Human liver quality is a dominant factor in the outcome of in vitro studies

Donated human liver in the form of precision-cut tissue slices or isolated hepatocytes, is increasingly being used to predict metabolism and toxicity of xenobiotics in man. These tissue slices or hepatocytes can also be cold-preserved and cryopreserved to prolong their use for biological experiments. The viability of human liver could substantially affect the outcome of such experimentation. The goal of this investigation was to assess the viability of donated human livers, in the form of tissue slices, as they were received and to determine how varying degrees of liver quality affect experimental outcomes. Over one hundred human livers were categorized according to initial viability, as assessed by ATP content, K⁺ retention, protein synthesis, and LDH leakage. Each liver was placed in a low-, a medium-, or a high-quality group. The results showed that 76% of transplant-grade tissue (procured for transplantation) fell into the high-viability classification while the majority of research-grade tissue (not procured for transplantation) fell into the lowest viability classification. It was also found that only tissue slices prepared from highly viable human liver could be cold-preserved and cryopreserved. Dichlorobenzene metabolism was also greater in slices from highly viable human livers as compared to less viable livers. This study showed that human liver tissue acquired for medical research substantially varies in its viability and that these differences will affect the experimental data obtained. This revised version was published online in July 2006 with corrections to the Cover Date.     

Theaflavin attenuates ischemia-reperfusion injury in a mouse fatty liver model

The incidence of non-alcoholic fatty liver disease (NAFLD) has been increasing, and there is a shortage of liver donors, which has led to the acceptance of steatotic livers for transplantation. However, steatotic livers are known to experience more severe acute ischemia-reperfusion (I/R) injury than normal livers upon transplantation. In the present study, we investigated the role of theaflavin, a polyphenol substance extracted from black tea, in attenuating acute I/R injury in a fatty liver model. We induced I/R in normal and steatotic livers treated with or without theaflavin. We also separated primary hepatocytes from the normal and steatotic livers, and applied RAW264.7 cells, a mouse macrophage cell line, that was pretreated with theaflavin. We observed that liver steatosis, oxidative stress, inflammation and hepatocyte apoptosis were increased in the steatotic liver compared to the normal liver, however, these changes were significantly decreased by theaflavin treatment. In addition, theaflavin significantly diminished the ROS production of steatotic hepatocytes and TNF-α production by LPS-stimulated RAW264.7 cells. We concluded that theaflavin has protective effects against I/R injury in fatty livers by anti-oxidant, anti-inflammatory, and anti-apoptotic mechanisms.     

Influence of Pre-, Intra- and Post-Operative Parameters of Donor Liver on the Outcome of Isolated Human Hepatocytes

The aim of the present study was to analyse, retrospectively on a large panel of patients (149), the influence of the donor liver characteristics on the outcome of human hepatocyte isolation obtained from resected liver biopsies from surgical waste after hepatectomy. Among the pre-operative parameters, the type of disease, age and sex of the patient, previous chemotherapy, alcohol or tobacco consumption did not affect the yield, viability, attachment rate and function of the isolated human hepatocytes. Pre-operative biological and anatomopathological data indicated that, while mild steatosis (≤10% steatotic hepatocytes) did also not affect the outcome of hepatocyte isolation, stronger steatosis (>10% steatotic hepatocytes) tended to decrease hepatocyte yield. Cholestasis, as assessed by γ-glutamyl transferase serum values, significantly negatively correlated with the percentage of digested liver and the yield of viable cells. Intra-operative clamping time, that is, warm ischaemia, longer than 30 min was found to decrease both the percentage of digested liver and cell yield. Among the post-operative parameters, the percentage of digested liver decreased when biopsy weights were higher than 100 g, the use of glue tended to increase both the percentage of digested tissue and the yield of viable cells. In conclusion, human diseased livers appear to be a valuable source of isolated functional human hepatocytes. We recommend, for an optimal isolation, to use liver biopsies weighing less than 100 g, to glue the section surfaces of the biopsies and to avoid the use of moderate steatotic livers (>10% steatotic hepatocytes) and cholestatic livers, as well as livers undergoing warm ischaemia or clamping during resection due to the decrease in cell yield. This revised version was published online in July 2006 with corrections to the Cover Date.     

