing cell delivery from the trachea and perfusion from both the PA and PV.

ACCEPTED MANUSCRIPT Decellularized left lung lobes were primed by PA perfusion at 0.5 ml/min with 100 ml ICM for 1h prior to cell seeding. After overnight culture in ICM, 1500 IEq were re-suspended in 50 ml of ICM and seeded with gravity (20mmHg) through the trachea, followed by infusion of 200 ml ICM through trachea at the same pressure. Seeded constructs (n=3) were cultured in ICM under static

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conditions at 37°C, 5% CO2 for 2 hours. For quantification of islet delivery, the constructs were divided along the long axis in 4 sections of similar length: Apex (A), Upper middle section (UMS), Lower middle section (LMS) and Base (B). Within each section, 10 histologic sections (150 µm

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were skipped between collected sections) were prepared and stained. The number of Ins+ islets, were counted for each histologic section. For analysis of islet location in the airway, the fraction of

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islets in the distal airway was assessed by insulin and collagen IV staining. Distal airway was considered alveolar space or respiratory bronchioles, which appear morphologically similar.

Culture medium test

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Freshly isolated islets were in vitro tested for survival with ICM, Angiogenic medium (AM), Stabilization Medium (SM) and Modified Stabilization Medium (MSM). AM was Medium 199 (Gibco-5.5mM glucose) supplemented with 1% insulin-transferrin-selenium (Gibco), ascorbic acid

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(50 µg/ml, STEMCELL Technologies), 1% P/S (Sigma-Aldrich), 10% FBS, recombinant human VEGF (40 ng/ml) and bFGF (40 ng/ml). Stabilization medium was Medium 199 (Gibco-5.5mM

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glucose) supplemented with 1% insulin- transferrin-selenium (Gibco), ascorbic acid (50 µg/ml, STEMCELL Technologies), 1% P/S (Sigma-Aldrich), 2% FBS, recombinant human VEGF (20 ng/ml), bFGF (20 ng/ml), forskolin (10 µM, Cayman Chemical) and hydrocortisone (110 nM, Sigma-Aldrich).31 Modified stabilization medium was Medium 199 (Gibco-5.5mM glucose) supplemented with 1% insulin-transferrin-selenium (Gibco), ascorbic acid (50 µg/ml, STEMCELL Technologies), 1% P/S (Sigma-Aldrich), 5% FBS, recombinant human VEGF (20 ng/ml), bFGF (20 ng/ml). Islets, isolated from the same batch, were divided into 300 IEq per testing condition (n=3) and cultured in untreated 6-well plates with ICM for one day. Islets were then cultured in AM

ACCEPTED MANUSCRIPT for additional four days, followed by SM or MSM for two days. Seven days culture of islets in ICM served as control. Dextran transwell permeability assay We measured dextran permeability across the HUVEC monolayer using Transwell polycarbonate

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membrane cell culture inserts (3.0 µm pore, 12-well format, Corning). HUVECs were plated on 0.1% gelatin-coated transwell inserts at a density of 75,000 cells/well and cultured in EGM-2 for 1 day, AM, SM and MSM were then cultured for an additional 2 days (0.5 ml upper chamber, 1.5 ml

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lower chamber). Completing the culture, 50 µl of FITC-conjugated 500-kDa dextran (5 mg/ml, Sigma-Aldrich) was added to the upper chamber and the the media fluorescence of the lower

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chamber was measured after 30 min of incubation at 37°C, 5% CO2 using SpectraMax Microplate Reader (Molecular Devices) set at 485 nm (excitation)/538 nm (emission). Fluorescence readings were normalized to dextran permeability in transwell inserts without HUVECs.

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Insulin secretion test of isolated islets

To assess insulin secretion after in vitro culture of isolated islets (300 IEq), an insulin secretion test (IST) was performed on day 7 of either ICM or 1d ICM followed by 4d AM, followed by 2d MSM

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suspension culture. The IST was performed as previously described.32 Briefly, isolated islets were loaded in a perfusion device containing a 40µm BD filter membrane (Bedford, MA). The flow rate

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was set to 1 ml/min and the assay was performed at 37°C in a 95% O2 and 5% CO2 environment. Islet preparations were initially perfused with an insulin-free perifusion buffer (3.3 mM glucose) for 20 minutes to allow islet equilibration and removal of culture medium. Next, islets were stimulated with insulin-free perifusion buffer at 16.7 mM glucose for 20 minutes. Samples of the effluent were collected at baseline and at minutes 1, 2, 3, 4, 5, 10, 15 and 20 minutes of stimulation. All samples were frozen at -20°C until assayed for insulin content (Insulin ELISA Kit, Abcam ab100578). The dynamic insulin release results were compared using the following parameters: insulin release as determined by the area under the curve (AUC, calculated by the linear trapezoid method expressed

ACCEPTED MANUSCRIPT in picograms insulin/equivalent islet/min), first insulin phase (mean of the peak at interval 0/+10), and second insulin phase (mean of the peak at interval +10/+20). In vitro seeding and culture of VIOs Decellularized left lung lobes were mounted in a custom-made bioreactor with PA cannula and PV

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cannula attached to individual perfusion circuits and the trachea cannula attached to a reservoir that was about 25 cm (20mmHg) above the level of the PA. The trachea was kept open to the inside of the bioreactor and closed to the reservoir through a three-way valve 5 cm above the level of PA.

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Scaffolds were primed from PA and PV perfusion at 0.5 ml/min with 50 ml AM 1h prior to cell seeding. For endothelial delivery through the PA and PV, 20 million HUVECs were re-suspended

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in two separate seeding chambers (each with 10 million HUVECs in 100 ml AM), and seeded simultaneously through the PA and PV under 30mmHg gravity. The organ was cultured under static conditions for two hours. Perfusion was initiated at 0.5 ml/min from both the PA and PV. For tracheal seeding, the PV cannula was released and the trachea opened to the reservoir and closed to

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the chamber on day 1 of culture. 1500 IEq (rat) or 500 IEq (mouse) were re-suspended with 5 million HUVECs in 50 ml of AM and seeded from the tracheal reservoir, followed by 200 ml AM with 5 million HUVECs. Ratio of vascular islet volume/lung volume (mm3/mm3) was 0.00252 and

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0.00084 respectively for rat and mouse islet seeding. Static culture for 2h completed airway seeding and before starting perfusion, we measured the amount of disperse islet in the culture media to

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evaluate islet loss. To re-initiate perfusion, the trachea was opened to the chamber and flow was set to 1 ml/min from the PA. VIOs were cultured for a total of 7d with the initial 5d in AM and subsequent 2d in MSM at 37°C in 5% CO2. VIOs were harvested for functional and histological assessment on day 1 (2h after islet seeding) and on day 7. For in vivo transplantation VIOs were used at day 7.

