Improved human islets' viability and functionality with mesenchymal stem cells and arg-gly-asp tripeptides supplementation of alginate micro-encapsulated islets in vitro.

INTRODUCTION: The extension of islet transplantation to a wider number of type 1 diabetes patients is compromised by severe adverse events related to the immunosuppressant therapy required for allogenic islet transplantation. In this context, microencapsulation offers the prospects of immunosuppressive-free therapy by physically isolating islets from the immune system. However, current biomaterials need to be optimized to: improve biocompatibility, guaranty the maintenance of graft viability and functionality, and prevent fibrosis overgrowth around the capsule in vivo. Accumulating evidence suggest that mesenchymal stem cells (MSCs) and anchor points consisting of tripeptides arg-gly-asp (RGD) have cytoprotective effects on pancreatic islets. Here, we investigated the effect of supplementing reference M-rich alginate microcapsules with MSCs and RGD-G rich alginate on bioprocessing as well as on human pancreatic islets viability and functionality. METHODS: We characterized the microcapsules components, and then for the new microcapsule composite product: we analyzed the empty capsules biocompatibility and then investigated the benefits of MSCs and RGD-G rich alginate on viability and functionality on the encapsulated human pancreatic islets in vitro. We performed viability tests by confocal microscopy and glucose stimulated insulin secretion (GSIS) test in vitro to assess the functionality of naked and encapsulated islets. RESULTS: Encapsulation in reference M-rich alginate capsules induced a reduction in viability and functionality compared to naked islets. This side-effect of encapsulation was in part counteracted by the presence of MSCs but the restoration was complete with the combination of both MSCs and the RGD-G rich alginate. CONCLUSIONS: The present findings show that bioprocessing a favorable composite environment inside the M-rich alginate capsule with both MSCs and RGD-G rich alginate improves human islets survival and functionality in vitro.

Oxygen generating biomaterial improves the function and efficacy of beta cells within a macroencapsulation device.

Tissue-engineered devices have the potential to significantly improve human health. A major impediment to the success of clinically scaled transplants, however, is insufficient oxygen transport, which leads to extensive cell death and dysfunction. To provide in situ supplementation of oxygen within a cellular implant, we developed a hydrolytically reactive oxygen generating material in the form of polydimethylsiloxane (PDMS) encapsulated solid calcium peroxide, termed OxySite. Herein, we demonstrate, for the first time, the successful implementation of this in situ oxygen-generating biomaterial to support elevated cellular function and efficacy of macroencapsulation devices for the treatment of type 1 diabetes. Under extreme hypoxic conditions, devices supplemented with OxySite exhibited substantially elevated beta cell and islet viability and function. Furthermore, the inclusion of OxySite within implanted macrodevices resulted in the significant improvement of graft efficacy and insulin production in a diabetic rodent model. Translating to human islets at elevated loading densities further validated the advantages of this material. This simple biomaterial-based approach for delivering a localized and controllable oxygen supply provides a broad and impactful platform for improving the therapeutic efficacy of cell-based approaches.

Activated platelet-rich plasma improves cartilage regeneration using adipose stem cells encapsulated in a 3D alginate scaffold.

In the current study, the effect of superimposing platelet-rich plasma (PRP) on different culture mediums in a three-dimensional alginate scaffold encapsulated with adipose-derived mesenchymal stem cells for cartilage tissue repair is reported. The three-dimensional alginate scaffolds with co-administration of PRP and/or chondrogenic supplements had a significant effect on the differentiation of adipose mesenchymal stem cells into mature cartilage, as assessed by an evaluation of the expression of cartilage-related markers of Sox9, collagen II, aggrecan and collagen, and glycosaminoglycan assays. For in vivo studies, following induction of osteochondral lesion in a rabbit model, a high degree of tissue regeneration in the alginate plus cell group (treated with PRP plus chondrogenic medium) compared with other groups of cell-free alginate and untreated groups (control) were observed. After 8 weeks, in the alginate plus cell group, functional chondrocytes were observed, which produced immature matrix, and by 16 weeks, the matrix and hyaline-like cartilage became completely homogeneous and integrated with the natural surrounding cartilage in the defect site. Similar effect was also observed in the subchondral bone. The cell-free scaffolds formed fibrocartilage tissue, and the untreated group did not form a continuous cartilage over the defect by 16 weeks.

Bioenergetics of lactate vs. acetate outside TCA enhanced the hydrogen evolution levels in two newly isolated strains of the photosynthetic bacterium Rhodopseudomonas.