Combined use of N-acetylcysteine and Liberase improves the viability and metabolic function of human hepatocytes isolated from human liver

Background aimsSuccessful hepatocyte isolation is critical for continued development of cellular transplantation. However, most tissue available for research is from diseased liver, and the results of hepatocyte isolation from such tissue are inferior compared with normal tissue. Liberase and N-acetylcysteine (NAC) have been shown separately to improve viability of isolated hepatocytes. This study aims to determine the effect of Liberase and NAC in combination on human hepatocyte isolation from normal and diseased liver tissues.MethodsHepatocytes were isolated from 30 liver specimens through the use of a standard collagenase digestion technique (original protocol) and another 30 with the addition of NAC and standard collagenase substituted by Liberase (new protocol). Viability and success, defined as maintenance of cell adhesion and morphology for 48 hours, were assessed. Metabolic function was assessed by means of albumin and urea synthesis.ResultsBaseline factors were similar for both groups. The delay to tissue processing was slightly shorter in the new protocol group (median, 2 versus 4 hours; P = 0.007). The success rate improved from 12 of 30 (40.0%) to 21 of 30 (70.0%) with the use of the new protocol (P = 0.037), and median viable cell yield increased from 7.3 × 10⁴ to 28.3 × 10⁴ cells/g tissue (P = 0.003). After adjusting for delay, success rate (P = 0.014) and viable cell yield/g tissue (P = 0.001) remained significantly improved. Albumin and urea synthesis were similar or superior in the new protocol group.ConclusionsNAC and Liberase improve the success of hepatocyte isolation, with a significantly higher yield of viable cells. The use of these agents may improve the availability of hepatocytes for transplantation and laboratory research.     

Progress in human hepatocytes: isolation,culture & cryopreservation

The quality of the hepatocytes used for clinical cell transplantation is very important, and depends to a large extent on the nature of the tissue used for isolation. The collagenase perfusion technique to isolate hepatocytes from animal livers has been further developed for isolation of human hepatocytes. As the donor organ pool is a scarce resource, marginal livers unsuitable for transplantation and segments from reduced grafts remain the main source of tissue for cell isolation. Use of livers from non-heart beating donors and foetal livers may further increase the tissue pool. With the limited supply of available tissue, improvements in the cryopreservation protocols are required to maintain cell viability on thawing and establish hepatocyte banks.     

Activation, isolation, identification and culture of hepatic stem cells from porcine liver tissues

Objectives: Utility of hepatic stem cells could provide a novel solution to the severe shortage of human donor livers, for treatment of liver‐related diseases, due to their ability to proliferate and differentiate into functional hepatocytes. Porcine liver tissues also offer an alternative source from human donor livers. However, morphology, phenotype, successful isolation and culture of porcine hepatic stem cells still require much investigation. Materials and methods: In the present study, we performed partial hepatectomy to activate hepatic oval cells and developed a procedure utilizing enzymatic digestion and density gradient centrifugation to isolate and purify oval cells derived from porcine livers. We identified ovoid cells by their morphological characteristics and phenotypic properties, thereby providing definitive evidence for the presence of hepatic stem cells in porcine livers. Moreover, we established a culture system, using various growth factors, to provide nourishment for these cells. Results and conclusions: By transmission electron microscopy, oval‐shaped cells with ovoid nuclei, a high nucleus/cytoplasm ratio and few organelles were demonstrated. Flow cytometry and immunocytochemistry showed that freshly isolated oval cells expressed albumin, cytokeratin 19, alpha fetoprotein (AFP) and OV6 at high levels. Immunofluorescence revealed that porcine hepatic oval cells after culture expressed stem‐cell factor, c‐kit, Thy‐1, CK19, OV6, and AFP. Taken together, this study provides a novel insight into morphological and phenotypic characteristics of porcine hepatic stem cells. Our ability for isolation and culturing porcine hepatic stem cells offers an abundant source of cells for transplantation and tissue engineering to help alleviate liver disease.     

