A thin‐walled polydimethylsiloxane bioreactor for high‐density hepatocyte sandwich culture

In vitro drug testing requires long‐term maintenance of hepatocyte liver specific functions. Hepatocytes cultured at a higher seeding density in a sandwich configuration exhibit an increased level of liver specific functions when compared to low density cultures due to the better cell to cell contacts that promote long term maintenance of polarity and liver specific functions. However, culturing hepatocytes at high seeding densities in a standard 24‐well plate poses problems in terms of the mass transport of nutrients and oxygen to the cells. In view of this drawback, we have developed a polydimethylsiloxane (PDMS) bioreactor that was able to maintain the long‐term liver specific functions of a hepatocyte sandwich culture at a high seeding density. The bioreactor was fabricated with PDMS, an oxygen permeable material, which allowed direct oxygenation and perfusion to take place simultaneously. The mass transport of oxygen and the level of shear stress acting on the cells were analyzed by computational fluid dynamics (CFD). The combination of both direct oxygenation and perfusion has a synergistic effect on the liver specific function of a high density hepatocyte sandwich culture over a period of 9 days. Biotechnol. Bioeng. 2013; 110: 1663–1673. © 2012 Wiley Periodicals, Inc.     

QSG‐7701 human hepatocytes form polarized acini in three‐dimensional culture

Hepatocytes are polarized and fulfill a variety of liver‐specific functions in vivo; but the polarized tissue structure and many of these functions are lost when the cells are cultured on plastic. To recapitulate the polarized structure and tissue‐specific function of liver cells in culture, we established a three‐dimensional (3D) culture assay with the human hepatocyte line QSG‐7701. In 3D Matrigel culture, QSG‐7701 cells formed polarized spheroids with a center lumen, which is reminiscent of bile canaliculi in the liver. Immunofluoresence analysis showed that F‐actin bundles and radixin were mainly located at the apical membrane and that α6 and β1 integrins were localized basally in 3D culture. Lumen formation was associated with the selective apoptosis of centrally located cells and was accompanied by proliferative suppression during acinar development. Compared to QSG‐7701 cells in 2D or agarose gel cultures, the cells in 3D Matrigel culture maintained a given direction of biliary excretion and acquired higher levels of cytochrome P450 and albumin expression. Our study shows that the immortal human hepatocytes, QSG‐7701, in 3D Matrigel culture reacquire cardinal features of glandular epithelium in vivo, providing an ex vivo model to study liver‐specific function and tumorigenesis. J. Cell. Biochem. 110: 1175–1186, 2010. Published 2010 Wiley‐Liss, Inc.     

Perfusion enhanced polydimethylsiloxane based scaffold cell culturing system for multi‐well drug screening platform

Conventional two‐dimensional cultures in monolayer and sandwich configuration have been used as a model for in vitro drug testing. However, these culture configurations do not present the actual in vivo liver cytoarchitecture for the hepatocytes cultures and thus they may compromise the cells liver‐specific functions and their cuboidal morphology over longer term culture. In this study, we present a three‐dimensional polydimethylsiloxane (PDMS) scaffold with interconnected spherical macropores for the culturing of rat liver cells (hepatocytes). The scaffolds were integrated into our perfusion enhanced bioreactor to improve the nutrients and gas supply for cell cultures. The liver‐specific functions of the cell culture were assessed by their albumin and urea production, and the changes in the cell morphology were tracked by immunofluorescence staining over 9 days of culture period. N‐Acetyl‐Para‐Amino‐Phenol (acetaminophen) was used as drug model to investigate the response of cells to drug in our scaffold‐bioreactor system. Our experimental results revealed that the perfusion enhanced PDMS‐based scaffold system provides a more conducive microenvironment with better cell‐to‐cell contacts among the hepatocytes that maintains the culture specific enzymatic functions and their cuboidal morphology during the culturing period. The numerical simulation results further showed improved oxygen distribution within the culturing chamber with the scaffold providing an additional function of shielding the cell cultures from the potentially detrimental fluid induced shear stresses. In conclusion, this study could serve a crucial role as a platform for future preclinical hepatotoxicity testing. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:418–428, 2014     

"Contact inhibition" of alpha-fetoprotein synthesis and junctional communication in adult mouse hepatocyte culture

