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. Author manuscript; available in PMC: 2019 Sep 1.
Published in final edited form as: Transplantation. 2018 Sep;102(9):e373–e381. doi: 10.1097/TP.0000000000002330

Macroporous dual compartment hydrogels for minimally invasive transplantation of primary human hepatocytes

Nailah Seale a, Suvasini Ramaswamy b, Yu-Ru Shih c, Inder Verma b, Shyni Varghese a,c,d,e,*
PMCID: PMC6102079  NIHMSID: NIHMS975264  PMID: 29916986

Abstract

Background

Given the shortage of available organs for whole or partial liver transplantation, hepatocyte cell transplantation has long been considered a potential strategy to treat patients suffering from various liver diseases. Some of the earliest approaches that attempted to deliver hepatocytes via portal vein or spleen achieved little success due to poor engraftment. More recent efforts include transplantation of cell sheets or thin hepatocyte laden synthetic hydrogels. However, these implants must remain sufficiently thin to ensure that nutrients can diffuse into the implant.

Methods

To circumvent these limitations, we investigated the use of a vascularizable dual compartment hydrogel system for minimally invasive transplantation of primary hepatocytes. The dual compartment system features a macroporous outer Polyethylene glycol diacrylate/Hyaluronic acid Methacrylate hydrogel compartment for seeding supportive cells and facilitating host cell infiltration and vascularization, and a hollow inner core to house the primary human hepatocytes.

Results

We show that the subcutaneous implantation of these cell-loaded devices in NOD/SCID mice facilitated vascular formation while supporting viability of the transplanted cells. Furthermore, the presence of human serum albumin in peripheral blood and the immunostaining of excised implants indicated that the hepatocytes maintained function in vivo for at least 1 month, the longest assayed time point.

Conclusion

Cell transplantation devices that assist the anastomosis of grafts with the host can be potentially used as a minimally invasive ectopic liver accessory to augment liver specific functions as well as potentially treat various pathologies associated with compromised functions of liver such as hemophilia B or alpha-1 antitrypsin deficiency.

Keywords: Primary hepatocyte transplantation, dual-compartment hydrogels, hepatic tissue engineering, subcutaneous implantation, vascularization

1. Introduction

Approximately 30 million people in the United State have some form of liver disease1. Because the only approved cure for end-stage liver disease or acute liver failure is whole or partial organ transplantation, there remains a significant demand on transplantable donor organs2. This lack of available organs has led to approximately 27,000 deaths annually in the United States alone1,3. A number of strategies, ranging from cell4–8 to engineered liver tissue transplantation, are currently under investigation to help alleviate the demand for donor organs9–13.

Regardless of the approach, the success of any cell therapy hinges on the long-term survival and function of the transplanted cells. Ectopic transplantation of single and multi-layer sheets of hepatocytes has shown some success in maintaining liver tissue function14. However, given space constraints, to reasonably scale up this technique, sheets would have to be stacked. This will inevitably result in the diffusion barrier being breeched, thus necessitating the incorporation of vasculature. Another approach has been the use of biomaterials as scaffolds to engineer 3D liver tissues15. However, for these to function, the implants must be sufficiently thin. Furthermore, studies have shown that such implants function better when placed in a heavily vascularized area15,16, which usually requires an invasive surgery.

In an effort to improve the efficacy and function of the engineered tissues, de-cellularized liver organs have been employed as scaffolds17,18. De-cellularized livers can provide the structural and biochemical cues necessary to maintain viability and function of primary hepatocytes19,20. Furthermore, if used as intact structures, they provide an existing architecture that facilitates vascularization and provides liver specific geometrical cues. In this approach, due to the existing shortage of human organs, porcine or other xenogeneic livers have to be used. It is necessary to ensure proper de-cellularization to preserve the extracellular matrix (ECM) while ensuring the destruction of xenogeneic deoxyribonucleic acid (DNA). While low DNA content (under 50 ng double stranded DNA per mg ECM) may not have triggered an immune response in tested animals, it can still carry the risk of immune rejection in humans21,22.

