Alterations in the Blood Parameters and Fecal Microbiota and Metabolites during Pregnant and Lactating Stages in Bama Mini Pigs as a Model.

This study was conducted to analyze plasma reproductive hormone and biochemical parameter changes, as well as fecal microbiota composition and metabolites in sows, at different pregnancy and lactation stages, using Bama mini pig as an experimental animal model. We found that plasma prolactin (PRL), progesterone, follicle-stimulating hormone (FSH), and estrogen levels decreased from day 45 to day 105 of pregnancy. Plasma total protein and albumin levels were lower in pregnant sows, while glucose, urea nitrogen, total cholesterol, and high-density lipoprotein-cholesterol, as well as fecal acetate, butyrate, valerate, total short-chain fatty acids, skatole, and tyramine levels, were higher in lactating sows. Interestingly, the lactating sows showed lower α-diversity and Spirochaetes and Verrucomicrobia relative abundances, while pregnant sows showed a higher Proteobacteria relative abundance. Notably, the Akkermansia relative abundance was highest on day 7 of lactation. Spearman analysis showed a positive correlation between plasma triglyceride and cholinesterase levels and Akkermansia and Streptococcus relative abundances. Moreover, Oscillospira and Desulfovibrio relative abundances were also positively correlated with plasma FSH, LH, and E2 levels, as well as PRL and LH with Bacteroides. Collectively, plasma reproductive hormones, biochemical parameters, and fecal microbiota composition and metabolite levels could alter along with pregnancy and lactation, which might contribute to the growth and development demands of fetuses and newborns.

Chemical and Nutraceutical Studies on Infertility of Albino Rats Induced by Cadmium Chloride.

BACKGROUND AND OBJECTIVE: Infertility in couples is rated one in every eight couple worldwide which affects 15% of couples and a male factor is found to be solely responsible or in conjunction with a female factor in 50% of cases. The natural chemicals found in rocca and red cabbage leaves breakdown into compounds like indole-3-carbinol, which has anti-cancer property. Flavonoids of the crop have good therapeutic potential in inflammation and pain. Meanwhile, this investigation aimed to evaluate the effect of rocca leaves and red cabbage leaves on male infertility rats. MATERIALS AND METHODS: Thirty-six adult male Sprague Dawley rats were divided into six groups. Group 1: Normal rats fed on basal diet as control negative (C-), Group 2: Control positive C+, in which infertility rats were fed on basal diet. Group 3: Infertility rats fed on basal diet and 5% rocca leaves. Group 4: Infertility rats fed on basal diet and 10% rocca leaves. Group 5: Infertility rats fed on basal diet and 5% red cabbage leaves. Group 6: Infertility rats fed on basal diet and 10% red cabbage leaves. At the end of experiment, after 28 days of feeding, all serum samples were analyzed for biochemical parameters. RESULTS: Injection with cadmium chloride caused a significant increase in the level of glucose, urea, creatinine, uric acid, AST, ALT, ALP, total cholesterol, triglycerides, LDLc, VLDLc, AI, Glob, TB, IB, DB and LH hormone while a significant decrease was recorded in HDLc, testosterone, FSH hormones, TP and Alb. Meanwhile, in infertility rats then treated with rocca leaves 5 and 10% and red cabbage leaves at the same doses 5 and 10% caused significant improvement in all tested parameters. CONCLUSION: The obtained results demonstrated that rocca leaves and red cabbage leaves had significant improvement in testosterone, Follicle-stimulating hormone, luteinizing hormone, total protein, albumin and lipids profile in cadmium chloride induced infertility in rats.

Use of 3D Human Liver Organoids to Predict Drug-Induced Phospholipidosis.