Synthesis of oncofetal serum protein alpha-fetoprotein (AFP) may be reexpressed in adult differentiated mouse hepatocytes both in regenerating liver and in primary monolayer culture of intact adult liver. We have found that appearance of AFP in these cultures was strongly correlated with the loss of junctional communication between hepatocytes as tested by the dye transfer method. When in hepatocyte culture the gradient of cell density was formed, and the cells in the center of the dense monolayer retained an epithelial morphology and junctional communication and were AFP-negative during 5 days of culture. At the periphery of the monolayer hepatocytes lost junctional communication by the third day of cultivation. They acquired fibroblast-like morphology, formed multilayered sheets, and started to produce AFP. These findings suggest that reexpression of AFP synthesis may be regulated through a process related to "contact inhibition" and junctional communication might play an important role in the phenomenon.     

Primary rat hepatocyte culture on 3D nanofibrous polymer scaffolds for toxicology and pharmaceutical research

Primary rat hepatocytes are a widely used experimental model to estimate drug metabolism and toxicity. In currently used two‐dimensional (2D) cell culture systems, typical problems like morphological changes and the loss of liver cell‐specific functions occur. We hypothesize that the use of polymer scaffolds could overcome these problems and support the establishment of three‐dimensional (3D) culture systems in pharmaceutical research. Isolated primary rat hepatocytes were cultured on collagen‐coated nanofibrous scaffolds for 7 days. Cell loading efficiency was quantified via DNA content measurement. Cell viability and presence of liver‐cell‐specific functions (albumin secretion, glycogen storage capacity) were evaluated. The activity of liver‐specific factors was analyzed by immunofluorescent staining. RNA was isolated to establish quantitative real‐time PCR. Our results indicate that primary rat hepatocytes cultured on nanofibrous scaffolds revealed high viability and well‐preserved glycogen storage. Albumin secretion was existent during the entire culture period. Hepatocytes remain HNF‐4 positive, indicating highly preserved cell differentiation. Aggregated hepatocytes re‐established positive signaling for Connexin 32, a marker for differentiated hepatocyte interaction. ZO‐1‐positive hepatocytes were detected indicating formation of tight junctions. Expression of cytochrome isoenzymes was inducible. Altogether the data suggest that nanofibrous scaffolds provide a good in vitro microenvironment for neo tissue regeneration of primary rat hepatocytes. Biotechnol. Bioeng. 2011; 108:141–150. © 2010 Wiley Periodicals, Inc.     

Modeling spatial distribution of oxygen in 3d culture of islet beta‐cells

Three‐dimensional (3D) scaffold culture of pancreatic β‐cell has been proven to be able to better mimic physiological conditions in the body. However, one critical issue with culturing pancreatic β‐cells is that β‐cells consume large amounts of oxygen, and hence insufficient oxygen supply in the culture leads to loss of β‐cell mass and functions. This becomes more significant when cells are cultured in a 3D scaffold. In this study, in order to understand the effect of oxygen tension inside a cell‐laden collagen culture on β‐cell proliferation, a culture model with encapsulation of an oxygen‐generator was established. The oxygen‐generator was made by embedding hydrogen peroxide into nontoxic polydimethylsiloxane to avoid the toxicity of a chemical reaction in the β‐cell culture. To examine the effectiveness of the oxygenation enabled 3D culture, the spatial‐temporal distribution of oxygen tension inside a scaffold was evaluated by a mathematical modeling approach. Our simulation results indicated that an oxygenation‐aided 3D culture would augment the oxygen supply required for the β‐cells. Furthermore, we identified that cell seeding density and the capacity of the oxygenator are two critical parameters in the optimization of the culture. Notably, cell‐laden scaffold cultures with an in situ oxygen supply significantly improved the β‐cells' biological function. These β‐cells possess high insulin secretion capacity. The results obtained in this work would provide valuable information for optimizing and encouraging functional β‐cell cultures. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:221–228, 2017     

The importance of physiological oxygen concentrations in the sandwich cultures of rat hepatocytes on gas‐permeable membranes

