Macroporous Dual-compartment Hydrogels for Minimally Invasive Transplantation of Primary Human Hepatocytes

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.

Conclusions: 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.

Figure 1

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.

Figure 2

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.

Figure 3

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

Figure 4

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.

Figure 5

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

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.