Bioengineered devices that can facilitate vascularization of the implant while supporting the viability and function of transplanted cells could be a potential solution to improve the outcome of cell transplantation23. Enabling vascularization of the implant will allow the implant size to be scaled and a large number of cells to be housed within the device, which is necessary to improve the therapeutic outcome. In this study, we describe the use of a dual compartment device for minimally invasive hepatocyte transplantation. Recently we used such an approach successfully to support bone marrow transplantation24. Building upon this dual compartment concept, we optimized the biomaterial composition and dimensions to develop constructs with higher cell carrying capacity, capable of maintaining long-term hepatocyte function and promoting vascularization. The dual compartment system consists of an outer interconnected, macroporous solid structure to promote vascularization and/or to house supporting cells and a hollow inner compartment to load the donor cells. When implanted subcutaneously in mice, the cell-loaded dual compartment device supported the viability and sustained function of transplanted primary human hepatocytes (from 2 different donors) for at least 1 month (the longest experimental time investigated).

2. Materials and Methods

2.1 Polyethylene glycol diacrylate (PEGDA) synthesis

PEGDA (Mn=10kDA) oligomer was prepared according to a previously reported method25. Briefly, 18.0 g of PEG was dissolved in 300 mL of toluene in a 500 mL round bottomed flask in an oil bath heated at 125 °C. The solution was refluxed for 4 h with vigorous stirring. Traces of water in the reaction mixture were removed by azeotropic distillation. Upon cooling the solution to room temperature, 3.262 g (32.2 mmol, 4.493 mL) of triethylamine was added to it with vigorous stirring. Then the flask was moved to an ice bath and stirred for 30 min. 2.918 g (32.2 mmol, 2.452 mL) of acryloyl chloride in 15 mL of anhydrous dichloromethane was then added to the reaction mixture dropwise over 30 min. After keeping the reaction mixture in the ice bath for another 30 min, the flask was heated to 45°C overnight. The reaction mixture was then cooled to room temperature and the quaternary ammonium salt was removed from the mixture by filtration. The filtrate was condensed using a rotary evaporator and precipitated in excess diethyl ether. The white precipitate was collected by filtration and vacuum dried at 40 °C for 24 h. The resultant PEGDA oligomer was purified by precipitation followed by column chromatography and dialysis prior to its usage. The purified PEGDA was lyophilized and stored at −20°C.

2.2 Hyaluronic acid Methacrylate (HAMA) synthesis

Sodium Hyaluronate (Lifecore Biomedical), Research Grade, 41KDa-65KDa Mw (500 mg) was dissolved in DI water (25 mL). Methacrylate (MA) anhydride (8 mL) was added into the HA solution (drop-by-drop manner), pH was adjusted to 8 and the reaction was carried out at 4°C for 24 hrs. pH was checked frequently and adjusted to 8 as needed. After 24 hrs the resulting mixture was purified using membrane dialysis (3.5-5 kDa) against Milli-Q water for 3 days, lyophilized, and stored at −20°C.

2.3 Porous scaffold formation

The porous scaffolds (either PEGDA or PEGDA/HAMA copolymer) were made by the leaching of polymethyl methacrylate (PMMA) beads to create the macroporous structure26. Briefly, 160 μm PMMA beads were packed in a 10mm diameter by 3mm height mold. 80 μl of 20% acetone solution (in ethanol) was added to the PMMA filled mold before it was placed in a 37°C oven for 10 min. To this PMMA filled mold a PEGDA/HAMA solution (10%/5% w/v mixture in PBS) or a 10% PEGDA (w/v mixture in PBS) containing 0.005% (w/v) Irgacure (ie, a photoinitiator) was added and UV polymerized for 10 min. Acetone was used to dissolve the PMMA beads to create the macroporous hydrogel structures (Figure 1A). The structure was sterilized using multiple ethanol washes, followed by multiple PBS washes.