Oxygen supply is a critical issue in the optimization of in vitro hepatocyte microenvironments. Although several strategies have been developed to balance complex oxygen requirements, these techniques are not able to accurately meet the cellular oxygen demand. Indeed, neither the actual oxygen concentration encountered by cells nor the cellular oxygen consumption rates (OCR) was assessed. The aim of this study is to define appropriate oxygen conditions at the cell level that could accurately match the OCR and allow hepatocytes to maintain liver specific functions in a normoxic environment. Matrigel overlaid rat hepatocytes were cultured on the polydimethylsiloxane (PDMS) membranes under either atmospheric oxygen concentration [20%‐O₂ (+)] or physiological oxygen concentrations [10%‐O₂ (+), 5%‐O₂ (+)], respectively, to investigate the effects of various oxygen concentrations on the efficient functioning of hepatocytes. In parallel, the gas‐impermeable cultures (polystyrene) with PDMS membrane inserts were used as the control groups [PS‐O₂ (?)]. The results indicated that the hepatocytes under 10%‐O₂ (+) exhibited improved survival and maintenance of metabolic activities and functional polarization. The dramatic elevation of cellular OCR up to the in vivo liver rate proposed a normoxic environment for hepatocytes, especially when comparing with PS‐O₂ (?) cultures, in which the cells generally tolerated hypoxia. Additionally, the expression levels of 84 drug‐metabolism genes were the closest to physiological levels. In conclusion, this study clearly shows the benefit of long‐term culture of hepatocytes at physiological oxygen concentration, and indicates on an oxygen‐permeable membrane system to provide a simple method for in vitro studies. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1401–1410, 2014     

Oxygen uptake rates and liver-specific functions of hepatocyte and 3T3 fibroblast co-cultures

Bioartificial liver (BAL) devices have been developed to treat patients undergoing acute liver failure. One of the most important parameters to consider in designing these devices is the oxygen consumption rate of the seeded hepatocytes which are known to have oxygen consumption rates 10 times higher than most other cell types. Hepatocytes in various culture configurations have been tested in BAL devices including those formats that involve co-culture of hepatocytes with other cell types. In this study, we investigated, for the first time, oxygen uptake rates (OUR)s of hepatocytes co-cultured with 3T3-J2 fibroblasts at various hepatocyte to fibroblast seeding ratios. OURs were determined by measuring the rate of oxygen disappearance using a ruthenium-coated optical probe after closing and sealing the culture dish. Albumin and urea production rates were measured to assess hepatocyte function. Lower hepatocyte density co-cultures demonstrated significantly higher OURs (2 to 3.5-fold) and liver- specific functions (1.6-fold for albumin and 4.5-fold for urea production) on a per cell basis than those seeded at higher densities. Increases in OUR correlated well with increased liver-specific functions. OURs (V(m)) were modeled by fitting Michaelis-Menten kinetics and the model predictions closely correlated with the experimental data. This study provides useful information for predicting BAL design parameters that will avoid oxygen limitations, as well as maximize metabolic functions.     

Ethanol inhibits hormone stimulated hepatocyte DNA synthesis

Insulin, glucagon, and epidermal growth factor (EGF) addition stimulated DNA synthesis in primary hepatocyte cell cultures prepared from adult rat liver. The addition of ethanol (20-200mM) to the culture medium resulted in a substantial reduction in DNA synthesis as measured by 3H-thymidine incorporation and autoradiography. This effect was specific for differentiated hepatocytes compared to fibroblasts and two other human hepatoma cell lines. These studies demonstrate in a cell culture system that one of the major properties of ethanol is the inhibition of hepatocyte DNA synthesis.     

Multilayer rat hepatocyte aggregates formed on expanded polytetrafluoroethylene surface