Drug-induced phospholipidosis (PL) is a storage disorder caused by the formation of phospholipid-drug complexes in lysosomes. Because of the diversity of PL between species, human cell-based assays have been used to predict drug-induced PL in humans. We established three-dimensional (3D) human liver organoids as described previously and investigated their liver characteristics through multiple analyses. Drug-induced PL was initiated in these organoids and in monolayer HepG2 cultures, and cellular changes were systemically examined. Organoids that underwent differentiation showed characteristics of hepatocytes rather than HepG2 cells. The organoids also survived under PL-inducing drug conditions for 48 h and maintained a more stable albumin secretion level than the HepG2 cells. More cytoplasmic vacuoles were observed in organoids and HepG2 cells treated with more potent PL-induced drugs, but to a greater extent in organoids than in HepG2 cells. Lysosome-associated membrane protein 2, a marker of lysosome membranes, showed a stronger immunohistochemical signal in the organoids. PL-distinctive lamellar bodies were observed only in amiodarone-treated organoids by transmission electron microscopy. Human liver organoids are thus more sensitive to drug-induced PL and less affected by cytotoxicity than HepG2 cells. Since PL is a chronic condition, these results indicate that organoids better reflect metabolite-mediated hepatotoxicity in vivo and could be a valuable system for evaluating the phospholipidogenic effects of different compounds during drug development.

Expansion of Transdifferentiated Human Hepatocytes in a Serum-Free Microcarrier Culture System.

BACKGROUND AND AIMS: Bioartificial livers (BALs) have attracted much attention as potential supportive therapies for liver diseases. A serum-free microcarrier culture strategy for the in vitro high-density expansion of human-induced hepatocyte-like cells (hiHeps) suitable for BALs was studied in this article. METHODS: hiHeps were transdifferentiated from human fibroblasts by the lentiviral overexpression of FOXA3, HNF1A, and HNF4A. Cells were cultured on microcarriers, their proliferation was evaluated by cell count and CCK-8 assays, and their function was evaluated by detecting liver function parameters in the supernatant, including urea secretion, albumin synthesis, and lactate dehydrogenase levels. The expressions of hepatocyte function-associated genes of hiHeps were measured by qRT-PCR in 2D and 3D conditions. The expression of related proteins during fibronectin promotes cell adhesion, and proliferation on microcarrier was detected by western blotting. RESULTS: During microcarrier culture, the optimal culture conditions during the adherence period were the use of half-volume high-density inoculation, Cytodex 3 at a concentration of 3 mg/mL, a cell seeding density of 2.0 × 105 cells/mL, and a stirring speed of 45 rpm. The final cell density in self-developed, chemically defined serum-free medium (SFM) reached 2.53 × 106 cells/mL, and the maximum increase in expansion was 12.61-fold. In addition, we found that fibronectin (FN) can promote hiHep attachment and proliferation on Cytodex 3 microcarriers and that this pro-proliferative effect was mediated by the integrin-ß1/FAK/ERK/CyclinD1 signaling pathway. Finally, the growth and function of hiHeps on Cytodex 3 in SFM were close to those of hiHeps on Cytodex 3 in hepatocyte maintenance medium (HMM), and cells maintained their morphology and function after harvest on microcarriers. CONCLUSIONS: Serum-free microcarrier culture has important implications for the expansion of a sufficient number of hiHeps prior to the clinical application of BALs.

4 in 1: Antibody-free protocol for isolating the main hepatic cells from healthy and cirrhotic single rat livers.

Liver cells isolated from pre-clinical models are essential tools for studying liver (patho)physiology, and also for screening new therapeutic options. We aimed at developing a new antibody-free isolation method able to obtain the four main hepatic cell types (hepatocytes, liver sinusoidal endothelial cells [LSEC], hepatic macrophages [HMΦ] and hepatic stellate cells [HSC]) from a single rat liver. Control and cirrhotic (CCl4 and TAA) rat livers (n = 6) were perfused, digested with collagenase and mechanically disaggregated obtaining a multicellular suspension. Hepatocytes were purified by low revolution centrifugations while non-parenchymal cells were subjected to differential centrifugation. Two different fractions were obtained: HSC and mixed LSEC + HMΦ. Further LSEC and HMΦ enrichment was achieved by selective adherence time to collagen-coated substrates. Isolated cells showed high viability (80%-95%) and purity (>95%) and were characterized as functional: hepatocytes synthetized albumin and urea, LSEC maintained endocytic capacity and in vivo fenestrae distribution, HMΦ increased expression of inflammatory markers in response to LPS and HSC were activated upon in vitro culture. The 4 in 1 protocol allows the simultaneous isolation of highly pure and functional hepatic cell sub-populations from control or cirrhotic single livers without antibody selection.

Effects of feed supplementation on growth, blood parameters and reproductive performance in Sanga and Friesian-Sanga cows grazing natural pasture.