Two local hydrogen-evolving strains of purple nonsulfur bacteria have been isolated, characterized, and identified as Rhodopseudomonas sp. TUT (strains Rh1 and Rh2). Lactate followed by succinate and malate supported the highest amounts of H2 production, growth (O.D.660nm, proteins and bacteriochlorphyll contents), nitrogenase activity, and uptake hydrogenase; the least of which was acetate. Alginate-immobilized cells evolved higher hydrogen amounts than free cell counterparts. Rh1 was more productive than Rh2 at all circumstances. Lactate-dependent hydrogen evolution was more than twice that of acetate, due to ATP productivity (2/-1, respectively), which is limiting to the nitrogenase activity. The preference of lactate over other acids indicates the feasibility of using these two strains in hydrogen production from dairy wastewater.

Synergistic enhancement of ectopic bone formation by supplementation of freshly isolated marrow cells with purified MSC in collagen-chitosan hydrogel microbeads.

PURPOSE: Bone marrow-derived mesenchymal stem cells (MSC) can differentiate osteogenic lineages, but their tissue regeneration ability is inconsistent. The bone marrow mononuclear cell (BMMC) fraction of adult bone marrow contains a variety of progenitor cells that may potentiate tissue regeneration. This study examined the utility of BMMC, both alone and in combination with purified MSC, as a cell source for bone regeneration. METHODS: Fresh BMMC, culture-expanded MSC, and a combination of BMMC and MSC were encapsulated in collagen-chitosan hydrogel microbeads for pre-culture and minimally invasive delivery. Microbeads were cultured in growth medium for 3 days, and then in either growth or osteogenic medium for 17 days prior to subcutaneous injection in the rat dorsum. RESULTS: MSC remained viable in microbeads over 17 days in pre-culture, while some of the BMMC fraction were nonviable. After 5 weeks of implantation, microCT and histology showed that supplementation of BMMC with MSC produced a strong synergistic effect on the volume of ectopic bone formation, compared to either cell source alone. Microbeads containing only fresh BMMC or only cultured MSC maintained in osteogenic medium resulted in more bone formation than their counterparts cultured in growth medium. Histological staining showed evidence of residual microbead matrix in undifferentiated samples and indications of more advanced tissue remodeling in differentiated samples. CONCLUSIONS: These data suggest that components of the BMMC fraction can act synergistically with predifferentiated MSC to potentiate ectopic bone formation. The microbead system may have utility in delivering desired cell populations in bone regeneration applications.

Versatile click alginate hydrogels crosslinked via tetrazine-norbornene chemistry.

Alginate hydrogels are well-characterized, biologically inert materials that are used in many biomedical applications for the delivery of drugs, proteins, and cells. Unfortunately, canonical covalently crosslinked alginate hydrogels are formed using chemical strategies that can be biologically harmful due to their lack of chemoselectivity. In this work we introduce tetrazine and norbornene groups to alginate polymer chains and subsequently form covalently crosslinked click alginate hydrogels capable of encapsulating cells without damaging them. The rapid, bioorthogonal, and specific click reaction is irreversible and allows for easy incorporation of cells with high post-encapsulation viability. The swelling and mechanical properties of the click alginate hydrogel can be tuned via the total polymer concentration and the stoichiometric ratio of the complementary click functional groups. The click alginate hydrogel can be modified after gelation to display cell adhesion peptides for 2D cell culture using thiol-ene chemistry. Furthermore, click alginate hydrogels are minimally inflammatory, maintain structural integrity over several months, and reject cell infiltration when injected subcutaneously in mice. Click alginate hydrogels combine the numerous benefits of alginate hydrogels with powerful bioorthogonal click chemistry for use in tissue engineering applications involving the stable encapsulation or delivery of cells or bioactive molecules.

Differential response of encapsulated nucleus pulposus and bone marrow stem cells in isolation and coculture in alginate and chitosan hydrogels.