Feasibility of using a macroporous membrane material, expanded polytetrafluoroethylene (ePTFE), for culturing hepatocytes on its surface was examined. Adult rat hepatocytes were attached to an ePTFE surface and cultured in a hormonally defined medium supplemented with or without fetal calf serum (FCS, 10%) or bovine serum albumin (BSA, 0.03–3%). When cultured in a FCS-suplemented medium, hepatocytes reorganized themselves into multilayer cell aggregates on an ePTFE surface. The morphological characteristics of hepatocytes were influenced by the modification of the ePTFE surface as well as the culture medium. Hepatocytes cultured on a polyvinylalcohol (PVA)-coated ePTFE surface formed many more multilayer cell aggregates than those cultured on an uncoated ePTFE surface. Such highly multilayered hepatocyte aggregates were also noted when the cells were cultivated in a BSA-supplemented medium. On the other hand, when cultured in a FCS- or BSA-free medium, hepatocytes formed cell monolayers on both PVA-coated and uncoated ePTFE surfaces as did the cells on a collagen-coated polystyrene surface. The hepatocytes in the aggregates exhibited high albumin expression capability and low DNA synthesis rate as compared with those in monolayer cultures. The multilayer hepatocyte aggregates, as immobilized on a PVA-coated ePTFE surface in a serum-supplemented medium, are shown to be not only morphologically, but functionally differentiated, and will provide us a model system for the development of a bioreactor using hepatocytes, particularly for a hybrid-type artificial liver. This revised version was published online in June 2006 with corrections to the Cover Date.     

Investigation of ifosfamide nephrotoxicity induced in a liver–kidney co‐culture biochip

In this article, we present a liver–kidney co‐culture model in a micro fluidic biochip. The liver was modeled using HepG2/C3a and HepaRG cell lines and the kidney using MDCK cell lines. To demonstrate the synergic interaction between both organs, we investigated the effect of ifosfamide, an anticancerous drug. Ifosfamide is a prodrug which is metabolized by the liver to isophosforamide mustard, an active metabolite. This metabolism process also leads to the formation of chloroacetaldehyde, a nephrotoxic metabolite and acrolein a urotoxic one. In the biochips of MDCK cultures, we did not detect any nephrotoxic effects after 72 h of 50 µM ifosfamide exposure. However, in the liver–kidney biochips, the same 72 h exposure leads to a nephrotoxicity illustrated by a reduction of the number of MDCK cells (up to 30% in the HepaRG‐MDCK) when compared to untreated co‐cultures or treated MDCK monocultures. The reduction of the MDCK cell number was not related to a modification of the cell cycle repartition in ifosfamide treated cases when compared to controls. The ifosfamide biotransformation into 3‐dechloroethylifosfamide, an equimolar byproduct of the chloroacetaldehyde production, was detected by mass spectrometry at a rate of apparition of 0.3 ± 0.1 and 1.1 ± 0.3 pg/h/biochips in HepaRG monocultures and HepaRG‐MDCK co‐cultures respectively. Any metabolite was detected in HepG2/C3a cultures. Furthermore, the ifosfamide treatment in HepaRG‐MDCK co‐culture system triggered an increase in the intracellular calcium release in MDCK cells on contrary to the treatment on MDCK monocultures. As 3‐dechloroethylifosfamide is not toxic, we have tested the effect of equimolar choloroacetaldehyde concentration onto the MDCK cells. At this concentration, we found a quite similar calcium perturbation and MDCK nephrotoxicity via a reduction of 30% of final cell numbers such as in the ifosfamide HepaRG‐MDCK co‐culture experiments. Our results suggest that ifosfamide nephrotoxicity in a liver–kidney micro fluidic co‐culture model using HepaRG‐MDCK cells is induced by the metabolism of ifosfamide into chloroacetaldehyde whereas this pathway is not functional in HepG2/C3a‐MDCK model. This study demonstrates the interest in the development of systemic organ–organ interactions using micro fluidic biochips. It also illustrated their potential in future predictive toxicity model using in vitro models as alternative methods. Biotechnol. Bioeng. 2013; 110: 597–608. © 2012 Wiley Periodicals, Inc.     

Development of a 3D Tissue‐Engineered Skeletal Muscle and Bone Co‐culture System

In vitro 3D tissue‐engineered (TE) structures have been shown to better represent in vivo tissue morphology and biochemical pathways than monolayer culture, and are less ethically questionable than animal models. However, to create systems with even greater relevance, multiple integrated tissue systems should be recreated in vitro. In the present study, the effects and conditions most suitable for the co‐culture of TE skeletal muscle and bone are investigated. High‐glucose Dulbecco's modified Eagle medium (HG‐DMEM) supplemented with 20% fetal bovine serum followed by HG‐DMEM with 2% horse serum is found to enable proliferation of both C2C12 muscle precursor cells and TE85 human osteosarcoma cells, fusion of C2C12s into myotubes, as well as an upregulation of RUNX2/CBFa1 in TE85s. Myotube formation is also evident within indirect contact monolayer cultures. Finally, in 3D co‐cultures, TE85 collagen/hydroxyapatite constructs have significantly greater expression of RUNX2/CBFa1 and osteocalcin/BGLAP in the presence of collagen‐based C2C12 skeletal muscle constructs; however, fusion within these constructs appears reduced. This work demonstrates the first report of the simultaneous co‐culture and differentiation of 3D TE skeletal muscle and bone, and represents a significant step toward a full in vitro 3D musculoskeletal junction model.     