Fluorescence microangiography

ACCEPTED MANUSCRIPT Fluorescence microangiography was performed on matured VIOs, which were submerged in PBS at 37°C. 5 ml of 1% low melting point agarose (Invitrogen) in PBS containing 1:10 diluted blue FluoSpheres (0.2 µm, 365/415 Invitrogen) was manually injected into the PA (about 5ml/min). PA perfusion pressure was below 20 mmHg (PressureMAT Single-Use Sensor). Immediately after

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microangiography, VIOs were moved to ice, to allow the agarose to solidify.

Lectin perfusion assay

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An ex vivo lectin perfusion assay was performed on immature VIOs cultured for 1 day (immature VIOs) and VIOs cultured for 7 days (mature VIOs) after insulin secretion tests were completed.

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First, VIOs were perfused at 1 ml/min from PA with 50 ml DMEM (GIBCO- 5.5 mM glucose) and 1% P/S for 30 minutes, followed by 25µg/ml biotinylated lectin (GSL I B4, Vector laboratories) in 20 ml DMEM at the same flow rate for 20 minutes. VIOs were then rinsed with 50 ml DMEM and

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1% P/S for additional 20 minutes at 1 ml/min and fixed for histological analysis.

Insulin secretion tests (IST) of VIOs

VIOs cultured for 1 (immature) and 7 days (mature) received 1500 IEq isolated from the same

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batch. We tested immature VIOs 2h after islet and HUVEC seeding at the end of static culture on day 1 and mature VIOs after 7 days of culture. VIOs were mounted on a custom-build ex-vivo

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perfusion device as followed: the VIO was placed on a platform in prone position with the PA cannula connected to a perfusion line, the PV cannula open to the level of the PA allowing collection of venous drainage, and with the trachea open 5 cm above the PA. Insulin secretion of VIOs was measured using a modified IST protocol.32 VIOs were perfused with the previously described perifusion buffer at 3.3 mM glucose for 20 minutes to allow equilibration and to remove remnants of culture medium. Next, islets were stimulated with 16.7 mM for 20 min. Samples were collected from venous drainage at baseline and at 1, 2, 3, 4, 5, 10, 15 and 20 minutes of stimulation. The IST was performed with 1ml/min perfusion at 37°C and 5% CO2. Repeated ISTs were

ACCEPTED MANUSCRIPT conducted on mature VIOs. At the end of the initial IST, VIOs were perfused with MSM (5.5 mM) at 1ml/min for 120 min, with the first 15 minutes single pass perfusion. VIOs were then tested with a second IST. All samples were frozen at -20°C until assayed for insulin content (Insulin ELISA Kit, Abcam). The dynamic insulin release results were compared using the following parameters:

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insulin release as determined by the area under the curve (AUC calculated by the linear trapezoid method expressed in picograms insulin/equivalent islet), first insulin phase (mean of the peak value

Diabetes induction and metabolic monitoring

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at interval 0/+5), and second insulin phase (mean of the peak value at interval +5/+20).

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Diabetes was induced by administrations of streptozotocin ( 65 mg/kg i.p., Sigma-Aldrich) in RNU rats on day -4 and day -2 and alloxan (65 mg/Kg i.v., Sigma-Aldrich) NSG on day -2 before transplantations.26,33,34 Animals with non-fasting glucose <450 mg/dl prior the transplantation were not considered diabetic and excluded from the study. RNU rats were resuscitated with subcutaneous

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saline on days -2 and -1 before VIOs implantation to compensate for osmolar diuresis according to the body weight lost from baseline. Rats were fasted for 12h prior to surgery. In long-term experiments, the reversal of diabetes was defined as two consecutive glycaemia ≤250

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mg/dl and maintained until study completion. Islet graft function was assessed through non-fasting blood glucose measurements, using a portable glucometer (OneTouch Ultra, Bayer ) daily following

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islet transplantation, in all groups transplanted. Exogenous insulin therapy (LinBit pellet; LinShin) was administered subcutaneously peri-transplant to maintain an acceptable health status35. The day after transplantation a suboptimal quantity of two pellets were implanted in all diabetic recipients with a not fasting glycaemia >250 mg/dl. No pellets were implanted in animals with a sustained function and not fasting glycaemia ≤250 mg/dl. One Pellet was removed at ten days after transplantation and the second one twenty days after transplantation.

VIOs implantation and intrahepatic islet transplantation in diabetic recipient rats

ACCEPTED MANUSCRIPT RNU diabetic rats (250-300g) were anesthetized with isoflurane (Forane 1–2%; Abbott Laboratories, Toronto, Canada) and intubated. The left external jugular vein (LEJ) was cannulated with a 24G catheter (IV Catheter Radiopaque, Jelco) for vascular access. All animals received a single administration of 1.5 ml/100g bodyweight 0.9% saline via this access in the beginning of the

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procedure.

For implantation of the VIO in diabetic recipient rats, a midline laparotomy was performed. The inferior vena cava (IVC) and aorta (AO) of recipient rats were dissected out, clamped proximally at

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the level of the gonadal vessels and ligated distally at the level of their bifurcation. Prior to implantation, the VIO was flushed with heparinized (10 units/ml) and 100 ml ice cold DMEM

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(GIBCO- 5.5 mM glucose) from the PA. Next, the VIO was carefully dissected from the heart and the trachea was ligated. PA and PV were prepared for anastomosis by attaching a 20 G and 16G cuff (Jelco), respectively. The VIO was kept on ice while the recipient was prepared for the implantation. The cuffed PA and PV of VIOs were inserted into the recipient AO and IVC,

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respectively, to create end-to-end anastomoses. The AO and IVC were dissected out proximally to the anastomoses and divided distally to the anastomoses to avoid kinking of the vasculature. The

the VIO.

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recipient animal was prepared for the IVGTT before the vascular clamps were released to perfuse

For intrahepatic islet transplantation RNU diabetic rats (250-300g) were anesthetized with

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isoflurane (Forane 1–2%; Abbott Laboratories, Toronto, Canada) and intubated. The left external jugular vein (LEJ) was cannulated with a 24G catheter (IV Catheter Radiopaque, Jelco) for vascular access. All animals received a single administration of 1.5 ml/100g bodyweight 0.9% saline via this access in the beginning of the procedure. A midline laparotomy was performed, the intestines were eviscerated from the peritoneal cavity onto dampened gauze and the portal vein was stretched and exposed. Freshly isolated 1500 IEq were suspended in 0.5 ml RPMI media and collected into an insulin syringe with a 29G 12.5 mm

ACCEPTED MANUSCRIPT needle. A vascular clamp was applied to the portal vein, the needle was then inserted into the portal vein and the plunger was gently advanced to slowly infuse the islets into the hepatic site.