This study determined the effects of feed supplementation during the postpartum period on the weight gain, milk yield, blood profiles and reproductive performance of Sanga and Friesian-Sanga cows grazing on natural pasture. 20 Sanga and 20 Friesian-Sanga cows were randomly allocated either to serve as a control on grazing only or to be supplemented with 2.5 kg of concentrate a day for 10 weeks during the dry season. Each week, all cows were weighed and scored for body condition. Partial milk yield of cows was determined daily. Plasma concentrations of blood metabolites were assessed fortnightly from weeks 1 to 10 postpartum. Resumption of postpartum ovarian activity was determined by measuring progesterone concentration in the plasma from weeks 1 to 10. Supplemented cows had a better body condition score (6.2 versus 5.8; P < 0.05) and higher partial milk yield (1.94 versus 1.55 L/day; P < 0.01) than non-supplemented cows. Sanga cows had a better body condition score (6.2 versus 5.8; P < 0.05) but lower milk yield (1.58 versus 1.92 L/day; P < 0.01) than the Friesian-Sanga crossbreds. Total protein (P < 0.05) and albumin (P < 0.01) concentrations were higher in the supplemented than in the non-supplemented cows. Sanga cows recorded higher globulin (P < 0.05) and total cholesterol (P < 0.01) but lower albumin (P < 0.01) concentrations than Friesian-Sanga crossbred cows. Feed supplementation did not affect (P < 0.05) the interval from calving to resumption of ovarian activity, and the days to resumption of ovarian activity in the Sanga and Friesian-Sanga cows were also similar (P > 0.05). The results demonstrate the beneficial effects of feed supplementation in terms of improved body condition and metabolic status and increased milk yield.

Systemic and vascular inflammation in an in-vitro model of central obesity.

Metabolic disorders due to over-nutrition are a major global health problem, often associated with obesity and related morbidities. Obesity is peculiar to humans, as it is associated with lifestyle and diet, and so difficult to reproduce in animal models. Here we describe a model of human central adiposity based on a 3-tissue system consisting of a series of interconnected fluidic modules. Given the causal link between obesity and systemic inflammation, we focused primarily on pro-inflammatory markers, examining the similarities and differences between the 3-tissue model and evidence from human studies in the literature. When challenged with high levels of adiposity, the in-vitro system manifests cardiovascular stress through expression of E-selectin and von Willebrand factor as well as systemic inflammation (expressing IL-6 and MCP-1) as observed in humans. Interestingly, most of the responses are dependent on the synergic interaction between adiposity and the presence of multiple tissue types. The set-up has the potential to reduce animal experiments in obesity research and may help unravel specific cellular mechanisms which underlie tissue response to nutritional overload.

Maintenance of drug metabolism and transport functions in human precision-cut liver slices during prolonged incubation for 5 days.

Human precision-cut liver slices (hPCLS) are a valuable ex vivo model that can be used in acute toxicity studies. However, a rapid decline in metabolic enzyme activity limits their use in studies that require a prolonged xenobiotic exposure. The aim of the study was to extend the viability and function of hPCLS to 5 days of incubation. hPCLS were incubated in two media developed for long-term culture of hepatocytes, RegeneMed®, and Cellartis®, and in the standard medium WME. Maintenance of phase I and II metabolism was studied both on gene expression as well as functional level using a mixture of CYP isoform-specific substrates. Albumin synthesis, morphological integrity, and glycogen storage was assessed, and gene expression was studied by transcriptomic analysis using microarrays with a focus on genes involved in drug metabolism, transport and toxicity. The data show that hPCLS retain their viability and functionality during 5 days of incubation in Cellartis® medium. Albumin synthesis as well as the activity and gene expression of phase I and II metabolic enzymes did not decline during 120-h incubation in Cellartis® medium, with CYP2C9 activity as the only exception. Glycogen storage and morphological integrity were maintained. Moreover, gene expression changes in hPCLS during incubation were limited and mostly related to cytoskeleton remodeling, fibrosis, and moderate oxidative stress. The expression of genes involved in drug transport, which is an important factor in determining the intracellular xenobiotic exposure, was also unchanged. Therefore, we conclude that hPCLS cultured in Cellartis® medium are a valuable human ex vivo model for toxicological and pharmacological studies that require prolonged xenobiotic exposure.