Potential treatment of liver-related disorders with in vitro expanded human liver precursors

Abstract Inherited deficiencies in critical components of metabolic pathways are the primary cause of many liver and lysosomal disorders, most of which are incurable. Stem cell transplantation may offer a new type of treatment for these diseases. We have isolated hepatocyte precursors from human fetal livers. These cells were highly proliferative in vitro in media with or without serum. Expanded hepatocyte precursors expressed endoderm and early hepatocyte markers. The precursors synthesized a large number of molecules related to human metabolic diseases and released some of them into the environment. In a homing test, these cells migrated preferentially into the liver. When transplanted into fetal sheep liver, they incorporated into the liver tissue and differentiated into hepatocytes. Transplantation of the liver precursors to α- l -iduronidase-deficient mice partially corrected the enzyme deficiency. Data from these studies suggest that in vitro expanded human liver precursor cells are a potential cell source for the treatment of liver- and lysosome-related disorders.     

Application of liver stem cells for cell therapy

The worldwide shortage of donor livers to transplant end stage liver disease patients has prompted the search for alternative cell therapies for intractable liver disease. Embryonic stem cells can be readily differentiated into hepatocytes, and their transplantation into animals has improved liver function in the absence of teratoma formation: their use in bioartificial liver support is an obvious application. In animal models of liver disease, adopting strategies to provide a selective advantage for transplanted foetal or adult hepatocytes have proved highly effective in repopulating recipient livers, but the poor success of today's hepatocyte transplants can be attributed to the lack of a clinically applicable procedure to force a similar repopulation of the human liver. The activation of bipotential hepatic progenitor cells is clearly vital for survival in many cases of acute liver failure, but surprisingly little progress has been made with these cells in terms of transplantation. Finally there is the controversial subject of autologous bone marrow, and while the contribution of these indigenous cells to liver turnover seems at best, trivial, results from a small number of phase 1 studies of transplantation of bone marrow to cirrhotic patients have been moderately encouraging.     

Establishment of a tightly regulated human cell line for the development of hepatocyte transplantation

Hepatocyte transplantation (HTX) could be an attractive treatment for patients with liver failure and liver-based metabolic disease. Human primary hepatocytes are ideal in this modality, but the shortage of human livers available for hepatocyte isolation severely limits the use of this form of therapy. A tightly regulated human hepatocyte cell line that grows economically in culture and exhibits differentiated liver functions would be an attractive alternative to the primary human hepatocytes. To test the feasibility, human hepatocytes were immortalized by a retroviral vector expressing simian virus 40 large T antigen and herpes simplex virus-thymidine kinase. A highly differentiated immortal hepatocyte line NKNT-3 was established. NKNT-3 cells grew in chemically defined serum-free medium, retained highly differentiated liver functions, and were sensitivity to ganciclovir as a prodrug. Essentially unlimited availability of NKNT-3 cells may be clinically useful for HTX and bioartificial liver.     