Intravenous glucose tolerance test

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VIOs were functionally assessed and compared to intrahepatic islet transplantation, using an intravenous glucose tolerance test (IVGTT). Islet-free, re-endothelialized scaffolds served as controls. Baseline blood glucose levels were taken (t = 0 min) from right external jugular vein

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(REJ) using a 29G insulin syringe before a glucose bolus was administered. 0.5 g/kg glucose bolus in 1ml 0.9% saline was administered via the left external jugular vein (LEJ). Once perfusion was

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initiated by releasing the clamps, venous blood from REJ was sampled after t = 1, 5, 10, 15, 20, 30, 45 and 60 minutes for glucose and insulin quantification. The area under the curve (AUC) and elimination of glucose (KG) 1-15 minutes and 1-60 minutes were calculated. The glucose tolerance was quantified from the glucose elimination constant (KG; expressed as percentage of elimination

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of glucose per minute) as the reduction in circulating glucose between 1 and 15 minutes (KG1-15) after intravenous administration, following logarithmic transformation of the individual plasma glucose values. A similar calculation was performed for the 1 to 60-minute glucose elimination rate

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(KG1-60). This value indicates the rate of glucose elimination during the whole test, when the

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delayed insulin effect is properly accounted for.

Plasma collection and Insulin quantification Blood samples obtained from REJ were collected into 1ml Lithium Heparin Tubes (MiniCollect, Greiner Bio-One GmbH). Plasma was obtained after centrifugation at 15000g for 13 min at room temperature. All samples were frozen at -80°C until assayed for insulin content (Insulin ELISA Kit, Abcam ab100578). The dynamic insulin release results were compared using the following parameters: insulin release as determined by the area under the curve (AUC, calculated by the linear

ACCEPTED MANUSCRIPT trapezoid method expressed in ng/ml/60 min), first insulin phase (peak at interval 0/+10), and second insulin phase (the peak at interval +10/+60).

Device-less subcutaneous transplant site generation and islet transplantation

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NSG recipient diabetic mice were anesthetized (22-26 gr) with Avertin (tribromoethanol) (intraperitoneal injection at a dose 0.75 mg/g; Sigma). As described by Pepper25, three to 6 weeks before islet transplant, the device-less (DL) site was created by implanting a 2cm segment of 5-

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French (Fr.) textured nylon radiopaque angiographic catheter (Cook Medical, Indiana, USA) subcutaneously into the lower left quadrant of 20–25 g male recipient NSG mice. At the time of

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transplantation, a small (4 mm) incision was made to gain access to the catheter. The tissue matrix surrounding the superior margin of the catheter was dissected to withdraw and remove the catheter. 500 IEq mouse islets with purity of 95% ± 5% were aspirated into polyethylene (PE-50) tubing using a micro-syringe, and centrifuged into a pellet suitable for transplantation. The PE-50 tubing

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and islet preparation was delivered within DL space, and islets carefully injects into the void using the microsyringe (Hamilton). Incision was closed with surgical silk suture (Ethicon, New Jersey,

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USA).

Unmodified subcutaneous islet transplantation

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NSG recipient diabetic mice were anesthetized (22-26 gr) with Avertin (tribromoethanol) (intraperitoneal injection at a dose 0.75 mg/g; Sigma). At the time of transplantation, a suspension of 500 IEq with purity of 95% ± 5% and 30x106 huvec cells were aspirate into polyethylene (PE50) tubing using a micro-syringe, and centrifuged into a pellet suitable for transplantation. The PE50 tubing and cells preparation was delivered within the subcutaneous lumen using the microsyringe. The incision was closed with a surgical silk suture (Ethicon, New Jersey, USA).

VIO implantations in diabetic recipient mice

ACCEPTED MANUSCRIPT NSG recipient diabetic were anesthetized (22-26 gr) with Avertin (tribromoethanol) (intraperitoneal injection at a dose 0.75 mg/g; Sigma). Prior to mice implantation, VIOs were flushed with 100 ml ice cold DMEM (GIBCO- 5.5 mM glucose) from the pulmonary artery (PA). Next, VIOs were carefully dissected from the heart and trachea and divided with sagittal incision. VIOs halves were

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implanted in two separate subcutaneous pockets generated on the dorsal left and right area. The single pocket was specifically created by two 10-mm lateral transverse incisions limiting a 2 cm tunneled area. An adequate void (1cm by 3 cm) was created. Once VIOs were placed in the

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dedicated space, the implant was ligated under the skin with two prolene sutures (Ethicon, New

USA).

Contrast enhanced Ultrasound

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Jersey, USA) by sides and skin incision was closed with surgical silk suture (Ethicon, New Jersey,

Contrast enhanced ultrasound (CEUS) was performed on NSG recipient mice as previously

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described with minimal changes36,37. Analysis was performed using a high-performance ultrasonographic scanner designed (Vevo 2100; FUJIFILM VisualSonics, Toronto, Canada). Mice were anesthetized with isoflurane (flurane; Isoba, Schering-Plough, USA, 4% in oxygen for

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induction, 2% for maintenance at a rate of 1liter/minute). Mice were positioned in a prone position on a MousePad (THM150 MousePad part of the VisualSonics Vevo Integrated Rail System III) and

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monitored ECG and respiration rate during the examination. During imaging, heart rate was 450±50 beats per minute (bpm) and body temperature was 37±1°C (rectal probe). CEUS studies were performed in contrast mode using the MS250 linear transducer (fc=21 MHz, 13-24 MHz; FUJIFILM, Visualsonics). The scanhead was fixed on the railing system to maintain the acoustic focus at the bottom of VIO, SC and DL implants at the level of the largest sagittal cross section. Untargeted ultrasound contrast agent (CA, MicroMarker, Bracco, Geneva, Switzerland; FUJIFILM VisualSonics) was prepared in agreement with the specifications of the producer. CA was injected using a syringe pump (Pump 11 Elite, Harvard Apparatus, Holliston, MA) as a bolus

ACCEPTED MANUSCRIPT via tail vein catheter at flow rate of 0.750 ml/min for 5 seconds (50µl bolus, 7.0x107 microbubbles). Sonographic data acquisition started immediately after CA injection and cine loops of contrast wash-in were acquired. Imaging parameters were held constant throughout each experiment to reduce variability (transmit power 4%; dynamic range 35 dB; frequency 18 MHz; frame rate 25;

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contrast gain 48 dB; gate 4; beam-width wide) with focus and depth optimized at the beginning of the study for each animal. Perfusion was determined using the appropriate VisualSonics software package. A region of interest (ROI) was drawn along the perimeter of different implants. Perfusion

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parameters calculated from the generated intensity graph were the Peak Enhancement (PE), defined the ratio of the plateau value to the baseline contrast value and this is a measure of relative blood

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volume; the Time to Peak (TTP) defined is the amount of time it takes the contrast intensity to reach a plateau value. This is a measure of relative blood velocity and the Area Under the Curve (AUC), calculated on 284 frames. All ultrasound examinations were performed by a single investigator with

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high experience in preclinical imaging.

