Cryopreservation of Hepatocyte Microbeads for Clinical Transplantation

Abstract

Intraperitoneal transplantation of hepatocyte microbeads is an attractive option for the management of acute liver failure. Encapsulation of hepatocytes in alginate microbeads supports their function and prevents immune attack of the cells. Establishment of banked cryopreserved hepatocyte microbeads is important for emergency use. The aim of this study was to develop an optimized protocol for cryopreservation of hepatocyte microbeads for clinical transplantation using modified freezing solutions. Four freezing solutions with potential for clinical application were investigated. Human and rat hepatocytes cryopreserved with University of Wisconsin (UW)/10% dimethyl sulfoxide (DMSO)/5% (300 mM) glucose and CryoStor CS10 showed better postthawing cell viability, attachment, and hepatocyte functions than with histidine-tryptophan-ketoglutarate/10% DMSO/5% glucose and Bambanker. The 2 freezing solutions that gave better results were studied with human and rat hepatocytes microbeads. Similar effects on cryopreserved microbead morphology (external and ultrastructural), viability, and hepatocyte-functions post thawing were observed over 7 d in culture. UW/DMSO/glucose, as a basal freezing medium, was used to investigate the additional effects of cytoprotectants: a pan-caspase inhibitor (benzyloxycarbonyl-Val-Ala-dl-Asp-fluoromethylketone [ZVAD]), an antioxidant (desferoxamine [DFO]), and a buffering and mechanical protectant (human serum albumin [HSA]) on RMBs. ZVAD (60 µM) had a beneficial effect on cell viability that was greater than with DFO (1 mM), HSA (2%), and basal freezing medium alone. Improvements in the ultrastructure of encapsulated hepatocytes and a lower degree of cell apoptosis were observed with all 3 cytoprotectants, with ZVAD tending to provide the greatest effect. Cytochrome P450 activity was significantly higher in the 3 cytoprotectant groups than with fresh microbeads. In conclusion, developing an optimized cryopreservation protocol by adding cytoprotectants such as ZVAD could improve the outcome of cryopreserved hepatocyte microbeads for future clinical use.

Keywords: acute liver failure; alginate encapsulation; apoptosis; clinical grade; cryopreservation; cytoprotectants; hepatocyte microbeads.

Fig. 1.

Effects of cryopreservation using the 4 different freezing solutions on human hepatocytes viability and function (N = 6). (A) Cell viability immediately after thawing assessed by trypan blue exclusion, (B) cell viability assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, (C) cell attachment assessed by sulforhodamine B assay, (D) albumin production, and (E) urea synthesis after thawed and plated for 24 h. Statistical significance as compared to fresh hepatocytes (controls): *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig. 2.

Representative images of human hepatocyte morphology after plating and maintenance in culture for 24 h (under light microscopy; 400×), (A) fresh hepatocytes (control). Cryopreserved hepatocytes using (B) University of Wisconsin solution/dimethyl sulfoxide (DMSO)/glucose, (C) CryoStorCS10, (D) histidine–tryptophan–ketoglutarate/DMSO/glucose, and (E) Bambanker.

Fig. 3.

Effects of 2 freezing solutions on cell viability and function of human hepatocyte microbeads (HMBs) after thawing and maintenance in culture for 7 d (N = 4); (A) cell viability, (B) albumin production, (C) urea synthesis, and (D) cytochrome P450 activity. Statistical significance: *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. Statistical significance compared to fresh HMBs: ^#P < 0.05, ^##P < 0.01, ^###P < 0.001, and ^####P < 0.0001.

Fig. 4.

Effects of 2 freezing solutions on cell viability and function of rat hepatocyte microbeads after thawing and 7 d maintenance in culture (N = 4). (A) Cell viability, (B) albumin production, (C) urea synthesis, and (D) cytochrome P450 family activity. Statistical significance compared to either immediately post thawing or compared to day 1: *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 and compared to fresh human hepatocyte microbeads: ^#P < 0.05 and ^##P < 0.01, ^###P < 0.001 and ^####P < 0.0001.

Fig. 5.

Transmission electron microscopy (TEM) of hepatocytes within microbeads after 24-h culture. Representative TEM images of normal healthy hepatocytes (left panel) and necrotic/apoptotic hepatocytes (right panel). (A, B) at magnification of 1,400×, and (C, D) at high magnification of 4,800×. cNC, condensed nuclear chromatin; G, Golgi apparatus; Ld, lipid droplet; Ly, lysosomes, Mt, mitochondria; N, nuclei; Nu, nucleoli; Nm, nuclear membrane; Pm, plasma membrane; RER, rough-surfaced endoplasmic reticulum; sMt, swelling mitochondria; V, vacuole.

Fig. 6.

Effect of cryopreservation with different cytoprotectants on viability and hepatocyte-specific functions of rat hepatocyte microbeads after thawing and maintenance in culture for 7 d (N = 4). (A) Cell viability, (B) albumin production, (C) urea synthesis, and (D) cytochrome P450 family activity. Statistical significance: *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Fig. 7.

Representative histograms of cell cycle analysis using fluorescence activated cell sorting (FACS). Cryopreserved rat hepatocyte microbeads were thawed and then cultured for 24 h. Hepatocytes were released from microbeads and analyzed using propidium iodide staining and FACs.