Mesenchymal-Epithelial Transition in Culture of Stromal Progenitor Cells Isolated from the Liver of a Patient with Alcoholic Cirrhosis.

The cells isolated from biopsy specimen of a patient with alcoholic liver cirrhosis and cultured under standard conditions for obtaining stromal cell culture clearly diverged during early passages into two morphologically and phenotypically different subtypes: epithelial and mesenchymal. Mesenchymal cells expressed CD90 and CD44 and epithelial cells expressed CD166, CD227, and hepatocyte growth factor receptor Met. Starting from passage 6, the culture underwent spontaneous morphological changes and by passages 8-10 contained only epithelium-like cells. CD90 and CD44 expression disappeared, CD166 and CD227 expression remained unchanged, and Met expression increased. A small fraction of cells expressed GATA-4, HNF3ß, HNF1α, and HNF4α. After addition of inducers of hepatogeneic differentiation, the cells started producing albumin.

[Effect of shift rotation culture on formation and activity of encapsulated hepatocytes aggregates].

Objective: To observe the effect of uniform and shift rotation culture on the formation and activity of the alginate-chitosan (AC) microencapsulated HepLL immortalized human hepatocytes and HepG2 cells aggregates. Methods: AC microcapsulated HepG2 and HepLL cells were randomly divided into two groups. Each group was divided into 3 subgroups according to uniform and shift rotation culture.The size and number of aggregates were observed and measured under laser confocal microscopy and inverted microscope dynamically. The amount of albumin synthesis was detected by ELISA, the clearance of ammonia was detected by colorimetry, and diazepam conversion function was detected by high performance liquid chromatography (HPLC). Results: On day 6, 8, 10, 12, 14 and 16, the number and size of the aggregates, albumin synthesis, diazepam clearance and ammonium clearance increased significantly in shift rotation culture group than in uniform group (all P<0.01). The albumin synthesis, diazepam clearance, and ammonium clearance in the microencapsulated HepLL groups were significantly higher than those of HepG2 cells at any time (all P<0.01). Conclusion: Shift rotation culture can significantly promote the formation and increase the activity of AC microencapsulated HepLL and HepG2 aggregates, and HepLL cells may be more suitable for bioartificial liver than HepG2.

Nanofibers promote HepG2 aggregate formation and cellular function.

Formation of hepatocyte spheroids is a necessary strategy for increasing liver-specific function in vitro. In this study, HepG2 cells showed good viability when grown on a polylactic acid-chitosan (PLA-CS) nanofiber and aggregated to form multicellular spheroids on the PLA-CS nanofibers with a diameter of approximately 100-200 mm in 5 days of culture, whereas no such aggregation was observed in cells cultured on 24-well plates. Hepatocyte spheroids formed on the PLA-CS nanofibers displayed excellent hepatic-related protein expression, such as albumin and urea, compared to HepG2 cells cultured on the 24-well plates. These results indicated that formation of the hepatocyte spheroids in nanofibers can increase and maintain hepatocyte functions for a longer time, supporting a new strategy for bioartificial liver development.

Enzymatically-gellable galactosylated chitosan: Hydrogel characteristics and hepatic cell behavior.

The influence of contents of galactose and phenolic hydroxyl (Ph) groups incorporated into chitosan was investigated on characteristics of the chitosan derivatives and the resultant gels as well as HepG2 cell attachment and growth behaviors. Introduction of galactose groups increased the solubility of the chitosan derivatives. The gelation time decreased with increasing content of Ph groups in the chitosan derivatives. The increase of galactose groups incorporated at a fixed content of Ph groups improved mechanical properties of the resultant gels. In vitro degradation rate of the resultant gels decreased by increasing Ph groups and decreasing galactose groups incorporated into the chitosan derivatives. The HepG2 cells formed dense spheroid cell clusters when the galactose groups were absent or incorporated at high level into chitosan (13.8mol%). However, the cells exhibited spreading morphology with spheroid formation on the gels containing 1.1 and 5.2mol% galactose groups. The albumin secretion level on a cellular basis also increased considerably when the galactose groups increased to 13.8mol%. The results demonstrated the potential of the chitosan derivative hydrogels for liver tissue engineering applications.