Histology and immunostaining

All samples were fixed overnight with 4% paraformaldehyde in PBS (PFA) unless stated otherwise.

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For paraffin-embedded samples, 5 µm sections were used for histological analysis. For H&E staining, sections were deparaffinized, rehydrated, stained with hematoxylin QS (Vector

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Laboratories) and eosin Y (Fisher Scientific), and mounted with Permount (Fisher Scientific) after dehydration. For immunofluorescence staining, sections were deparaffinized, rehydrated, put through heat-induced antigen retrieval, permeabilized with 0.1% Triton X-100 in PBS, washed three times in PBS and blocked with 1% bovine serum albumin in PBS. Sections were incubated in primary antibodies overnight at 4 °C. Primary antibodies included antibodies against human CD31 (1:200, DAKO, M082301-2), rat CD34 (1:400; LSBio, LS-C150289), murine CD34 (1:300; Biolegend, 119302), rat Insulin (1:100; Abcam, ab181547 and ab7842), rat Glucagon (1:100; Abcam, ab10988), rat Collagen IV (1:200; Abcam, ab6586). After overnight incubation, sections

ACCEPTED MANUSCRIPT were washed three times with PBS and then incubated in A594, A488 or Cy5-conjugated secondary antibodies (1:500; Molecular Probes) for 40 min at room temperature, followed by three washes in PBS. Sections were mounted with DAPI Fluoromount-G (SouthernBiotech) and imaged on a Nikon Eclipse TE200 microscope. Samples for immunohistochemistry after primary antibody labeling

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were stained with Rabbit-on-Rodent (for CD31) Horseradish peroxidase (HRP) polymer detection system (Biocare medical) and DAB Substrate-Chromogen (DAKO, Carpinteria, CA, USA) were used for detection. Samples labeled with biotinylated lectin were stained with a streptavidin Alexa

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Fluor conjugate (1:500; Molecular Probes). Microangiography samples were fixed in PFA for 2 h, equilibrated in 30% sucrose in PBS overnight, and embedded in O.C.T. compound (Tissue-Tek). 50

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µm cryo-sections were prepared for staining. Sections were mounted with Fluoromount-G and imaged on a Nikon Eclipse Ti confocal microscope.

Statistical analysis

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Values are summarized as mean ± SD (or SEM, where indicated) or median according to their distribution. Statistical analysis was performed by Mann Whitney U tests, Student’s t-tests (two-tail comparisons) ANOVA (Bonferroni Post-hoc correction) and general linear model. Gain of

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normoglycaemia was evaluated using Kaplan-Meier analysis and the significance was estimated using the log-rank test. Statistically significant differences were defined as P < 0.05. Microsoft

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Excel 2011 (Microsoft Corporation, https://www.microsoft.com), Graphpad Prism v6 (GraphPad Software, http://www.graphpad.com) and SPSS v20 (statistical package for Windows SPSS Inc.) were used for data management, statistical analysis and graph generation.

Results An ideal scaffold for engineering a vascularized islet organ contains a vascular compartment to enable perfusion and an islet compartment to enable delivery and engraftment of endocrine cells. The islet compartment should provide suitable ECM cues for islet engraftment and adequate space

ACCEPTED MANUSCRIPT to host whole islets. The vascular compartment should provide a hierarchical vascular network with two large vessels for arterial and venous anastomosis and a dense capillary network that provides blood supply to every islet, like the endocrine pancreas. Finally, the bio-engineered vasculature should integrate with the islets’ own capillary network ex vivo to allow efficient glucose sensing

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and insulin secretion upon implantation38 (Fig. 1A). Given our experience with native scaffolds, we hypothesized that a bio-engineered scaffold from decellularized lungs would satisfy these requirements. The microscopic architecture of native lungs has two anatomically independent

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compartments for air and blood volume. Moreover, alveoli are readily accessible via the large airways and well embedded in the pulmonary vascular network 39. The vascular supply mirrors that

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of pancreatic tissue, where islets are preserved in a highly vascularized niche (Fig. 1B). In native rat pancreas, CD34 staining showed a capillary network surrounding and integrating with islets and connecting them to the main vasculature (Fig. 1B). In native rat lung, CD34 staining showed a similarly developed capillary network surrounding and connecting the alveolar space to the main

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vasculature (Fig. 1B). To generate a scaffold, we decellularized rat lungs via detergent perfusion as previously reported27. Through this process, lung tissue lost all cellular components but retained an intact ECM structure. The composition of islet specific pancreatic ECM, in particular the basal

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lamina constituents laminin 5 and collagen IV, are critical to islet survival and function5. To characterize the components of rat pancreas derived ECM, we conducted proteomic analysis of

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decellularized pancreatic scaffolds and then compared proteome overlap with decellularized rat lung scaffolds (Fig. 1C). Among 33 ECM proteins identified by comparison to the UniProt database, there were 22 proteins common to both scaffolds, including laminin 5 and collagen IV; nine proteins were unique to the lung and only one protein was unique to the pancreas (Fig.1C). Because of the similarities between lung and pancreas matrix composition and architecture, we hypothesized that islets could be delivered through the larger airways to engraft in the alveolar spaces of decellularized lungs, which would then be connected to a repopulated vascular bed and ultimately would sustain islet viability and endocrine function. We first determined whether islets

ACCEPTED MANUSCRIPT could be delivered to the alveolar spaces, to be near the capillary bed. We cultured freshly isolated islets overnight in conventional islet culture medium (ICM) and subsequently infused 1500 ± 90 islet equivalents (IEq) through the trachea into the decellularized left lung applying a perfusionpressure of 20 mmHg. Islets were delivered across the entire lobe with predominance to the upper

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and lower middle section compared to apex and base following a Gaussian distribution (Fig.2A). Moreover, islets were preferentially delivered to the alveolar space (97.52±0.2%), appeared morphologically intact and were surrounded by a preserved collagen IV positive alveolar membrane

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(Fig.2B and C).