Re-evaluation of liver stem/progenitor cells

Liver stem/progenitor cells (LPCs) are defined as cells that supply two types of liver epithelial cells, hepatocytes and cholangiocytes, during development, cellular turnover, and regeneration. Hepatoblasts, which are fetal LPCs derived from endoderm stem cells, robustly proliferate and differentiate into hepatocytes and cholangiocytes during fetal life. Between mid-gestation and the neonatal period, some cholangiocytes function as LPCs. Although LPCs in adult livers can be enriched in cells positive for cholangiocyte markers, their tissue localization and functions in cellular turnover remain obscure. On the other hand, it is well known that liver regeneration under conditions suppressing hepatocyte proliferation is supported by LPCs, though their origin has not been clearly identified. Recently many groups took advantage of new techniques including prospective isolation of LPCs by fluorescence-activated cell sorting and genetic lineage tracing to facilitate our understanding of epithelial supply in normal and injured livers. Those works suggest that, in normal livers, the turnover of hepatocytes mostly depends on duplication of hepatocytes. It is also demonstrated that liver epithelial cells as well as LPCs have great plasticity and flexible differentiation capability to respond to various types of injuries by protecting or repairing liver tissues.     

Re-evaluation of liver stem/progenitor cells

Liver stem/progenitor cells (LPCs) are defined as cells that supply two types of liver epithelial cells, hepatocytes and cholangiocytes, during development, cellular turnover, and regeneration. Hepatoblasts, which are fetal LPCs derived from endoderm stem cells, robustly proliferate and differentiate into hepatocytes and cholangiocytes during fetal life. Between mid-gestation and the neonatal period, some cholangiocytes function as LPCs. Although LPCs in adult livers can be enriched in cells positive for cholangiocyte markers, their tissue localization and functions in cellular turnover remain obscure. On the other hand, it is well known that liver regeneration under conditions suppressing hepatocyte proliferation is supported by LPCs, though their origin has not been clearly identified. Recently many groups took advantage of new techniques including prospective isolation of LPCs by fluorescence-activated cell sorting and genetic lineage tracing to facilitate our understanding of epithelial supply in normal and injured livers. Those works suggest that, in normal livers, the turnover of hepatocytes mostly depends on duplication of hepatocytes. It is also demonstrated that liver epithelial cells as well as LPCs have great plasticity and flexible differentiation capability to respond to various types of injuries by protecting or repairing liver tissues.     

Effect of patient, operative and isolation factors on subsequent yield and viability of human hepatocytes for research use

It is widely accepted that the model of choice for pharmacotoxicological studies are human hepatocytes. There is therefore a demand for these cells, but quality must be maintained for their widespread use. We present a retrospective review of the isolation of hepatocytes from both surgically resected tissue and livers rejected for transplantation, and evaluated patient, operative and isolation variables to ascertain which may affect the viability and yield of cells. Seven clinically rejected whole livers and 60 surgically resected specimens (from two distinct operating centres) were isolated. For surgically resected tissue we found that decreasing age, securing the perfusing cannulae with suture rather than reforming Glissons capsule with glue and steatotic livers improved viability. No significant correlation could be found with pre-operative blood results, disease, type of operation, presence or absence of Pringle manoeuvre, weight of tissue isolated, time of digestion with collagenase and cold ischaemic time. There was a reduction in mean yield and viability when hepatocyte isolations were performed in livers rejected for transplant, compared to surgically resected tissue although this did not reach significance. Human hepatocytes can be successfully and consistently isolated from surgically resected tissue and appear to be superior to those isolated from rejected for transplant livers. From our study, there are few parameters that significantly affect the quality of isolated hepatocytes, which increases the possible pool of tissue that hepatocytes can be isolated from.     

Differentiated human adipose‐derived stem cells exhibit hepatogenic capability in vitro and in vivo