Albumin expression distinguishes bile duct adenomas from metastatic adenocarcinoma.

AIMS: Bile duct adenomas may be difficult to distinguish from metastatic carcinomas, particularly well-differentiated pancreatic ductal adenocarcinoma. Prior studies have evaluated the utility of various immunohistochemical markers, although these markers are notable for low sensitivity and/or specificity. The aim of this study was to investigate the utility of albumin and BRAFV600E expression in distinguishing between metastatic pancreatic adenocarcinoma and bile duct adenoma. METHODS AND RESULTS: We studied 26 bile duct adenomas, three bile duct hamartomas, and 158 pancreatic ductal adenocarcinomas. Branched-chain in-situ hybridization (bISH) for albumin was performed; bISH is based on the branched DNA technology, wherein signal amplification is achieved via a series of sequential steps. Additionally, BRAFV600E immunohistochemistry (IHC) was performed on a subset of cases. Twenty-three of 25 (92%) bile duct adenomas were positive for albumin; 18 (72%) showed diffuse staining, and five showed focal staining (20%), including two challenging examples. Two bile duct hamartomas also stained positively. All pancreatic adenocarcinomas were negative for albumin. Seven of 16 (44%) bile duct adenomas and five of 106 (5%) pancreatic ductal adenocarcinomas were positive for BRAFV600E by IHC. The sensitivity and specificity of expression of albumin, as detected by bISH, for distinguishing bile duct adenomas from metastatic pancreatic adenocarcinomas were 92% and 100%, respectively; the sensitivity and specificity of BRAFV600E IHC for distinguishing bile duct adenomas from metastatic pancreatic adenocarcinomas were 43.8% and 95.3%, respectively. CONCLUSIONS: Diagnostically challenging examples of bile duct adenoma may be distinguished from metastatic pancreatic adenocarcinoma by the use of albumin bISH.

Development of complex-shaped liver multicellular spheroids as a human-based model for nanoparticle toxicity assessment in vitro.

The emergence of human-based models is incontestably required for the study of complex physiological pathways and validation of reliable in vitro methods as alternative for in vivo studies in experimental animals for toxicity assessment. With this objective, we have developed and tested three dimensional environments for cells using different types of hydrogels including transglutaminase-cross-linked gelatin, collagen type I, and growth-factor depleted Matrigel. Cells grown in Matrigel exhibited the greatest cell proliferation and spheroid diameter. Moreover, analysis of urea and albumin biosynthesis revealed that the created system allowed the immortalized liver cell line HepG2 to re-establish normal hepatocyte-like properties which were not observed under the conditions of conventional cell cultures. This study presents a scalable technology for production of complex-shaped liver multicellular spheroids as a system which improves the predictive value of cell-based assays for safety and risk assessment. The time- and dose-dependent toxicity of nanoparticles demonstrates a higher cytotoxic effect when HepG2 cells grown as monolayer than embedded in hydrogels. The experimental setup provided evidence that the cell environment has significant influence on cell sensitivity and that liver spheroid is a useful and novel tool to examine nanoparticle dosing effect even at the level of in vitro studies. Therefore, this system can be applied to a wide variety of potentially hostile compounds in basic screening to provide initial warning of adverse effects and trigger subsequent analysis and remedial actions.

Oxygenated Static Preservation of Donation after Cardiac Death Liver Grafts Improves Hepatocyte Viability and Function.