To develop a functional pre-vascularized organ, we matured scaffolds, seeded with endothelial cells

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and islets, over seven days under perfusion culture. To develop culture media that supported both endothelial maturation and islet engraftment, we examined whether a two-phase endothelial culture protocol with angiogenic medium (AM) followed by stabilization medium (SM) was compatible with the culture of isolated islets

31

. We did not observe a difference in islet survival between

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conventional ICM and AM (phase 1) but a significant decline in islet survival at the end of phase 2 in SM (p=0.02) (Supplementary Fig. 1A). Thus, we developed and validated a modified stabilization medium (MSM) for phase 2 that supported islet survival and retained its barrier-

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strengthening function for endothelial cells. Compared to conventional culture in ICM, the newly developed two-phase medium was equivalent in supporting islet survival (% of islets,

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ICM=18.33±3.5 and ICM+AM+MSM=16±2., p=ns) (Supplementary Fig. 1A). Additionally, MSM significantly improved endothelial barrier function, compared to AM (% Leakage, AM = 0.85±0.11 and MSM = 0.28±0.13, p=0.001) (Supplementary Fig. 1B). Based on these results, we tested the overall insulin release after 7 days of islet culture with the newly developed media protocol. The mean area under the curve (AUC) showed no statistically significant difference between islets cultured in ICM and ICM+AM+MSM. (AUC: ICM= 22.67±10.04, ICM+AM+MSM= 18.25±1.82, p=ns) (Fig. 2D). After identifying the optimal ex vivo culture media combination for islet viability and function, we applied this combination to bioengineered VIOs. First, we repopulated acellular

ACCEPTED MANUSCRIPT lung matrixes by delivering human umbilical vein endothelial cells (HUVECs) from both the pulmonary artery (PA) and pulmonary vein (PV) and cultured the re-endothelialized scaffolds in AM (Fig. 2E). On the same day, we isolated 1500 islet equivalents (IEq) from cadaveric rat pancreata and cultured them overnight in ICM. The following day, we delivered a suspension of

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both islets and HUVECs through the trachea. During this step, we did not observe any islet spillage from the lung. We cultured the resulting constructs for an additional four days in AM, followed by two days in MSM (Fig. 2E). By the end of this culture period, we observed higher spatial

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organization of distinct tissue compartments. A continuous human CD31+ endothelial cell network throughout the bio-engineered organ formed with islets embedded in the vascular bed (Fig. 3A).

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Capillaries developed which surrounded islets in proximity and integrated into islet clusters (Fig. 3B). Moreover, the VIOs displayed preserved pancreatic islet morphology with the presence of insulin positive ß-cells and glucagon positive α-cells in the islet periphery (Fig. 3C). To investigate whether the newly formed vasculature would allow direct perfusion of islets, we perfused the PA

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with 0.2-µm microspheres at the end of culture and observed perfusable neo-vasculature embedded within islets (Fig. 3D). Confocal microscopic examination of mature VIOs, revealed vascular continuity from the surrounding human CD31+ endothelium to the rodent derived CD34+ capillary

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network within islets (Fig. 3E and Movie S1). We further confirmed this by performing a lectin perfusion assay (Supplementary Fig. 2). In mature VIOs, histological analysis revealed lectin+ rat

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CD34+ endothelium within single islets. In contrast, early stage VIOs, two hours after islet seeding, showed absence of lectin in rat CD34+ endothelium within islets. Taken together, these findings suggest that VIOs provide direct perfusion of islet parenchyma due to functional integration of islets in the bio-engineered vascular architecture. After confirmation of anatomic integration and perfusion of the bioengineered vascular bed and the islet’s vascular bed, we examined the endocrine function of the engineered VIOs. We assessed the VIO’s ex vivo endocrine function with an insulin secretion test (IST) (Fig. 4A). We tested immature

ACCEPTED MANUSCRIPT VIOs at day one and matured VIOs at day seven of culture using a modified standardized glucose response assay32. When exposed to supra-physiologic arterial glucose levels, VIOs demonstrated a physiologic biphasic insulin release from the vein. Matured, 7 days cultured VIOs released significantly higher

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insulin levels during both phases as shown by point-by-point comparison (Fig. 4B). The peak insulin release of matured VIOs during the first and second phase of insulin response was 2.6±0.54, and 6.9±4.85 fold higher than from immature VIOs, respectively (Supplementary Fig. 3). Mature

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VIOs released significantly more total insulin than immature VIOs (AUC: 7d VIO = 408.24± 89.83 pg/islet/min; 1d VIO=109.16±51.8 pg/islet/min, p=0.041; Fig. 4C). To rule out bias related to islet

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quality, we only compared batch matched islet preparations in immature and mature VIOs, therefore only evaluating the impact of the maturation process on the VIO’s insulin secretion. As previously reported, isolated islets rapidly loose endocrine function and undergo apoptosis during ex vivo culture40. When insulin release of isolated islets was compared to immature VIOs seeded

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with fresh islets prior to testing, both performed similarly. However, mature VIOs were superior to isolated islets cultured for the same period under the same culture media conditions (AUC Day 1: Islet vs. VIO: 75.09±1.89 and 109.16±51.84, p=ns; AUC Day 7: Islet vs. VIO 22.67±10.04 and

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408.24±89.83, p= 0.034) (Fig. 4D). Thus, the beta cell death during VIOs maturation did not exceed 0.4% of total seeded islet mass (data not shown). In vivo, VIOs would be exposed to fluctuations in

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blood glucose (BG) during the course of a day. To determine whether VIOs could respond adequately to rapid changes in glucose levels, we challenged mature VIOs with two consecutive ISTs41. A two-hour interval between glucose stimulation mimicked lower BG between meals. VIOs maintained their physiologic biphasic insulin release in response to both glucose stimulation cycles with no significant difference in total insulin released (Supplementary Fig. 4). To test VIOs under pathophysiologic conditions in vivo, we induced severe diabetes in rats and mice via streptozocin injection. We subsequently evaluated VIOs performance in two independent transplant models. First, we examined acute function over one hour after vascular anastomosis. We

ACCEPTED MANUSCRIPT compared VIOs to intrahepatically transplanted islets as the current standard of care (Fig. 5A). We generated VIOs, from 1500±89 rat IEq, transplanted them into the abdomen of diabetic rats and anastomosed the VIO’s artery and vein to the recipient aorta (Ao) and inferior vena cava (IVC), respectively (Fig. 5B). To generate a standard of care control, we transplanted an equivalent

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quantity of freshly isolated rat islets (1500±109 IEq) via portal vein injection into the liver of diabetic rats (intrahepatic). Islet-free VIOs (VIO-islet) served as a control. We then evaluated islet function in all three groups with a peri-transplant intravenous glucose tolerance test (IVGTT) and

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recorded blood glucose (BG) and plasma insulin levels at specific time points over 60 minutes (Fig. 5C-5E). Insulin was detected in the systemic circulation of recipient rats within the first minute after

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reperfusion of VIOs (Fig. 5C), indicating efficient arterial to venous blood flow in the VIO. Compared to intrahepatic islet transplantation, VIOs responded to the glucose bolus with a significantly higher initial insulin release (min 5; ng/ml: VIO 9.94±2.37 vs. Intrahepatic 2.98±1.47, p=0.006) and a more distinctive second phase of insulin release. Finally, the total insulin release of

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VIOs, was significantly higher than that of intra-hepatically transplanted islets (AUC x 105: VIO 2.157±0.226 ng/ml/60min vs. Intrahepatic 1.549±0.485 ng/ml/60min, p=0.038). As expected, animals that received VIO-islet showed an absence of plasma insulin at all time points (Fig. 5C-5D).