The availability of suitable human livers for transplantation falls short of the number of potential patients. In addition, the availability of primary human hepatocytes for cell‐therapy and drug development applications is significantly limited; less than 700 livers per year are available for such studies. However, the majority of these organs cannot be utilized due to pathological infections (e.g., HepB, HepC, or HIV) or excessive levels of steatosis. Thus, the number of cells needed for cell therapy applications far exceeds the number of cells available from donated livers. The ability to implant progenitor cell populations that can form liver tissue in situ, or can be differentiated in vitro would be a major advance in current cell‐based therapies. In addition, and importantly for this application, the ability to utilize a non‐hepatic progenitor cell to mimic hepatocytes in vitro would enable the scale‐up production of cells for bioartifical liver assist devices, cell‐therapy and drug discovery applications. We demonstrate the feasibility of inducing adipose‐derived stromal (ASC) cells to express several features of human hepatocytes such as glycogen storage and expression of liver specific genes. Importantly, we also show that undifferentiated ASCs and ASC‐derived hepatic cells engraft robustly into the liver in a mouse model of toxic injury. These data indicate a significant potential for the use of undifferentiated ASCs and ASC‐derived hepatic cells as novel and valuable products for cell therapy. J. Cell. Physiol. 225: 429–436, 2010. © 2010 Wiley‐Liss, Inc.     

Investigation of Functional and Morphological Integrity of Freshly Isolated and Cryopreserved Human Hepatocytes

There is a pressing need for alternative therapeutic methods effective in the treatment of patients with liver insufficiency. Isolated human hepatocytes may be a viable alternative or adjunct to orthotopic liver transplantation in such patients. The purpose of this study was to evaluate the viability and functional integrity of freshly isolated and cryopreserved human hepatocytes, in preparation for a multi-center human hepatocyte transplantation trial. We are currently processing transplant-grade human parenchymal liver cells from nondiseased human livers that are obtained through a network of organ procurement organizations (OPOs). Thus far, sixteen hepatocyte transplants have been performed using hepatocytes processed by our methods. At the time of referral all specimens were deemed unsuitable for transplantation due to anatomical anomalies, high fat content, medical history, etc. Hepatocytes were isolated from encapsulated liver sections by a modified two-step perfusion technique. Isolated cells were cryopreserved and stored in liquid nitrogen for one to twelve months. The total yield of freshly isolated hepatocytes averaged 3.7×10⁷ cells per gram of wet tissue. Based on trypan blue exclusion, fresh preparations contained an average of 85% viable hepatocytes vs. 70% in cryopreserved samples. The plating efficiencies of cells seeded immediately after isolation ranged from 87% to 98%, while those of cryopreserved/thawed cells were markedly lower. Flow cytometry analysis of cells labeled with 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) showed that there was no significant difference in viability compared with trypan blue staining. Both freshly isolated hepatocytes and those recovered from cryopreservation showed typical and intact morphology as demonstrated by light and electron microscopy. The product of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reaction was always expressed more intensely in cultures of freshly isolated hepatocytes. Measurements of lactate dehydrogenase (LDH) leakage were inversely correlated with trypan blue exclusion and CFSE labeling. Energy status, evaluated by the intracellular ATP concentration measurements, and various liver-specific functions such as urea synthesis and metabolism of 7-ethoxycoumarin were maintained both in fresh and cryopreserved/thawed hepatocytes. However, the activities were expressed at different levels in thawed cells. These data illustrate the importance and feasibility of human hepatocyte banking. In addition, it is clear that further refinements in the methods of hepatocyte isolation and cryopreservation are needed to utilize more fully these valuable cells in the clinic.     

The isolation of primary hepatocytes from human tissue: optimising the use of small non-encapsulated liver resection surplus

Two-step perfusion is considered the gold standard method for isolating hepatocytes from human liver tissue. As perfusion may require a large tissue specimen, which is encapsulated and has accessible vessels for cannulation, only a limited number of tissue samples may be suitable. Therefore, the aim of this work was to develop an alternative method to isolate hepatocytes from non-encapsulated and small samples of human liver tissue. Healthy tissue from 44 human liver resections were graded for steatosis and tissue weights between 7.8 and 600 g were used for hepatocyte isolations. Tissue was diced and underwent a two-step digestion (EDTA and collagenase). Red cell lysis buffer was used to prevent red blood cell contamination and toxicity. Isolated hepatocyte viability was determined by trypan blue exclusion. Western blot and biochemical analyses were undertaken to ascertain cellular phenotype and function. Liver tissue that weighed ≥50 g yielded significantly higher (P < 0.01) cell viability than tissue <50 g. Viable cells secreted urea and displayed the phenotypic hepatocyte markers albumin and cytochrome P450. Presence of steatosis in liver tissue or intra-hepatocellular triglyceride content had no effect on cell viability. This methodology allows for the isolation of viable primary human hepatocytes from small amounts of “healthy” resected liver tissue which are not suitable for perfusion. This work provides the opportunity to increase the utilisation of resection surplus tissue, and may ultimately lead to an increased number of in vitro cellular studies being undertaken using the gold-standard model of human primary hepatocytes.     