Figure 1.

Figure 1

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Fabrication of dual compartment device. A: Creating PEGDA/HAMA outer porous compartment via acetone leeching of PMMA beads. B: Assembly of the dual compartment device. Primary hepatocytes encapsulated in fibrin are loaded into the inner compartment, while supporting cells are seeded into the outer compartment. After 3 days in vitro structures are subcutaneously implanted in NOD/SCID mice. C: Dimensions of the dual compartment device. Gross structure of the device before and 28 days after implantation. Scale 0.25 cm.

2.4 Dual compartment assembly

A 7 mm punch was used to cut out the center of the porous gels to leave a hollow ring. The 7 mm inner portion that was removed was sliced to make caps for the hollow ring. Both the hollow rings and the caps were sterilized with ethanol then washed multiple times with PBS. Under sterile conditions, the ring and the caps were dried to remove the solvent from the pores. Then, supporting cells (some combination of HUVECS, hMSCs or MEFs) were loaded into the outer compartment and the caps. The primary human hepatocytes were thawed in thawing media (MCHT50; Lonza) and centrifuged for 10 minutes at 100 g. Next, fibrinogen (8 mg/ml) and thrombin (2 U/ml) were added to the cell pellet. Fibrin was allowed to form at 37 °C and the system was maintained at this temperature for up to 30 min. to complete the reaction. Once the gel was formed it was placed into the inner cored out compartment of the porous gel (now consisting of the hollow ring and the bottom cap). The top cap is then placed to seal the dual compartment system. This resulted in a cell-laden fibrin gel inner compartment, encased by a macroporous gel outer compartment (Figure 1 B and C). Fibrin was then used around the caps as an added precaution to seal them in place. Each device was loaded with approximately 5 million hepatocytes (or 5-20 million when testing loading capacity) and approximately 5×105 supporting cells. Human Umbilical Vein Endothelial Cells (HUVECS), Human Bone Marrow Stromal Cells (hMSCs) and Mouse embryonic Fibroblasts (MEFs) were used either alone (5×105 HUVECS) or in combination (2.5×105 HUVECS plus 2.5×105 hMSCS or MEFs) as supporting cells. In order to determine the effect of the scaffold to facilitate in vivo vascularization, acellular PEGDA and PEGDA/HAMA constructs were used.

2.5 Swelling Ratio

Dual compartment systems made from PEGDA macroporous hydrogels and PEGDA/HAMA macroporous hydrogels were assembled as described above, however, without the inner fibrin compartment. Gels were flash frozen and lyophilized for 2 days. Then 5 gels for each group were weighed to get the dry weight. Gels were then placed in PBS and weighed periodically over a 48 hour time period to determine the wet weight. The wet weight was divided by the dry weight to get the swelling ratio and swelling kinetics27.

2.6 Scanning Electron Microscopy

The microstructure of the PEGDA/HAMA hydrogels was examined using a scanning electron microscope (SEM). Briefly, samples were thinly sectioned, flash frozen, and lyophilized for 2 days. Then using a sputter coater (Emitech, K575X), Iridium was coated onto samples for 7 s. The iridium-coated samples were imaged using an SEM machine (Phillips XL30 ESEM). The diameter of interconnected pores was measured using ImageJ by randomly selecting 10 pores from each of 3 different SEM and bright-field images, respectively, and presented as mean ± standard deviation (n = 30).

2.7 Cell Culture

Primary human hepatocytes from 2 different donors (Donor 1, HUM4100 and Donor 2 HUM4113) were acquired from LONZA (formerly TRL). The cells were thawed in thawing media (MCHT50, LONZA) and immediately encapsulated into fibrin gel and loaded into scaffolds. The cells encapsulated in fibrin gel were loaded as described above. The cell-laden dual compartment scaffolds were cultured in 4 parts maintenance media (MM250, LONZA) and 1 part HUVEC media (components described below).