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To investigate how the VIOs’ insulin release affected blood glucose homeostasis in the recipient animals, we monitored the glycaemia profile during the IVGTT (Fig. 5E). After glucose

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stimulation, a more rapid reduction of blood glucose was observed in animals that had received VIOs compared to intrahepatic transplantation. As shown by point-by-point analysis (VIO vs. Intrahepatic: 20 min p=0.041; 30 min, p=0.006; 45 min, p=0.002 and 60 min p=0.033). The glucose reduction profile revealed an increase in the glucose elimination constant (KG) over 15 minutes and a statistically significant increase over 60 minutes (KG 1-60: VIO=0.0069±0.0011 vs. Intrahepatic =0.0047±0.0006; p= 0.032, VIO vs. VIO-islet, p= 0.0140) (Fig. 5F). After completion of IVGTT VIOs’ morphology was immunohistochemically assessed. VIOs exposed to arterial blood pressure for 60 minutes showed preserved vascular and islet organization as well as an absence of red blood

ACCEPTED MANUSCRIPT cell (RBC) accumulation outside the vascular compartment (Fig. 5G). Additionally, RBCs were present within the human CD31+ vascular network and in solid islets, which confirmed functional pre-vascularization of islets during ex vivo culture. After confirming VIO function in a short-term transplant model, we then assessed graft function

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over a period of 30 days. We implanted VIOs subcutaneously into diabetic NSGmice and compared their blood glucose levels against fresh islet transplantation into pre-vascularized subcutaneous pouches

25

. We generated mature VIOs from 500IEq mouse islets and 3x106 HUVECs. We then

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divided mature VIOs into halves and implanted them into two subcutaneous pouches under the skin of a diabetic recipient (treatment group, VIO) (Fig. 6A). In a second group, we transplanted an

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equivalent quantity of freshly isolated batch-matched islets into a pre-vascularized subcutaneous device-less site (DL) as previously reported

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(Fig. 6A). The third group, a non-prevascularized

group, received batch-matched islets and HUVECs (the equivalent amount used for VIOs) in an unmodified subcutaneous transplantation site (SC). Recipients with a blood glucose level greater

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than 250 mg/dl on day 1 post-implantation received 2 insulin pellets for suboptimal glycemic control. The first pellet was removed at day 10, the second one at day 20 post-implantation. We recorded graft function over 30 days by measuring daily blood glucose levels. (Fig. 6A). There was

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a significant difference in daily not-fasting glycemic levels of VIO implanted mice compared to DL and SC treated mice (p=0.005 VIO vs DL, p=0.037 VIO vs SC, p=ns SC vs DL) (Fig. 6B).

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Importantly, 38.5% of VIOs (5/13) never required pellet implantation due to sufficient graft function after implantation. In contrast, all animals from DL and SC required suboptimal glucose control with pellets (p=0.026, chi-square test). Within 25 days VIOs reversed diabetes in 92.3% of recipients (12/13), while DL and SC achieved 40% (2/5) and 0% (0/4), respectively. Notably, 50% of VIOs achieved euglycemia within an average of 2±0.238 (median±SEM) days (Fig. 6C). Diabetes was cured in fewer than 50% of mice treated with DL and SC islet transplantation. One month after transplantation we compared vascular perfusion of the different islet transplantation sites. Contrast enhanced ultrasound (CEUS) performed on VIO, DL and SC

ACCEPTED MANUSCRIPT implantation sites showed significantly different perfusion levels. Compared to DL and SC, VIOs implanted mice showed a significantly higher contrast enhancement during the initial phase (median values of PE: VIO 3.87 vs DL 1.35 p=0.036 and VIO 3.87 vs SC 1.41 p=0.007) and a more distinctive overall blood perfusion (median values of AUC: VIO 165.02 vs DL 87.13 p=0.042 and

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VIO 165.02 vs SC 81.20 p=0.027) (Fig. 6D). Interestingly, vascular perfusion in the DL group correlated with endocrine graft function (blood glucose level ≤250 mg/dl). An increased vascular perfusion was detected in DL recipients with normoglycemia at day 30 after transplantation

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(Supplementary Fig.5). Morphological analysis of implanted VIOs was performed during early engraftment at day 8 and 14. Consistent with early graft function, VIOs showed a preserved

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bioengineered vascular network (human CD31+) and intact insulin positive islets. (Fig. 7A). Fourteen days after implantation histology revealed tissue remodeling by integration of recipient vasculature (murine CD34+) into the preserved vascular architecture of bioengineered VIOs. (Fig.

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7B).

Conclusions

Intrahepatically transplanted islets are deprived of their microenvironment and are vulnerable to

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Instant Blood Mediated Inflammatory Reaction (IBMIR), thrombosis and tissue ischemia42-44. To overcome these limitations, we designed a bioengineered vascularized islet organ (VIO). We

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assessed functionality ex vivo and in vivo in diabetic rodents and compared it to the standard in preclinical and clinical practice.

To provide a suitable microenvironment for islets, we explored the use of native extracellular lung matrix as a platform for VIOs. While acellular pancreas and kidney scaffolds have been repopulated with pancreatic islets16,45 and a beta cell line21, respectively, the approach to combine whole islets with a re-purposed vascularized organ scaffold is novel. Differently from other organ scaffolds with only vascular access, the unique two-compartment architecture of the lung offers the possibility to separately seed endothelial cells via the native vascular structure and pancreatic islets via the

ACCEPTED MANUSCRIPT trachea, into alveolar space. This avoids islet injury and disruption of scaffold microarchitecture. Additionally, seeding can be performed at different times over the in vitro maturation culture. We observed that almost all islets could be delivered to the alveolar space despite their variability in size (50 to 400 µm)46. We suspect this is due to the high elastin content of lung ECM, which is

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largely preserved after decellularization47. Compliance and elastic recoil allow the alveolar basal lamina to adapt to islet size and maintain proximity to promote engraftment. We demonstrated that the ECM composition of decellularized lungs largely overlap with pancreatic ECM. Laminin 5 and

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collagen IV, located in the alveolar membrane as well as in a basal lamina of pancreatic islets serve as ligands to ß-integrins expressed by islet cells and play a critical role in modulating islet viability 5,48

. Islet adhesion to appropriate ECM components has been reported to support their

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and function

function and survival 38,49. However, to date, this approach has focused on improving the immediate microenvironment of individual islets, not on providing the larger scale architecture of a perfusable organ like structure. To be clinically relevant, the scaffold must support an adequate number of

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islets to treat T1D. The lung scaffold offers the unique feature of containing hundreds of millions of alveoli, each of them serving as a functional unit. Taken together these findings suggest that ECM

isolated islets.