Density-dependent growth control of adult rat hepatocytes in primary culture

Adult rat hepatocytes in primary culture, which show various liver functions, did not show any mitosis at confluent cell density, although they entered the S phase and remained in the G2 phase, judging by cytofluorometry, when insulin and epidermal growth factor (EGF) were added to 2-day cultures (Tomita, Y., Nakamura, T., & Ichihara, A. (1981) Exp. Cell Res. 135, 363-371). However, when the cell density was decreased by half or one third, the number of nuclei and cell number increased to 1.5-2.0 times that after culture for 35 h with insulin and EGF. Moreover, at these lower densities, DNA synthesis started much earlier, although at the usual high density DNA synthesis with these two hormones did not start until the hepatocytes had been cultured for over 40 h. These results suggest that proliferation of mature rat hepatocytes is regulated by the cell density. First, cells in G0 enter the G1 phase density-dependently; then cells in the G1 phase seem to be stimulated to enter the S phase by insulin and EGF, and a low cell density may permit cells after DNA synthesis to enter the M phase. DNA synthesis of rat hepatocyte cultures at low cell density was strongly inhibited by co-culture with a dense culture. Therefore, the density-dependent mechanism of hepatocyte proliferation seems to involve regulation by a soluble inhibitor(s) secreted by the hepatocytes into the culture medium.     

Colony isolation and secondary culture of fetal porcine hepatocytes on STO feeder cells

Summary The secondary culture of non-transformed parenchymal hepatocytes has not been possible. STO feeder cell-dependent secondary cultures of fetal pig hepatocytes were established by colony isolation from primary cultures of 26-d fetal livers. The liver cells had the typical polygonal morphology of parenchymal hepatocytes. They also spontaneously differentiated to form small biliary canaliculi between individual cells or progressed further to large multicellular duct-like structures or cells undergoing gross lipid accumulation and secretion. The secondary hepatocyte cultures expressed alpha-fetoprotein (AFP), albumin, and β-fibrinogen mRNA, and conditioned medium from the cells contained elevated levels of transferrin and albumin. STO feeder cell co-culture may be useful for the sustainable culture of hepatocytes from other species.     

Heterotypic interactions in the preservation of morphology and functionality of porcine hepatocytes by bone marrow mesenchymal stem cells in vitro

Temporary replacement of specific liver functions with extracorporeal bioartificial liver has been hampered by rapid de‐differentiation of porcine hepatocytes in vitro. Co‐cultivation of hepatocytes with non‐parenchymal cells may be beneficial for optimizing cell functions via mimicry of physiological microenvironment consisting of endogenous matrix proteins. However, the underlying mechanisms remain to be elucidated. A randomly distributed co‐culture system composed of porcine hepatocytes and bone marrow mesenchymal stem cells was generated, and the morphological and functional changes of varying degrees of heterotypic interactions were characterized. Furthermore, contributions of extracellular matrix within this co‐culture were evaluated. A rapid attachment and self‐organization of three‐dimensional hepatocyte spheroids were encouraged. Studies on hepatocyte viability showed a metabolically active, viable cell population in all co‐culture configurations with occurrence of few dead cells. The maximal induction of albumin production, urea synthesis, and cytochrome P4503A1 activities was achieved at seeding ratio of 2:1. Immunocytochemical detection of various extracellular matrix confirmed that a high level of matrix proteins synthesis within distinct cells was involved in hepatocyte homeostasis. These results demonstrate for the first time that cell–matrix has synergic effects on the preservation of hepatic morphology and functionality in the co‐culture of porcine hepatocytes with mesenchymal stem cells in vitro, which could represent a promising tool for tissue engineering, cell biology, and bioartificial liver devices. J. Cell. Physiol. 219: 100–108, 2009. © 2008 Wiley‐Liss, Inc.     