Human Umbilical Vein Endothelial Cells (HUVECs) were obtained from ATCC and cultured in HUVEC medium (HM) containing 79% M199 medium (Gibco), 10% FBS (Gibco), 10% endothelial cell growth medium (Cell Application, Inc.), and 1% penicillin/streptomycin (Gibco). HUVECs used in this study were limited to cells between passages 3 and 5.

Mouse embryonic Fibroblasts (MEFs) were cultured in growth medium (GM), composed of Dulbecco’s Modified Eagle’s high glucose medium (Hyclone) supplemented with 10% fetal bovine serum (FBS, Gibco) and 1% penicillin/streptomycin (Gibco). The cells were grown on 0.1% (w/v) Gelatin coated dishes to 70% confluency.

Human Bone Marrow Stromal Cells (hMSCs) were acquired from the Institute for Regenerative Medicine, Texas A&M University (Donor 8013L). Cells were cultured in GM composed of Dulbecco’s Modified Eagle’s high glucose medium (Hyclone) supplemented with 16.5% fetal bovine serum (FBS, Gibco) and 1% penicillin/streptomycin (Gibco). The cells were passaged at 70% confluence and used for experiments at passages 4-5.

2.8 Subcutaneous implantation of devices

All animal procedures were approved by the Institutional Animal Care and Use Committee of the University of California, San Diego and performed in accordance with the NIH and national and international guidelines for laboratory animal care. Subcutaneous implantation of the cell-laden dual compartment devices was performed 3 days after assembly and culture in vitro. Recipient mice were administered with ketamine (100 mg/kg) and xylazine (10 mg/kg), and the fur on the back was shaved. Mice were then placed on a heating pad and a 1 cm-long incision was made in the back of the mice, and 1 subcutaneous pouch was inserted by blunt dissection using a 1 cm-wide spatula on the left side of the mouse. The process was repeated on the right side. A total of 2 devices were implanted per mouse. The skin was sutured once the devices were implanted. After the surgery, mice were housed in separated cages.

2.9 Albumin measurement

Sandwich enzyme linked immunosorbent assay (ELISA) was performed to assess the albumin production of the implanted cells as a function of time (over 28 days). In short, once a week, blood was collected from the mice via the tail vein using heparinized capillary tubes. The blood was then centrifuged at 14 900 g at 4°C for 15 min. to separate and extract the serum. The serum was assessed for human albumin by using the Human Albumin ELISA Quantitation Set (Bethyl Labs, Catalogue no. E80-129) according to the manufacturer’s protocol.

2.10 Vessel quantification

Acellular implants were placed in mice for 3, 7, 14 and 28 days to assess cell infiltration and vascularization over time. At each time point the implants were retrieved and washed with PBS. Then gross images of the implant were taken before fixing them for immunostaining. The number of visible vessels that contained blood were counted and divided by the total surface area.

2.11 Immunofluorescent staining

Post implantation constructs were retrieved, washed with PBS, and fixed with 4% paraformaldehyde (PFA, Sigma Aldrich) at 4 °C overnight. Samples were then incubated in OCT (Tissue-Tek O.C.T. Compound; Sakura, Torrance, CA) at 4 °C overnight on a rocker. Samples were transferred to a mold and frozen with 2-methylbutane and liquid nitrogen, and stored at −80°C until sectioning. For cryo-sectioning, frozen tissue blocks were sectioned with a cryotome cryostat (at −20 °C) to 20-μm thicknesses. For immunofluorescent staining, sections were treated with 20 μg/mL proteinase K, permeabilized with 0.5% Triton X-100 [4 min, room temperature (RT)], treated with NABH4 (30 min, RT), and blocked with 3% (w/v) bovine serum albumin (BSA; 60 min, RT) in PBS. Sections were stained for either CD31 (platelet endothelial cell adhesion molecule (PECAM-1); 1:100; Santa Cruz) or FITC conjugated human albumin (1:100; Bethyl Labs) or CK18 (R&D Systems) overnight at 4 °C. An appropriate secondary antibody Alexa Fluor 488 (1:200; Thermo Fisher) along with Hoechst 33342 (2 μg/mL; Thermo Fisher) was used to bind primary antibodies for 1 h at RT. Then samples were imaged with a fluorescent microscope.