BACKGROUND: Cell therapy, such as hepatocyte transplantation (HTx), is promising for the treatment of metabolic liver diseases or as a bridge to orthotopic liver transplantation in patients with fulminant liver failure. However, one of the limitations of this therapy is the shortage of donors. The present study aims to investigate whether the two-layer method (TLM) of cold preservation with oxygenation improves the viability and activity of hepatocytes from rat donation after cardiac death (DCD) donors compared with results obtained with the University of Wisconsin (UW) solution. Moreover, we evaluated the hepatocyte function after culture or transplantation into the spleen. MATERIALS AND METHODS: We used male Sprague-Dawley rats for this study. The DCD model was induced by phrenotomy after injecting heparin. We assigned rats based on warm ischemia times of 15 and 30 min to groups S and L, respectively. Each group (n = 5) was then subdivided as follows: (1) group S: not preserved (S/N), preserved by TLM for 3 h (S/TLM3) and 12 h (S/TLM12), and in the UW solution for 3 h (S/UW3) and 12 h (S/UW12), and (2) group L: not preserved (L/N), preserved by TLM for 3 h (L/TLM3) and 12 h (L/TLM12), and in the UW solution for 3 h (L/UW3) and 12 h (L/UW12). The cell viability and function of isolated DCD hepatocytes were analyzed for culture or HTx into the spleen. RESULTS: The viability and ATP levels of DCD hepatocytes significantly improved after TLM compared with the values after preservation in cold UW solution in group S/N (p < 0.059). The levels of albumin production and urea synthesis by hepatocytes after culture were significantly higher in groups S/TLM3 and S/TLM12 than in groups S/UW3 and S/UW12 (p < 0.05), respectively. Further, serum albumin levels after HTx were also markedly higher in groups S/TLM3 and S/TLM12 than in groups S/UW3 and S/UW12. The morphological features revealed that cultured and transplanted hepatocytes remained clearly viable and maintained an expression for specific hepatic function, such as the production of albumin and glycogen. CONCLUSION: This novel method of oxygenated cold preservation of DCD livers can expand the hepatocyte donor pool for HTx and establish a wider application of this developing technique.

Engraftment of human induced pluripotent stem cell-derived hepatocytes in immunocompetent mice via 3D co-aggregation and encapsulation.

Cellular therapies for liver diseases and in vitro models for drug testing both require functional human hepatocytes (Hum-H), which have unfortunately been limited due to the paucity of donor liver tissues. Human pluripotent stem cells (hPSCs) represent a promising and potentially unlimited cell source to derive Hum-H. However, the hepatic functions of these hPSC-derived cells to date are not fully comparable to adult Hum-H and are more similar to fetal ones. In addition, it has been challenging to obtain functional hepatic engraftment of these cells with prior studies having been done in immunocompromised animals. In this report, we demonstrated successful engraftment of human induced pluripotent stem cell (iPSC)-derived hepatocyte-like cells (iPS-H) in immunocompetent mice by pre-engineering 3D cell co-aggregates with stromal cells (SCs) followed by encapsulation in recently developed biocompatible hydrogel capsules. Notably, upon transplantation, human albumin and α1-antitrypsin (A1AT) in mouse sera secreted by encapsulated iPS-H/SCs aggregates reached a level comparable to the primary Hum-H/SCs control. Further immunohistochemistry of human albumin in retrieved cell aggregates confirmed the survival and function of iPS-H. This proof-of-concept study provides a simple yet robust approach to improve the engraftment of iPS-H, and may be applicable to many stem cell-based therapies.

A comparison of the chemical and liver extract-induced hepatic differentiation of adipose derived stem cells.

Adipose-derived stem cells (ADSCs) have been put forward as promising therapeutics for end-stage liver disease (ESLD). In the present study, we compared the effects of defined chemicals and liver extract on the hepatic differentiation of ADSCs. ADSCs were isolated according to the method described in our previously published study. Subsequently, the differentiation of ADSCs was induced separately by chemicals (including hepatic growth factor (HGF), fibroblast growth factor (FGF), and oncostatin M (OSM)) and liver extract (30 µg/ml) in a total period of 21 d. The efficiency of hepatic differentiation was evaluated by changes in the cell morphology, gene expression, and cellular function. The results showed that the liver extract promoted the hepatic differentiation of ADSCs to a significantly greater extent than the chemicals. In the group of ADSCs treated with liver extract, changes in the cell morphology began sooner, and the expression of alpha-FP and albumin genes was higher than that in the chemically treated group. The ADSCs in both the groups stained positive for anti-alpha trypsin (AAT) and albumin markers. The cells also exhibited glycogen storage capacity. Therefore, we concluded that the liver extract could efficiently induce the differentiation of ADSCs into hepatocyte-like cells. This study reveals the potential of mesenchymal stem cell differentiation in the liver extract, which supports further preclinical and clinical research on the application of ADSCs in ESLD treatment.

Cold Preservation of Human Adult Hepatocytes for Liver Cell Therapy.