Isolation and culture of hepatocytes from human liver of immediate autopsy

Summary Human livers were removed at immediate autopsy (IA) from brain death patients within 1 h after cessation of cardiac function. Viable hepatocytes were isolated successfully from these IA livers by perfusion of an intack lobe with collagenase or by digestion of a small tissue wedge with collagenase-dispase. The yields of hepatocytes ranged from 1 to 3 × 10⁶ cells/g liver in the five cases studied. Approximately 70 to 90% of the cells excluded trypan blue dye. In the isolated hepatocytes, 632 pmol/mg protein of cytochromep ₄₅₀ and 536. pmol/mg protein cytochromeb ₅ were measured. The cells attached to the dishes in 4 h and produced monolayer cultures with a high success rate. The cells maintained in primary cultures for several days and developed ultrastructural features characteristic of human hepatocytes in vivo. The cultured hepatocytes can hydroxylate benzo[a]pyrene, conjugate the metabolites, and have a benzo[a]pyrene hydroxylase activity of 48.7 pmol/mg DNA per h, which is comparable to that of rat hepatocytes. The liver cells repaired DNA damage caused by exposures to aminofluorene and acetylaminofluorene in culture. This work was supported by EPA Grants R-809835-01-1, R-809599010 and DOE Contract DE-A505-83ER60158. Cobtribution no. 1762 from the Cellular Pathobiology Laboratory, University of Maryland School of Medicine.     

The internal bioartifical liver

A possible alternative to liver transplantation is the internal bioartificial liver (IBAL) consisting in the transplantation of isolated encapsulated hepatocytes. The goal of IBAL is to allow an auxiliary liver until native liver regeneration. The hepatocytes could be allogeneic or xenogeneic (animal origin). Large animal source of liver cells should allow immediate isolation of fresh hepatocytes when IBAL is required. The peritoneal cavity is probably the site of implantation of IBAL. In the future, IBAL could be envisioned as the treatment of metabolic deficiencies or acute and chronic liver failure. It could ensure a therapeutic bridge until transplantation.     

Cytochrome 3A and 2E1 in human liver tissue: Individual variations among normal Japanese subjects

AimsThe metabolism of drugs, xenobiotic compounds, and other endogenous/exogenous substrates generally begins with their oxidation through cytochrome P450 (CYP). The results of recent pharmacogenetic analyses have demonstrated CYP's polymorphisms to be related to individual differences in metabolism, but only a limited number of CYP3A4 and CYP2E1 variant alleles influence enzymatic activities. Therefore, CYP gene expression profiling of both normal and pathological human livers should provide critical information for an evaluation of the biological significance of CYPs.Main methodsIn our present study, we first characterized the individual differences in CYP3A4 and CYP2E1 expression levels among Japanese normal or non-pathological liver tissue obtained from autopsy or surgery using immunohistochemistry and quantitative RT-PCR array of phase I metabolic enzymes with combined laser capture microscopy and qPCR analysis.Key findingsBoth CYP3A4 and CYP2E1 mRNA and proteins were predominantly detected in hepatocytes surrounding central veins in normal liver, but there were marked individual differences in both CYP3A4 and CYP2E1 mRNA and proteins among the 23 Japanese subjects examined. Individual differences in CYP3A and CYP2E1 subtypes were also detected in the livers obtained from monozygotic neonatal Japanese female twins with different survival periods. CYP3A and CYP2E1-positive cells were decreased in number in non-pathological hepatocytes of diseased livers compared to those in disease-free livers from autopsy.SignificanceThe above results suggest that individual differences in CYP3A4 and CYP2E1 exist among normal human liver tissues and in non-pathological hepatocytes between diseased and normal liver, and these differences may be important in evaluating the pharmacodynamics of various substances.     