Cell-based therapies may hold significant promise for the treatment of early stage degeneration of the intervertebral disc (IVD). Given their propensity to proliferate and ability to form multiple tissue types, mesenchymal stem cells (MSCs) have been proposed as a potential cell source to promote repair of the nucleus pulposus (NP). However, for any successful cell-based therapy, a carrier biomaterial may be essential for targeted delivery providing key biophysical and biochemical cues to facilitate differentiation of MSCs. Two widely used biomaterials for NP regeneration are chitosan and alginate. The primary objective of this study was to assess the influence of alginate and chitosan hydrogels on bone marrow stem cells (BM) and NP cells in isolation or in coculture. A secondary objective of this study was to investigate coculture seeding density effects of BM and NP cells and simultaneously explore which cell type is responsible for matrix formation in a cocultured environment. Porcine NP and BM cells were encapsulated in alginate and chitosan hydrogels separately at two seeding densities (4×10(6) and 8×10(6) cells/mL) or in coculture (1:1, 8×10(6) cells/mL). Constructs (diameter=5 mm, height=3 mm) were maintained under IVD-like conditions [low-glucose, low (5%) oxygen] with or without transforming growth factor-ß3 (TGF-ß3) supplementation for 21 days. Results demonstrated differential viability depending on hydrogel type. NP cells remained viable in both biomaterial types whereas BM viability was diminished in chitosan. Further, hydrogel type was found to regulate sulfated glycosaminoglycan (sGAG) and collagen accumulation. Specifically, alginate better supports sGAG accumulation and collagen type II deposition for both NP and BM cell types compared with chitosan. Having identified that alginate more readily supports cell viability and matrix accumulation, we further explored additional effects of seeding density ratios (NP:BM--1:1, 1:2) for coculture studies. Interestingly, in coculture conditions, the BM cell population declined in number while NP cells increased, indicating that MSCs may in fact be signaling NP cells to proliferate rather than contributing to matrix formation. These findings provide exciting new insights on the potential of MSCs for NP tissue regeneration strategies.

Novel bacterial bioassay for a high-throughput screening of 4-hydroxyphenylpyruvate dioxygenase inhibitors.

Plant 4-hydroxyphenylpyruvate dioxygenase (HPPD) is the molecular target of a range of synthetic ß-triketone herbicides that are currently used commercially. Their mode of action is based on an irreversible inhibition of HPPD. Therefore, this inhibitory capacity was used to develop a whole-cell colorimetric bioassay with a recombinant Escherichia coli expressing a plant HPPD for the herbicide analysis of ß-triketones. The principle of the bioassay is based on the ability of the recombinant E. coli clone to produce a soluble melanin-like pigment, from tyrosine catabolism through p-hydroxyphenylpyruvate and homogentisate. The addition of sulcotrione, a HPPD inhibitor, decreased the pigment production. With the aim to optimize the assay, the E. coli recombinant clone was immobilized in sol-gel or agarose matrix in a 96-well microplate format. The limit of detection for mesotrione, tembotrione, sulcotrione, and leptospermone was 0.069, 0.051, 0.038, and 20 µM, respectively, allowing to validate the whole-cell colorimetric bioassay as a simple and cost-effective alternative tool for laboratory use. The bioassay results from sulcotrione-spiked soil samples were confirmed with high-performance liquid chromatography.

Drug susceptibility of matrix-encapsulated Candida albicans nano-biofilms.

The rise in the use of biomedical devices and implants has seen a concomitant surge in the advent of device-related nosocomial (hospital-acquired) infections of bacterial and fungal origins. The most common nosocomial fungal infection is candidiasis caused mainly by Candida albicans biofilms. Candidiasis is associated with an unacceptably high mortality rate, and there is an urgent need for the discovery of new antifungal drugs that prevent or control biofilm formation. To this end, we recently developed an ultra-high-throughput microarray platform consisting of nano-scale biofilms of C. albicans encapsulated in collagen or alginate hydrogel matrices for antifungal drug screening. Here, we report that the choice of matrix influences the apparent susceptibility of C. albicans to the common antifungal drugs, amphotericin B, and caspofungin. While amphotericin B is equally effective against biofilms grown in collagen and alginate matrices, caspofungin is effective only against biofilms grown only in alginate, but not in collagen. We demonstrate differences in the distribution of the drugs in the two matrices may contribute to the susceptibility of C. albicans nano-biofilms. In a larger context, our results highlight the importance of the choice of matrix as a parameter in 3D cell encapsulation, and suggest a screening strategy to predict drug performance in vivo.

Simultaneous species-specific PCR detection and viability testing of poly(vinyl alcohol) cryogel-entrapped Rhodococcus spp. after their exposure to petroleum hydrocarbons.