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scaffolds of decellularized lung lobes could serve as an innovative new platform to efficiently host

Maintenance of organ viability, glucose sensing and endocrine secretion of insulin require a robust

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vascular network of the VIO, like that in a pancreas. Within 7 days of culture, VIOs formed a dense vascular network from seeded HUVECs and interconnected with islet-derived endothelium. Interestingly, this chimeric vascular composition mirrors in vivo revascularization of islets with vasculature derived from both recipient endothelial cells and islet native endothelium

22

. The

findings of lectin+ rat CD34+ within islets, the presence of microspheres in vascular beds embedded in islets, and histologic endothelial continuity between human CD31+ endothelium and rodent CD34+ endothelium within islets each suggest that the seeded pancreatic islets were functionally integrated into the bio-engineered vascular architecture. In this setting, islets do not depend on

ACCEPTED MANUSCRIPT oxygen and nutrient delivery by diffusion but instead by direct perfusion of islet parenchyma. To test in vivo function, we performed implantations of VIOs in an acute transplant model with surgical vascular anastomosis, and a chronic transplant model in subcutaneous position. In the acute model, VIOs withstood systemic arterial blood pressure and thus provided a partial oxygen pressure

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(pO2) more favorable to islet function than the liver 50. In vivo, VIOs more rapidly and efficiently reduced BG compared to intrahepatic transplantation of fresh islets, the standard clinical care. We suspect that the observed superior performance of VIOs was caused by pre-transplantation

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establishment of a perfusable vascular tree integrated with islet vasculature. By eliminating the avascular phase after islet cell delivery, we believe that the absolute islet mass needed to achieve

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normoglycaemia could be reduced. When VIOs were repeatedly challenged with supraphysiological glucose levels in an ex vivo isolated organ perfusion assay, we observed physiologic bi-phasic insulin release. This finding indicates a precise glucose triggered insulin release rather than stress-induced insulin dumping by damaged islets. Similar findings have been observed in 51

. Importantly, VIOs could be functionally

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native rat pancreata repeatedly exercised ex vivo

assessed prior to implantation without compromising islet function. While microvascular anastomosis of VIOs is theoretically clinically feasible, a lesser invasive

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approach would be favorable to facilitate clinical translation, minimize patient burden and procedural risks, and enable graft assessment and possibly retrieval. To meet this demand, we

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evaluated VIO function in a chronic subcutaneous implant model. In this model, we confirmed rapid reversal of hyperglycaemia after VIO implantation, and maintenance of endocrine function. In this context, we directly compared the recently reported pre-vascularized device-less implantation site to our approach of functional integrated vasculature to dissect the real impact of these alternative technologies on islet engraftment. Even without surgical anastomosis, biofabricated VIOs showed significantly better immediate function after subcutaneous implantation. In line with prior reports 25, islet implantation in a pre-vascularized site was able to reverse diabetes and sustain islet function, however with a significant delay when compared to VIOs. The preformed fully

ACCEPTED MANUSCRIPT integrated vasculature of VIOs facilitated rapid adaptive function, while the observed recipient mouse CD34+ ingrowth and robust vascular perfusion strongly demonstrated further integration into the recipient. Efficient in vitro derivation of adult19 or human pluripotent stem cells (hPSCs) into functional

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endothelial and pancreatic β-cells has recently been reported52-55. HPSCs technology will offer the possibility to obtain endothelial and beta cells from the same donor to avoid double MHC mismatch (endothelial and islet from different sources) and thus to clinical extend the use of VIOs. In current

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preclinical models of T1D, hPSCs derived β-cells are transplanted into extra-vascularized sites resulting in reduced engraftment and delayed insulin response after implantation56. Indeed, VIOs

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offer a new pre-vascularized implantation site that might increase the insulin responsiveness of hPSCs derived β-cells. In addition, implantation of solid organs composed of hPSCs derived cells would allow retrieval of VIOs in case of adverse events and may help to advance the clinical use of hPSCs derived tissue.

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In summary, we describe a novel biofabrication technique to generate a vascularized endocrine organ and show its advantages over currently available techniques in a clinically relevant in vivo

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endocrine organs.

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model. This concept could serve as a platform technology for engineering of other bio-artificial

Author contributions

AC and PTM contributed equally to design, conduction, and analysis of all experiments, and prepared the manuscript; ED assisted with in vivo experiments, ultrasound evaluation and contributed to the manuscript preparation. TKR assisted with in vivo experiments and contributed to the manuscript preparation. XR assisted with organ re-endothelialization. JMC assisted with data analysis and figure preparation; DEL assisted with scaffold preparation for mass spectrometry and proteomic analysis; BKP assisted with manuscript preparation and data interpretation. AP assisted

ACCEPTED MANUSCRIPT with preliminary experiments. FM assisted with immunofluorescence acquisition. SP helped with mouse islet isolation. HCO and LP oversaw study design, experimental conduction, data analysis, and manuscript preparation.

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Acknowledgments This study was supported by the Charles and Sara Fabrikant MGH Research Scholarship, Fondazione Banca del Monte di Lombardia - Progetto Professionalità Ivano Becchi fellowship, by

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grant from the European Commission (H2020 681070) and by grant from the Italian Ministry of Health referred to 5X1000 campaign of 2014 ‘OSR seed Grant’. The authors thank the MGH

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Center for Skeletal Research Core (NIH P30 AR066261) for histological processing. San Raffaele Scientific Institute carried out part of immunofluorescence acquisition in ALEMBIC. We thank Valentina Pasquale and Martina Policardi for the helpful technical contribution in in vivo mouse

Conflict of Interest

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experiments.

HCO is founder and stockholder of IVIVA Medical Inc. This relationship did not affect study

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design, execution, and data analysis and interpretation.

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Figure 1 - Design of a VIO

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Figures

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(A) Diagram of design and characteristics of a bioengineered vascularized islet organ for transplantation. (B) Histology of native rat pancreas (upper panel) and rat lung (lower panel). Left Panel: H&E of native rat pancreas and lung tissue. Arrows identify pancreatic islet, A: Artery; B: Bronchiole; AV: Alveolus. Right panel: Immunofluorescence of native pancreatic and lung endothelial architecture (rat CD34, purple; DAPI, blue). A pancreatic islet (Insulin, green; DAPI, blue) is integrated in native vasculature. (C) Proteomic overlap of ECM proteins in decellularized lung (L, n=3) and pancreatic (P, n=3) scaffolds. Black square indicates the presence of protein.

Figure 2 - In vitro assembly of a VIO.

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(A) Assessment of islet distribution across the scaffold (n=3). A: Apex; UMS: Upper Middle Section; LMS: Lower Middle Section; B: Base. Islets per scaffold cross section: Apex=1.55±0.965; UMS=7.82±2.9; LMS=5.79±0.92; Base=3.66±0.72. (B) Percentage of islets delivered to the distal

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(Dist.) and proximal (Prox.) airways of decellularized left lung lobes (n=3) (C) Representative immunofluorescence image of islets delivered to the alveolar space. White arrows indicate intact

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alveolar membrane surrounding islets. (Ins, red; Coll IV, green; DAPI, blue). (D) Insulin secretion test (IST) of islets after 7 days in suspension culture. Comparison of ICM (green, n=3) with ICM (1d) + AM (4d) and MSM (2d) (green + blue + red, n=3). AUC: ICM= 22.67±10.04, ICM + AM + MSM= 18.25±1.82 pg/islet/20min, p=ns. (E) Left panel: Schematic drawing of a bioreactor for seeding and culture of VIOs. Right panel: Steps in assembly of VIOs. Scale bars in µm. Values presented as Mean ± s.d.