2.12 Statistical analysis

All experiments were independently repeated at least twice with replicate samples as indicated in figure captions. Statistical analysis was performed using one-way ANOVA and Tukey’s post hoc test for group comparisons to determine statistical significance (p < 0.05). Errors bars represent SEM. GraphPad Prism 5 software was used to determine all statistical analysis.

3. Results

3.1 Development and characterization of the dual compartment system

The dual compartment device was developed as a cell-carrying device to successfully transplant primary human hepatocytes (Figure 1A & B). The dual compartment structure consisted of a 3 mm height by 5 mm radius, interconnected macroporous hydrogel (outer compartment), with a 1.5 mm height by 3.5 mm radius hollow interior (inner compartment) for cell loading (Figure 1C). Macroporous hydrogels with interconnected pores were fabricated by PMMA templating of PEGDA or PEGDA-co-HAMA crosslinked networks. SEM images were used to verify the presence of an interconnected macroporous network. The SEM images suggest that the gels had both larger pores, which were about 140 microns in diameter, and smaller pores, which were about 85 microns in diameter (Figure 2A) with an average pore size of 120 microns. We also determined the swelling ratio of the macroporous hydrogels (PEGDA/HAMA and PEGDA) (Figure 2B). PEGDA/HAMA macroporous hydrogels had a higher swelling ratio compared to PEGDA alone structures. Similarly, PEGDA/HAMA structures swelled and equilibrated faster compared to PEGDA alone structures.

Figure 2.

Figure 2

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Material Characterization. A. SEM images of porous network in the PEGDA/HAMA outer compartment. B. Swelling ratio and kinetics of PEGDA/HAMA porous hydrogel compared to PEGDA porous hydrogel.

3.2 Porous PEGDA/HAMA facilitates vascular formation

To examine the ability of the macroporous structures to promote vascularization, both PEGDA and PEGDA/HAMA structures were implanted in vivo without the presence of any exogenous cells. In addition to the appearance, we used immunofluorescent staining for DAPI (stains nucleus) and CD31 (a vascular marker) to assess host cell infiltration and vascular formation as a function of postimplantation time (3-28 days). Figure 3 shows the representative whole-mount images of the excised implants and the staining results. Analyses of the implants after 3 days of subcutaneous implantation showed minimal to no vascularization. While the sections were positive for DAPI staining, indicating that host cells had infiltrated the implant, no positive CD31 was observed. At the next experimental time point, day 7, the presence of vascular structures could be clearly seen in the PEGDA/HAMA constructs. The constructs appeared pink with clearly visible vascular structures filled with blood. The presence of vascular structures was further confirmed by the positive CD31 staining. As the days increased, increased vessel formation (Figure 3 A) as well as more CD31 positive cells was observed (Figure 3 B). By day 14, larger blood vessels filled with blood were observed while multiple smaller vessels filled with blood were seen at day 28. Interestingly, no such vascularization was observed with PEGDA constructs (Figure S1). The PEGDA constructs appeared as transparent as before implantation.

Figure 3.

Figure 3

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Vascularization of Acellular Structures. A. Top Panel: Gross structure of implants after retrieval from subcutaneous implantation in NOD/SCID mice. White arrowheads point to visible vasculature. Scale: 0.25 cm. Middle Panel: CD31 (green) and DAPI (blue) staining to visualize vasculature. Scale: 10μm. Lower panel: Magnified image of DAPI and CD 31 staining. Scale: 10μm. Each time point had n=6 or more constructs. B. Quantification of blood vessels. Data are presented as mean ± SE obtained from 6 engineered constructs (n = 6) per group. One-way ANOVA with Tukey post hoc test (on day 7, 14 and 28). ***P < 0.0001. **P < 0.001