A method of simultaneous species-specific PCR detection and viability testing of poly(vinyl alcohol) cryogel-entrapped Rhodococcus spp. was developed that allowed the estimation of immobilized Rhodococcus opacus and Rhodococcus ruber survival after their exposure to petroleum hydrocarbon mixture. Spectrophotometric INT assay revealed high tolerance of gel-immobilized rhodococci to petroleum hydrocarbons, while among two Rhodococcus strains studied, R. ruber tolerated better to hydrocarbons compared to R. opacus. These findings were confirmed by respirometry results that showed increased respiratory activity of gel-immobilized Rhodococcus strains after 10-day incubation with 3% (v/v) petroleum hydrocarbon mixture. Moreover, jointly incubated rhodococcal strains demonstrated higher oxidative activities toward petroleum hydrocarbons than individual strains. Both Rhodococcus species were recovered successfully in cryogel granules using 16S rDNA-targeted PCR, even though the granules were previously stained with INT and extracted with ethanol. The method developed can be used for rapid detection and monitoring of gel-immobilized bacterial inocula in bioreactors or contaminated soil systems.

Optimization of ethanol production from carob pod extract using immobilized Saccharomyces cerevisiae cells in a stirred tank bioreactor.

In this study, optimization of ethanol production from carob pod extract was carried out by immobilized Saccharomyces cerevisiae. Results showed that Ca-alginate concentration and the amount of immobilized cells had significant effects on yield. Optimum conditions for ethanol fermentation were determined to be 2% Ca-alginate concentration, 150 rpm agitation rate, 5% yeast cells entrapped in beads and pH 5.5. After validation experiments; ethanol concentration, yield, production rate and sugar utilization rate were respectively 40.10 g/L, 46.32%, 3.19 g/L/h and 90.66%; and the fermentation time was decreased to 24 h. In addition, the immobilized cells were shown to be reusable for five cycles, though a decrease in yield was observed. Finally, carob pod extract was used for ethanol fermentation by controlled and uncontrolled pH without any enrichment, and the results suggest that carob extract can be utilized effectively by immobilized-cell fermentation without the use of enrichments to facilitate yeast growth.

Silk ionomers for encapsulation and differentiation of human MSCs.

The response of human bone marrow derived human mesenchymal stem cells (hMSCs) encapsulated in silk ionomer hydrogels was studied. Silk aqueous solutions with silk-poly-L-lysine or silk-poly-L-glutamate were formed into hydrogels via ultrasonication in situ with different net charges. hMSCs were encapsulated within the hydrogels and the impact of matrix charge was assessed over weeks in osteogenic, adipogenic and maintenance growth media. These modified silk charged polymers supported cell viability and proliferative potential, and the hMSCs were able to differentiate toward osteogenic or adipogenic lineages in the corresponding differentiation media. The silk/silk-poly-L-lysine hydrogels exhibited a positive effect on selective osteogenesis of hMSCs, inducing differentiation toward an osteogenic lineage even in the absence of osteogenic supplements, while also inhibiting adipogenesis. In contrast, silk/silk fibroin-poly-L-glutamate hydrogels supported both osteogenic and adipogenic differentiation of hMSCs when cultured under induction conditions. The results demonstrate the potential utility of silk-based ionomers in gel formats for hMSCs encapsulation and for directing hMSCs long term functional differentiation toward specific lineages.

Bilayered constructs aimed at osteochondral strategies: the influence of medium supplements in the osteogenic and chondrogenic differentiation of amniotic fluid-derived stem cells.

The development of osteochondral tissue engineered interfaces would be a novel treatment for traumatic injuries and aging associated diseases that affect joints. This study reports the development of a bilayered scaffold, which consists of both bone and cartilage regions. On the other hand, amniotic fluid-derived stem cells (AFSCs) could be differentiated into either osteogenic or chondrogenic cells, respectively. In this study we have developed a bilayered scaffolding system, which includes a starch/polycaprolactone (SPCL) scaffold for osteogenesis and an agarose hydrogel for chondrogenesis. AFSC-seeded scaffolds were cultured for 1 or 2 weeks in an osteochondral-defined culture medium containing both osteogenic and chondrogenic differentiation factors. Additionally, the effect of the presence or absence of insulin-like growth factor-1 (IGF-1) in the culture medium was assessed. Cell viability and phenotypic expression were assessed within the constructs in order to determine the influence of the osteochondral differentiation medium. The results indicated that, after osteogenic differentiation, AFSCs that had been seeded onto SPCL scaffolds did not require osteochondral medium to maintain their phenotype, and they produced a protein-rich, mineralized extracellular matrix (ECM) for up to 2 weeks. However, AFSCs differentiated into chondrocyte-like cells appeared to require osteochondral medium, but not IGF-1, to synthesize ECM proteins and maintain the chondrogenic phenotype. Thus, although IGF-1 was not essential for creating osteochondral constructs with AFSCs in this study, the osteochondral supplements used appear to be important to generate cartilage in long-term tissue engineering approaches for osteochondral interfaces. In addition, constructs generated from agarose-SPCL bilayered scaffolds containing pre-differentiated AFSCs may be useful for potential applications in regeneration strategies for damaged or diseased joints.