Hepatocyte transplantation is a promising alternative therapy for the treatment of hepatic failure, hepatocellular deficiency, and genetic metabolic disorders. Hypothermic preservation of isolated human hepatocytes is potentially a simple and convenient strategy to provide on-demand hepatocytes in sufficient quantity and of the quality required for biotherapy. In this study, first we assessed how cold storage in three clinically safe preservative solutions (UW, HTS-FRS, and IGL-1) affects the viability and in vitro functionality of human hepatocytes. Then we evaluated whether such cold-preserved human hepatocytes could engraft and repopulate damaged livers in a mouse model of liver failure. Human hepatocytes showed comparable viabilities after cold preservation in the three solutions. The ability of fresh and cold-stored hepatocytes to attach to a collagen substratum and to synthesize and secrete albumin, coagulation factor VII, and urea in the medium after 3 days in culture was also equally preserved. Cold-stored hepatocytes were then transplanted in the spleen of immunodeficient mice previously infected with adenoviruses containing a thymidine kinase construct and treated with a single dose of ganciclovir to induce liver injury. Engraftment and liver repopulation were monitored over time by measuring the blood level of human albumin and by assessing the expression of specific human hepatic mRNAs and proteins in the recipient livers by RT-PCR and immunohistochemistry, respectively. Our findings show that cold-stored human hepatocytes in IGL-1 and HTS-FRS preservative solutions can survive, engraft, and proliferate in a damaged mouse liver. These results demonstrate the usefulness of human hepatocyte hypothermic preservation for cell transplantation.

Construction of bone marrow mesenchymal cells-derived engineered hepatic tissue and its therapeutic effect in rats with 90% subtotal hepatectomy.

Engineered hepatic tissue (EHT) is considered as a promising strategy for healing acute liver failure (ALF), therefore, in the present study we evaluated the therapeutic potential of the EHT which engaged with bone marrow mesenchymal cells (BMSCs) derived hepatocytes (BMSCs­Hepas) in ALF rats. After characterization of isolated BMSCs, we seeded passage 3 BMSCs which have being cultured in medium containing 20 ng/ml hepatocyte growth factor (HGF) and 10 ng/ml epidermal growth factor (EGF) for 14 days on three scaffolds individually in Transwell system, and then cultured for more than 3 days to construct three kinds of EHT named EHT1, EHT2, and EHT3. Based on morphology and urea production assays, we chose an optimal one and transplanted it into ALF rat with 90% subtotal hepatectomy and assessed its therapeutic potential by survival time, hepatic encephalopathy score (HES) and related liver function test. The remnant liver was acquired, sectioned and identified by con-focal scanning microscopy. The isolated cells possessed basic properties of BMSCs, when cultured in hepatogenic medium for 2 weeks, BMSCs would restore to the functional properties of primary rats' hepatocytes, expressing albumin (ALB) and alpha fetoprotein (AFP) simultaneously. Transplantation of EHT3 significantly prolonged the survival time, increased HES, and ameliorated the liver function. BMSC will be a newly cell source for the construction of EHT. Importantly, the EHT transplantation may be an effective strategy to treat ALF in clinic.

Rapid large-scale culturing of microencapsulated hepatocytes: a promising approach for cell-based hepatic support.

INTRODUCTION: The efficacy of any bioartificial liver device requires both rapid production and proper bioactivity of the cells for the bioreactor. The goal of this study was to observe the effect of spinner speed and cell density on the proliferation of microencapsulated immortalized human hepatocytes (HepLL) and human hepatoma (HepG2) cells. MATERIALS AND METHODS: Alginate-chitosan microcapsulated HepG2 and HepLL cells were randomly divided into 2 groups, and each group was further divided into 8 subgroups according to embedded cell density and spinner speed. The growth, metabolism, and functions of the encapsulated cells in each group were evaluated. RESULTS: In each group, the cell number, ammonium removal, albumin synthesis, and diazepam clearance increased significantly with the spinner speed, whereas embedded cell density had no impact. Albumin synthesis, removal of ammonium, and diazepam clearance were significantly higher in the microencapsulated HepLL groups than in HepG2 cells at any time point, without any significant difference in cell numbers. CONCLUSIONS: Spinner culture significantly promoted microencapsulated HepLL and HepG2 cell bioactivity. Wrapped cells had optimal function on day 10 in rolling culture groups. These data show that HepLL cells would be a promising candidate for cell-based liver support therapy.