3.4 Effect of supporting cells on function in vivo

Since the supporting cells could play a key role in maintaining hepatocyte function15,28, we have compared the effect of i) MEFs plus HUVECs and ii) hMSC plus HUVECs on the functionality of hepatocytes, based on albumin secretions. At 1 week posttransplantation, both MEFs and hMSCs-supported implants showed similar levels of albumin secretion, which was only slightly higher compared to the HUVECs only group (Figure 4 A & B). All groups showed significantly higher albumin productions compared to week 1. Amongst the different groups, implants loaded with MEFs or hMSCs produced more albumin than the HUVECs only group. Between the hMSCs and MEFs, the group containing hMSCs produced more albumin than the group containing MEFs. Even though there was no statistically significant difference between the albumin secretions amongst the week 3 hMSCs plus HUVECs group and the MEFs plus HUVECs, all subsequent experiments were carried out using a combination of HUVECs and hMSCs as the supporting cells. In addition to presence of human albumin in the host peripheral blood, we also stained the excised implants and they were positive for human specific albumin (Figure 4 A).

Figure 4.

Figure 4

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Effect of supporting cells on albumin production. A. Albumin staining of scaffolds with hMSCs and HUVECS supporting cells at low and high magnifications. Blue represents DAPI and green represents albumin. Scale: 100μm and 10μm respectively. B. Secretion of human serum albumin for supporting cells HUVECS only, HUVECs and hMSCs and HUVECS and MEFs at week 1 and week 3 post implantation. Data are presented as mean ± SE obtained from 6 engineered constructs (n = 6). One-way ANOVA with Tukey post hoc test. *P < 0.05. **P < 0.01.

3.4 Donor independent function of Dual compartment system in vivo

After characterizing and developing the dual compartment system, we wanted to ensure that the device could support cells from multiple donors. To this end, we used the dual compartment system to transplant cells from 2 different donors (Table S1). The implant function was assessed for functionality via albumin secretions over a 1-month period postimplantation (Figure 5 and 6). Albumin analysis of host serum indicated the presence of human specific albumin in the circulation of the host at day 7 postsubcutaneous implantation. The amount of albumin increased from day 7 to day 15 and remained more or less stable for the rest of the month (Figure 5B). On day 28 the implants were retrieved and analyzed. The gross picture of both the implants indicated vascular formation (Figure 6B column 1). DAPI and albumin staining of the implant (Figure 5A and 6B column 2) showed that in all implants, the albumin staining was concentrated within the inner compartment. The cells in the inner compartment of the implant were also positive for CK18, another hepatocyte specific marker (Figure 6B column 3). The implants were also positive for CD31 staining, which was used to identify vascular cells (Figure 6B column 4). The staining results corroborated the presence of vascular networks observed earlier by us in the whole-mount images.

Figure 5.

Figure 5

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Assessing donor cell function in vivo via human albumin secretion. A. Albumin staining for donor 1 & 2 after 28 days in vivo. Blue represents DAPI and green represents albumin. Scale: 100μm and 10μm respectively. B. ELISA analysis of human serum albumin secretions of donor 1 and 2 in NOD/SCID mice over 3 time points, day 7, day 15 and day 28. Data are presented as mean ± SE obtained from 6 engineered constructs (n = 6). One-way ANOVA with Tukey post hoc test. *P < 0.05. **P < 0.01. ***P < 0.001.

Figure 6.

Figure 6

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Immunofluorescent staining of retrieved implants. A. Experimental timeline. B. Column 1: Donor 1 and 2 gross structure of device 28 days post implantation, with white arrows pointing at visible vascular networks. Scale: 0.25 cm. Column 2: DAPI (blue) and Albumin (green) staining to visualize a slice of the dual compartment system. Scale: 200μm. Column 3: Ck18 (green) and DAPI (blue) staining. Scale: 50μm and 10μm. Column 4: CD31 (red) and DAPI (blue) staining to visualize vasculature. Scale: 50μm and 10μm. At least 3 mice were used per donor, each containing 2 implants.