Analysis of cell viability and differential activity of mouse hepatocytes under 3D and 2D culture in agarose gel

Three-dimensional (3D) and two-dimensional (2D) cultures of hepatocytes in various concentrations (0.3–0.7%) of agarose gel revealed that the hepatocytes under 3D cultures in 0.3% agarose gel possess long-term (>3 weeks) viability, significant self-assembly to form tissue like aggregates, low lactate dehydrogenase release and high albumin synthesis. These were in contrast to 2D culture of hepatocytes. Our results suggest that the 3D culture of hepatocytes in agarose gel favors aggregate formation of functionally active cells and would be useful for liver transplantation as well as to analyze hepatocytes biology.     

Functional 3‐D cardiac co‐culture model using bioactive chitosan nanofiber scaffolds

The in vitro generation of a three‐dimensional (3‐D) myocardial tissue‐like construct employing cells, biomaterials, and biomolecules is a promising strategy in cardiac tissue regeneration, drug testing, and tissue engineering applications. Despite significant progress in this field, current cardiac tissue models are not yet able to stably maintain functional characteristics of cardiomyocytes for long‐term culture and therapeutic purposes. The objective of this study was to fabricate bioactive 3‐D chitosan nanofiber scaffolds using an electrospinning technique and exploring its potential for long‐term cardiac function in the 3‐D co‐culture model. Chitosan is a natural polysaccharide biomaterial that is biocompatible, biodegradable, non‐toxic, and cost effective. Electrospun chitosan was utilized to provide structural scaffolding characterized by scale and architectural resemblance to the extracellular matrix (ECM) in vivo. The chitosan fibers were coated with fibronectin via adsorption in order to enhance cellular adhesion to the fibers and migration into the interfibrous milieu. Ventricular cardiomyocytes were harvested from neonatal rats and studied in various culture conditions (i.e., mono‐ and co‐cultures) for their viability and function. Cellular morphology and functionality were examined using immunofluorescent staining for alpha‐sarcomeric actin (SM‐actin) and gap junction protein, Connexin‐43 (Cx43). Scanning electron microscopy (SEM) and light microscopy were used to investigate cellular morphology, spatial organization, and contractions. Calcium indicator was used to monitor calcium ion flux of beating cardiomyocytes. The results demonstrate that the chitosan nanofibers retained their cylindrical morphology in long‐term cell cultures and exhibited good cellular attachment and spreading in the presence of adhesion molecule, fibronectin. Cardiomyocyte mono‐cultures resulted in loss of cardiomyocyte polarity and islands of non‐coherent contractions. However, the cardiomyocyte‐fibroblast co‐cultures resulted in polarized cardiomyocyte morphology and retained their morphology and function for long‐term culture. The Cx43 expression in the fibroblast co‐culture was higher than the cardiomyocytes mono‐culture and endothelial cells co‐culture. In addition, fibroblast co‐cultures demonstrated synchronized contractions involving large tissue‐like cellular networks. To our knowledge, this is the first attempt to test chitosan nanofiber scaffolds as a 3‐D cardiac co‐culture model. Our results demonstrate that chitosan nanofibers can serve as a potential scaffold that can retain cardiac structure and function. These studies will provide useful information to develop a strategy that allows us to generate engineered 3‐D cardiac tissue constructs using biocompatible and biodegradable chitosan nanofiber scaffolds for many tissue engineering applications. Biotechnol. Bioeng. 2013; 110: 637–647. © 2012 Wiley Periodicals, Inc.     