Propagation and functional characterization of serum-free cultured porcine hepatocytes for downstream applications

Hepatocyte transplantation is considered an alternative to whole organ transplantation. However, the availability of human cadaveric livers for the isolation of transplantation-quality hepatocytes is increasingly restricted. Xenogeneic porcine hepatocytes may therefore serve as an alternate cell ressource. The propagation of hepatocytes is often necessary to yield a sufficient cell number for downstream applications in xenotransplantation and in, for example, bioartificial liver support or pharmacological and toxicological studies. Our goal has been to propagate primary porcine hepatocytes in vitro and to determine the functional maintenance of the propagated cells. Porcine hepatocytes were cultured under serum-free conditions in the presence of hepatocyte growth factor and epidermal growth factor and passaged several times. The viability, proliferation and maintenance of liver-specific functions were determined as culture proceeded. Total cell number increased by 12-fold during four sequential passages, although the proliferative capacity was higher in primary cells and early passages as compared with late passages. Xenobiotics metabolism and urea synthesis gradually decreased with ongoing culture but could be restored by treatment with appropriate stimuli such, as β-naphthoflavone and cAMP. The expression of hepatocyte-specific genes was generally lower at the beginning than at later time-points of culture of individual passages. Porcine hepatocytes can thus be propagated in vitro. The partial loss of hepatocyte function may be restored in vitro by appropriate stimuli. This may also be achieved in a recipient liver after hepatocyte transplantation provided that the proper physiological environment for the maintenance of the differentiated hepatocyte phenotype is present. This study was supported by grants to B. Christ from the German Ministry of Education and Research (01 ZZ 0109 and NBL3-NG4) as well as by grants from the Federal State of Saxonia-Anhalt through the Wilhelm-Roux-Program at the Medical Faculty of the Martin-Luther-University of Halle-Wittenberg to B. Christ (09/07 and 04/03).     

Induction of three-dimensional assembly of human liver cells by simulated microgravity

Summary The establishment of long-term cultures of functional primary human liver cells (PHLC) is formidable. Developed at NASA, the Rotary Cell Culture System (RCCS) allows the creation of the unique microgravity environment of low shear force, high-mass transfer, and 3-dimensional cell culture of dissimilar cell types. The aim of our study was to establish long-term hepatocyte cultures in simulated microgravity. PHLC were harvested from human livers by collagenase perfusion and were cultured in RCCS. PHLC aggregates were readily formed and increased up to 1 cm long. The expansion of PHLC in bioreactors was further evaluated with microcarriers and biodegradable scaffolds. While microcarriers were not conducive to formation of spheroids, PHLC cultured with biodegradable scaffolds formed aggregates up to 3 cm long. Analyses of PHLC spheroids revealed tissue-like structures composed of hepatocytes, biliary epithelial cells, and/or progenitor liver cells that were arranged as bile duct-like structures along nascent vascular sprouts. Electron microscopy revealed groups of cohesive hepatocytes surrounded by complex stromal structures and reticulin fibers, bile canaliculi with multiple microvilli, and tight cellular junctions. Albumin mRNA was expressed throughout the 60-d culture. A simulated microgravity environment is conducive to maintaining long-term cultures of functional hepatocytes. This model system will assist in developing improved protocols for autologous hepatocyte transplantation, gene therapy, and liver assist devices, and facilitate studies of liver regeneration and cell-to-cell interactions that occur in vivo.