4. Discussion

This study describes the application of a dual compartment device, containing an outer vascularizable layer and an inner cell-loading compartment, for hepatocyte transplantation. The hollow core structure of the inner compartment enables loading of a large number of cells. At the current dimensions, the inner compartment can be easily loaded with up to 20 million hepatocytes (largest number tested for loading, Figure S2). Furthermore, our results demonstrated that the vascularization of the implant in the subcutaneous space supported donor cell function for 1 month.

The design of the dual compartment system, especially the hydrogel composition and architecture played an integral role in host cell infiltration, and device vascularization. Previously, we have shown that mineralized macroporous hydrogels promote host cell infiltration24,29. The findings that the PEGDA macroporous hydrogels were not able to promote vascularization in vivo suggest that pore architecture alone is not sufficient to facilitate vascular formation. The addition of HA to the PEGDA allowed cell infiltration and implant vascularization. This could be due to various reasons such as the biological functions of HA and its ability to interact with cell surface receptor CD4430,31. Furthermore, the addition of HA could have promoted vascular formation, as studies have shown that HA fragments exhibit pro-angiogenic effects30,32,33. While vascularization of the implant enables long-term maintenance of donor cells, the faster swelling-kinetics of the macroporous dual compartment device could be playing an important role in maintaining the viability of the donor cells in the initial days of the implantation (ie, before the implant was vascularized) through enhanced nutrient transport to the cells. Since these implants are large and thick, to avoid necrotic cores, fast swelling is important to ensure absorption and transport of nutrients throughout the structure.

While the liver has many functions, here we chose to use human serum albumin secretions to characterize the function of the implants. This allowed us to monitor function of the same implant, with minimal interference, over multiple time points. For our proof of concept study, ELISA analysis of albumin secretions for both donors followed the trend of rising after day 7 and then remaining stable for the rest of the experiment duration. Based on albumin synthesis and secretions, there were no statistically significant differences between the cells from different donors.

Although this proof of concept study used primary hepatocytes, the dual compartment system could be extended towards other cells. The modular assembly of the device allows parallel optimization of the outer and inner compartments to increase overall function of the device. Tuning pore size or material composition to accelerate host cell infiltration and vascularization can optimize the outer compartment. Similarly, the dimensions of the inner compartment could be increased to improve the number of donor cells that can be housed, while maintaining the overall outer dimensions. Increasing the dimensions of the inner compartment without changing the overall dimensions of the device involves decreasing the thickness of the outer compartment, which could improve diffusion of nutrients to the cells in the inner compartments. Further studies are needed to determine the upper cell-loading limit of these devices without compromising their viability or function. It is estimated that a delivery of 1-10 billion functioning cells is needed to achieve therapeutic effects in leu of solid organ transplantation34. Even with the vascularization, housing such a large number of cells within a single device could be challenging. However, since the dimensions of the device can be tuned, it is possible to increase the dimension of the inner compartment to accommodate more cells. If a diffusion limitation is reached leading to death and compromised function of the transplanted cells, then multiple devices could be transplanted. Another key parameter that determines successful cell transplantation is the longevity of the donor cells. The diminished function of the transplanted cells with time is often thought to be associated with lack of vascularization. Studies have shown that cell transplantation approaches that incorporated vascularization resulted in improvements to the viability and function of the transplanted cells. The results described in this study show the viability and function of the transplanted cells for a month. Though the vascularization of the implant suggests the possibility of survival and function of the transplanted cells beyond a month, additional studies are needed to assess the potential of the device to support long-term viability and function of the transplanted cells. Nonetheless, the dual compartment system described in this study offers a promising tool for cell transplantation. Furthermore, its function can be easily extended for applications in drug screening, personalized medicine and as a platform to screen for key components (eg, ECM composition, stiffness) of the microenvironment that are necessary to maintain long-term function of donor cells. It can also be used as an in vivo experimental tool to study how donor phenotype can affect transplantation success.