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Figure 3 - Spatial organization of VIOs.

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(A) Stitched image of a VIO’s cross-section after 7 d culture (human CD31, red; Insulin, green; DAPI, blue). (B) Immunofluorescence image of islets (Insulin, green; DAPI, blue) embedded in a

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re-generated endothelial network (human CD31, red; DAPI, blue). White arrows indicate smalldiameter vasculature integrated with the islets. (C) Immunofluorescence image of morphologically

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intact islet stained for insulin (green) and glucagon (purple) after 7 days culture (DAPI, blue). (D) Fluorescence microsphere perfusion. 0.2-µm microspheres (MS, blue) present in newly formed micro vasculature (human CD31, red) and surrounded by islets (insulin, green). Insert: location of CD31 (white). 50 µm section. (E) 15 µm z-stack image of vascular connection between regenerated human endothelium (human CD31, red) and rat capillary network (rat CD34, purple) of an islet (Insulin, green). Dotted: Immunofluorescent image shows transition of CD31+ to CD34+ vasculature (white arrow).

Figure 4 – Endocrine function of VIOs.

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(A) Culture protocol for IST. VIOs received batch-matched islets (1538±122 IEq) and were tested for insulin secretion on either day 1 (two hours after islet seeding) or day 7 of culture. (B) IST of

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VIOs after 1 and 7 days of culture (n=3). Values are expressed as pg/ml and normalized to the number of IEq seeded. Day 7 vs. day 1: 1 min: 43.8±3.21 vs. 17.05±3.21 p=0.0005; 2 min:

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36.6±3.58 vs. 13.53±2 p=0.001; 3 min: 29.77 ±7.9 vs. 13.81±2.3 p=0.029; 4 min: 17.07±17.59 vs. 6.15±4.08 p=ns; 5 min: 17.36±4,8 vs. 4.43±3.77 p=0.021; 10 min: 23.59±5.5 vs. 4.4±2.6 p=0.006;

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15 min: 16.7±3.8 vs. 2.89±2.13 p=0.005 and 20 min: 10±1.64 vs. 2.36±2.03 p=ns * p<0.05, ** p<0.01 and *** p<0.001. (C) Total insulin release (AUC) of VIOs cultured for 1 and 7 days (n=3). (D) Comparison of total insulin release (AUC) between VIOs and suspension cultured islets at day 1 and 7. Scale bars in µm. Values presented as Mean ± s.d.

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ACCEPTED MANUSCRIPT Figure 5 – Short term in vivo performance of VIOs (A) Diagram of experimental protocol. (B) Left panel: Photograph of a mature VIO ready for transplantation; Scale bar = 1 cm. Right panel: VIO transplanted into the abdominal cavity of a diabetic rat. PA and PV were connected to aorta (Ao) and inferior vena cava (IVC), respectively.

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(C-F) Comparison of intrahepatic islet transplantation (1500±89 IEq), VIO (1500±109 IEq) and VIO-islet (n=3). (C) Systemic plasma insulin during IVGTT (60 min post transplantation) in diabetic rats. * VIO vs. Intrahepatic p<0.05 (D) Total insulin release (AUC) over 60 minutes. (E) Point-by-

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point analysis of systemic blood glucose levels after IVGTT in diabetic rats. Pre-fasting blood glucose: Intrahepatic 473±10 mg/dl, VIO 487±11 mg/dl and VIO-islet 505±40 mg/dl mg/dl. VIO vs.

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Intrahepatic: 20 min: 461±32 vs. 516.±6 p=0.041; 30 min: 423±23 vs. 495.3±7.37, p=0.006, 45 min: 391±20 vs. 478±5, p=0.002 and 60 min: 359±34 vs. 440±28, p=0.033). * VIO vs. Intrahepatic p<0.05; ** p<0,01 (F) Glucose elimination constant (KG). KG 1-15 min: VIO 0.0077±0.003, Intrahepatic 0.0091±0.002, VIO-islet 0.0034±0.0021, VIO vs. Intrahepatic p=ns; KG 1-60 min: VIO

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0.0069±0.0011, Intrahepatic 0.0047±0.0006, VIO-islet =0.0027±0.0011; VIO vs. Intrahepatic p= 0.032, VIO vs. VIO-islet p= 0.0140 (G) Immunofluorescence image of VIO after 60 minutes of arterial blood perfusion. Islets (Insulin, green; DAPI, blue) and vascular network (human CD31,

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purple DAPI, blue) were preserved. Red blood cells (RBC, red) were found within the vascular bed

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and islet (insert). Scale bars in µm. Values presented as Mean ± s.d.

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Figure 6– Long term in vivo performance of VIOs. (A) Diagram of experimental protocol. (B) Daily not fasting glycaemia profile comparison between VIOs (n=13), DL (n=5) and SC (n=5). 30 days follow up. (* VIO vs. DL p=0.037; ** VIO vs. SC

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p=0.005; general linear model, repeated measures). (C) Kaplan Mayer analysis for gain of

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normoglycaemia (≤250 mg/dl). Differences between VIOs (n=13), DL (n=5) and SC (n=5) were estimated by Log Rank test. (**VIO vs DL p=0.0074 and **VIO vs SC p=0.0025). (D) Vascular perfusion performance comparison 30 days after transplantation. Left Panel: time intensity graph of VIOs (n=5), DL (n=5) and SC (n=3); Values present as mean ± SEM. Right panel: box plots represent median and 5-95 percentile of peak enhancement (PE) and total blood perfusion (AUC) respectively of VIOs (n=5), DL (n=5) and SC (n=3). (PE: *VIO vs DL p<0.05, *VIO vs SC p<0.01; AUC: *VIO vs DL p<0.05, *VIO vs SC p<0.05).

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Figure 7 – In vivo histological VIOs evaluation.

(A) Immunofluorescence images of VIO 8 days after transplantation. Islets (Insulin, green; DAPI, blue) human vascular network (human CD31, red; DAPI, blue) were preserved at 8 days. Scale bars in µm. (B) VIO preTx and gross pathology 14 days after transplantation. Upper dotted

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square:immunofluorescence of VIO edge show murine CD34+ vascular ingrowth in the close proximity of islet and human CD31+ area (Insulin, green; human CD31, red and murine CD34

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purple). Lower dotted square: murine vascular network (Insulin, green; human CD31, red; murine CD34 purple; DAPI, blue) was detected at 14 days after transplantation deep inside VIO. White

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arrows show presence of murine CD34+ vasculature. Scale bar in µm.