Chondrogenic differentiation of neonatal human dermal fibroblasts encapsulated in alginate beads with hydrostatic compression under hypoxic conditions in the presence of bone morphogenetic protein-2.

In the present work, neonatal human dermal fibroblasts (nHDFs) were evaluated as a potential cell source for intervertebral disc repair. Chondrogenic differentiation of nHDFs was studied in the presence or absence of hydrostatic compression under normal and hypoxic conditions. In addition, the potentially synergistic effects of mechanical stimulation and bone morphogenetic protein (BMP)-2 on the chondrogenic differentiation of nHDFs were assessed. Mechanical stimulation was applied to the cells encapsulated in alginate beads using a custom-built bioreactor system for either a 1- or 3-week period at a frequency of 1 Hz for 4 h/day. In general, after 21 days of culture, high cell viability was observed for all the groups, with the exception of the groups exposed to intermittent mechanical stimulation for 3 weeks. Long-term intermittent application of mechanical stimulation under low O(2) conditions resulted in elevated collagen biosynthesis rate from day 0. Inclusion of BMP-2 for this group improved the chondrogenic differentiation of nHDFs, as indicated by elevated aggrecan gene expression and an increased collagen production rate compared to the day 0 group. Thus, the combination of hypoxia, BMP-2 supplementation, and long-term intermittent application of dynamic hydrostatic pressure was found to be a potent promoter of the chondrogenic differentiation of nHDFs.

Thermoresponsive, in situ cross-linkable hydrogels based on N-isopropylacrylamide: fabrication, characterization and mesenchymal stem cell encapsulation.

Hydrogels that solidify in response to a dual, physical and chemical, mechanism upon temperature increase were fabricated and characterized. The hydrogels were based on N-isopropylacrylamide, which renders them thermoresponsive, and contained covalently cross-linkable moieties in the macromers. The effects of the macromer end group, acrylate or methacrylate, and the fabrication conditions on the degradative and swelling properties of the hydrogels were investigated. The hydrogels exhibited higher swelling below their lower critical solution temperature (LCST). When immersed in cell culture medium at physiological temperature, which was above their LCST, hydrogels showed constant swelling and no degradation over 8 weeks, with the methacrylated hydrogels showing greater swelling than their acrylated analogs. In addition, hydrogels immersed in cell culture medium under the same conditions showed lower swelling compared with phosphate-buffered saline. The interplay between chemical cross-linking and thermally induced phase separation affected the swelling characteristics of the hydrogels in different media. Mesenchymal stem cells encapsulated in the hydrogels in vitro were viable over 3 weeks and markers of osteogenic differentiation were detected when the cells were cultured with osteogenic supplements. Hydrogel mineralization in the absence of cells was observed in cell culture medium with the addition of fetal bovine serum and ß-glycerol phosphate. The results suggest that these hydrogels may be suitable as carriers for cell delivery in tissue engineering.

Reversible removal of SO2 at low temperature by Bacillus licheniformis immobilized on γ-Al2O3.

Bacillus licheniformis R08 biomass was immobilized on γ-Al2O3 and the effects of R08 biomass loading, SO2 concentration, water vapor, oxygen and temperature on removal of SO2 were investigated. The experimental results indicated that SO2 saturation capacity increased with increasing R08 biomass loading and SO2 concentration, but decreased with increasing adsorption temperature. Water vapor activated the adsorbent and promoted SO2 removal. An increase in oxygen concentration from 5 to 10% had little effect on SO2 removal. FTIR analysis revealed that the R08 biomass bound to γ-Al2O3 mainly by forming R-CO-O-Al bonds. X-ray photoelectron spectroscopy analysis indicated that γ-Al2O3 reacted with SO2 and formed aluminum sulfate in the presence of oxygen when R08 biomass loading was 13.8%, but that amido groups of the R08 biomass reacted with SO2 and formed sulfite when biomass loading was 32.4%. Ten continuous adsorption-desorption cycles showed that the adsorbent had an excellent regeneration performance.