Characterization of the three-compartment gel-entrapment porcine hepatocyte bioartificial liver

A hybrid bioartificial liver device supporting a large mass of cells expressing differentiated hepatocyte metabolic capabilities is necessary for the successful treatment of fulminant hepatic failure. The three-compartment gel-entrapment porcine hepatocyte bioartificial liver was designed to provide "bridge" support to transplantation or until native liver recovery is achieved for patients with acute liver failure. The device is an automated mammalian cell culture system supporting 6-7 × 109 porcine hepatocytes entrapped in a collagen matrix and inoculated into the capillary lumen spaces of two 100 kDa molecular mass cut-off hollow fiber bioreactors. Gel contraction recreates a small lumen space within the hollow fiber which allows for the delivery of a nutrient medium. This configuration supported hepatocyte viability and differentiated phenotype as measured by albumin synthesis, ureagenesis, oxygen consumption, and vital dye staining during both cell culture and ex vivo application. The hollow fiber membrane was also shown to isolate the cells from xenogenic immunoglobulin attack. The gel-entrapment bioartificial liver maintained a large mass of functional hepatocytes by providing a three-dimensional cell culture matrix, by delivering basal nutrients through lumen media perfusion, and by preventing rejection of the xenocytes. These features make this device a favorable candidate for the treatment of clinical fulminant hepatic failure.     

Development of a 3D human in vitro skin co‐culture model for detecting irritants in real‐time

Tissue engineered materials for clinical purposes have led to the development of in vitro models as alternatives to animal testing. The aim of this study was to understand the paracrine interactions arising between keratinocytes and fibroblasts for detecting and discriminating between an irritant‐induced inflammatory reaction and cytotoxicy. We used two irritants [sodium dodecyl sulphate (SDS) and potassium diformate (Formi®)] at sub‐toxic concentrations and studied interleukin‐1 alpha (IL‐1α) release from human keratinocytes and activation of NF‐κB in human fibroblasts. NF‐κB activation in fibroblast 2D cultures required soluble factors released by prior incubation of keratinocytes with either SDS or Formi®. Neither cell type responded directly to either agent, confirming a paracrine mechanism. Fibroblasts were then cultured in 3D microfiber scaffolds and transfected with an NF‐κB reporter construct linked to GFP. Findings for 3D cultures were similar to those in 2D in that soluble factors released by prior incubation of keratinocytes with SDS or Formi® was required for NF‐κB activation in fibroblasts. Similarly, direct incubation with either agent did not directly activate NF‐κB. A technical advantage of using transfected cells in 3D was an ability to detect NF‐κB activation in live fibroblasts. To confirm paracrine signaling a twofold increase in IL‐1α was measured in keratinocyte‐conditioned medium after incubation with SDS or Formi®, which correlated with fibroblast NF‐κB activity. In summary, this work has value for developing 3D tissue engineered co‐culture models for the in vitro testing of irritant chemicals at sub‐toxic concentrations, as an alternative to in vivo models. Biotechnol. Bioeng. 2010;106: 794–803. © 2010 Wiley Periodicals, Inc.     

Functional engineering of hepatocytes via heterocellular presentation of a homoadhesive molecule, E-cadherin.

Engineering functional activity of liver cell cultures requires the modulation of specific cell-cell interactions. We have investigated the quantitative role of systematically varied presentation of the cell-cell adhesion molecule, E-cadherin, on the differentiated function of cocultured parenchymal liver cells, hepatocytes. Specifically, we incorporated different proportions of E-cadherin transfected L-929 chaperone cells and untransfected chaperone cells, within cultures of primary rat hepatocytes on a collagen substrate. By using a strongly adhesive substrate that restricted cadherin-induced variations in cell spreading and growth-arresting chaperone cells, we could carefully isolate the potential role of cell-cell adhesion on cell differentiation. Using immunofluorescence microscopy, we confirmed that cadherins expressed at hepatocyte-hepatocyte contacts as well as hepatocyte-chaperone contacts were crossreactive. However, hepatocytes cocultured with cadherin-presenting chaperone cells had a 55-65% increase in longterm function over hepatocytes cocultured with control, nonpresenting chaperone cells. Notably, the cadherin-induced increase in function occurred over and above the basal, coculture-induced functional elevation. Further, we quantified the stoichiometric importance of cadherin contacts by comparing established markers of hepatocyte functional activity across a graded range of E-cadherin presentation. At low levels of cadherin-mediated contacts, the induction of differentiated function was weak, while high levels of contacts elicited a marked increase in function. Thus, hepatocyte biochemical functions (albumin and urea secretion) were biphasically governed by the degree of cadherin-based contacts presented during culture. Overall, our results demonstrate the unequivocal role of cell-cell adhesion molecules in hepatocyte functional engineering, through the graded use of cadherin presentation from functionally incompetent, heterotypic chaperone cells.