Conclusion

In conclusion, developing successful biomaterial devices for transplantable liver cell therapies requires an approach that can transplant large numbers of cells and be integrated with the host to facilitate formation of the functional vasculature needed to maintain the viability and function of transplanted cells. To meet these criteria, we have utilized a dual compartment biomaterial device for the transplantation of human primary hepatocytes. This device enabled minimally invasive (subcutaneous implant) cell transplantation and maintained the function of transplanted hepatocytes for at least 1 month. The dual compartment device described here is robust and scalable. Furthermore, the modular assembly of the device can be used as a tool to create and optimize scalable vascularized 3D liver tissues for extended applications in creating humanized tissue models for investigating disease pathology, drug testing and personalized medicine.

Supplementary Material

Supplemental Digital Content to Be Published _cited in text_

Figure S1: Addition of HAMA to promote vascular formation. A. Schematic of porous biomaterial implanted into NOD/SCID mice. B. Appearance of HAMA/PEGDA(left) and PEGDA (right) macroporous hydrogels after 2 weeks subcutaneous implantation in NOD/SCID mice.

Figure S2: Cell loading. Gross structure images of dual compartment gels with increasing number of cells (5, 10 and 20 million hepatocytes) loaded in the inner compartment. Scale: 0.25 cm

Acknowledgments

Funding

S.V and I.V acknowledges the generous financial support from California Institute of Regenerative Medicine grants (RT3-07907 and TR4-06809). N.M.S. Acknowledges the funding support of the National Science Foundation Graduate Research Fellowship Program.

List of Abreviations

3D

Three-dimensional

ANOVA

Analysis of variance

CD31

platelet endothelial cell adhesion molecule (PECAM-1)

CK18

Cytokeratin-18

DA

daltons

DAPI

4′,6-diamidino-2-phenylindole

DNA

deoxyribonucleic acid

ECM

extracellular matrix

ELISA

enzyme linked immunosorbent assay

FBS

fetal bovine serum

GM

growth medium

HA

hyaluronic acid

HAMA

hyaluronic acid Methacrylate

HM

human umbilical vein endothelial cells medium

HUVECS

human umbilical vein endothelial cells

hMSCs

human bone marrow stromal cells

MA

methacrylate

MEFs

mouse embryonic fibroblasts

NaBH3

sodium borohydride

NIH

national institute of health

OCT

optimum cutting temperature

PBS

phosphate buffered saline

PEG

polyethylene glycol

PEGDA

polyethylene glycol diacrylate

PMMA

polymethyl methacrylate

RT

room temperature

SEM

scanning electron microscope

Footnotes

Authorship

Nailah Seale: Participated in research design, performance of the research, analysis and writing of the manuscript

Suvasini Ramaswamy: Conceived the idea, participated in research design, and editing the manuscript

Yu-Ru Shih: Participated in animal surgeries and editing of the manuscript.

Inder Verma: Conceived the idea and editing the manuscript

Shyni Varghese: Conceived the idea, participated in experimental design, data interpretation, and writing the manuscript.

Disclosures

None.

References

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Supplementary Materials

Supplemental Digital Content to Be Published _cited in text_

Figure S1: Addition of HAMA to promote vascular formation. A. Schematic of porous biomaterial implanted into NOD/SCID mice. B. Appearance of HAMA/PEGDA(left) and PEGDA (right) macroporous hydrogels after 2 weeks subcutaneous implantation in NOD/SCID mice.

Figure S2: Cell loading. Gross structure images of dual compartment gels with increasing number of cells (5, 10 and 20 million hepatocytes) loaded in the inner compartment. Scale: 0.25 cm

NIHMS975264-supplement-Supplemental_Digital_Content_to_Be_Published__cited_in_text_.pdf (791.8KB, pdf)

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