Effect of aluminum phosphate on alkaline phosphatase activity of polyurethane foam immobilized cyanobacteria.

The impact of insoluble phosphorus such as aluminum and rock phosphate on alkaline phosphatase activity of polyurethane foam immobilized cyanobacteria was assessed. Polyurethane foam immobilized Nodularia recorded the highest alkaline phosphatase activity of 9.04 (m. mol p-nitrophenol released h(-1) mg(-1) protein) in vitro. A higher concentration of aluminum phosphate was recorded a 25% reduction in alkaline phosphatase activity, ammonia content, and available phosphorus in culture filtrate of polyurethane foam immobilized cyanobacteria. In general, immobilized cyanobacteria exhibited a higher alkaline phosphatase activity in rock phosphate than aluminum phosphate.

Study of immobilized and extracellular aminopeptidases of watermelon.

Watermelon cells after permeabilization in Tween 80 were immobilized by glutaraldehyde without any carrier. Cells immobilized by cross-linking demonstrated significantly lower aminopeptidase activities than untreated cells. Pectate and alginate hydrogels were used successfully for immobilization of watermelon cells which retained activities of some aminopeptidases. A simple and rapid procedure for determination of extracellular aminopeptidases was developed using a synthetic substrate. p-Nitroanilides of amino acids were used as substrates for the determination of the extracellular and intracellular enzymatic activities. The culture medium (without cells) was used for the identification and determination of extracellular enzyme activities, whereas intracellular activities were estimated from the cell suspension.

Biodegradation of crude oil across a wide range of salinities by an extremely halotolerant bacterial consortium MPD-M, immobilized onto polypropylene fibers.

The bacterial consortium MPD-M, isolated from sediment associated with Colombian mangrove roots, was effective in the treatment of hydrocarbons in water with salinities varying from 0 to 180 g L(-1). Where the salinity of the culture medium surpassed 20 g L(-1), its effectiveness increased when the cells were immobilized on polypropylene fibers. Over the range of salinity evaluated, the immobilized cells significantly enhanced the biodegradation rate of crude oil compared with free-living cells, especially with increasing salinity in the culture medium. Contrary to that observed in free cell systems, the bacterial consortium MPD-M was highly stable in immobilized systems and it was not greatly affected by increments in salinity. Biodegradation was evident even at the highest salinity evaluated (180 g L(-1)), where biodegradation was between 4 and 7 times higher with immobilized cells compared to free cells. The biodegradation of pristane (PR) and phytane (PH) and of the aromatic fraction was also increased using cells immobilized on polypropylene fibers.

Cytoprotective influence of ZVAD-fmk and glycine on gel-entrapped rat hepatocytes in a bioartificial liver.

BACKGROUND: This study was designed to determine if an anti-necrotic compound, glycine, and/or an anti-apoptotic agent, ZVAD-fmk, improved the viability and function of hepatocytes in a bioartificial liver. METHODS: Isolated rat hepatocytes were entrapped in collagen gel (1.0-10.0 x 10(6) cells/mL) and cultured in serum-free medium (1:10 ratio of gel:media) supplemented with glycine alone, ZVAD-fmk alone, or glycine and ZVAD-fmk. The cytoprotective effects of glycine and ZVAD-fmk on gel-entrapped rat hepatocytes (GERH) were determined after anoxic exposure (0-20 hours). Cell functionality (measured by urea production), cell viability (quantitated by vital staining with fluorescein diacetate:ethidium bromide [FDA:EB]), and the mechanism of cell death (verified by electron microscopy and DNA fragmentation studies) were determined for each condition. RESULTS: The viability of GERH declined gradually and then stabilized 12 hours after hepatocyte isolation. The rate of urea production by GERH was directly proportional to the number of viable hepatocytes. Apoptotic death predominated at low cell density, and necrotic cell death became significant at high cell density. Hepatocyte necrosis became more significant after exposure to longer periods of anoxia (4, 8, 12, and 20 hours). ZVAD-fmk provided dose-dependent cytoprotection to GERH with an optimum benefit at a concentration of 60 mumol/L. After anoxic exposure or under high cell density culture, glycine demonstrated a maximum benefit of inhibiting necrosis at a concentration of 3 mmol/L. The beneficial effects of glycine and ZVAD-fmk were additive. CONCLUSIONS: The metabolic activity of a hepatocyte bioartificial liver may benefit from the use of cytoprotective agents such as ZVAD-fmk and glycine.