Galactosylated PVDF membrane promotes hepatocyte attachment and functional maintenance

One of the major challenges in BLAD design is to develop functional substrates suitable for hepatocyte attachment and functional maintenance. In the present study, we designed a poly(vinylidene difluoride) (PVDF) surface coated with galactose-tethered Pluronic polymer. The galactose-derived Pluronic F68 (F68-Gal) was adsorbed on PVDF membrane through hydrophobic-hydrophobic interaction between PVDF and the polypropylene oxide segment in Pluronic. The galactose density on the modified PVDF surface increased with the concentration of the F68-Gal solution, reaching 15.4 nmol galactosyl groups per cm2 when a 1 mg/ml of F68-Gal solution was used. The adsorbed F68-Gal remained relatively stable in culture medium. Rat hepatocytes attachment efficiency on F68-Gal modified PVDF membrane was similar to that on collagen-coated surface. The attached hepatocytes on PVDF/F68-Gal membrane self-assembled into multi-cellular spheroids after 1 day of culture. These attached hepatocytes in spheroids exhibited higher cell functions such as albumin synthesis and P450 1A1 detoxification function compared to unmodified PVDF membrane and collagen-coated surface. These results suggest the potential of this galactose-immobilized PVDF membrane as a suitable substrate for hepatocyte culture.     

Preparation of lactose-silk fibroin conjugates and their application as a scaffold for hepatocyte attachment

We prepared glycoconjugates (Lac-CY-SF) by the homogeneous chemical modification of solubilized silk fibroin (SF) with lactose bearing the galactose residue using cyanuric chloride (CY) as a coupling spacer and examined the usefulness of their application as a scaffold for hepatocyte attachment. The covalent immobilization of lactose into SF was assessed by several criteria including 1H-NMR measurements and reactions with lectins such as Ricinus communis agglutinin (RCA(120)) and fluorescein isothiocyanate-labeled RCA(120) (FITC-RCA(120)). The 1H-NMR spectrum of Lac-CY-SF showed new broad peaks attributed to methine and methylene protons of lactose, and by the integrated intensities of the peaks the weight ratio of lactose to SF in Lac-CY-SF was determined to be 0.20. Addition of RCA(120) to Lac-CY-SF solution caused an increase in the turbidity of the Lac-CY-SF solution. Incubation of Lac-CY-SF films with FITC-RCA(120) showed that the fluorescence emitted from the whole surface of Lac-CY-SF films. Furthermore, we examined the effect of the Lac-CY-SF conjugate-coating onto polystyrene culture dishes on the attachment of rat hepatocytes and their morphology. Attachment of hepatocytes onto the 0.1% (w/v) conjugate-coated dishes showed about an 8-fold increase as compared with that on uncoated dishes, being comparable to that on collagen-coated dishes, whereas the attachment on the SF-coated dishes was lower than that on uncoated dishes. Hepatocytes cultured onto the conjugate-coated dishes for 2.5 h showed smaller round-shaped morphology compared to those on collagen-coated dishes. After 2 days of culture in medium containing 100 nM insulin and 100 nM dexamethasone, hepatocytes on the conjugate-coated dishes formed monolayer islands with a slightly dispersed morphology, while hepatocytes cultured on collagen-coated dishes were uniformly spread flat. These results indicate that the Lac-CY-SF conjugates are useful as a scaffold for hepatocyte attachment, but the morphology of hepatocytes cultured on the conjugate-coated dishes is different from that on collagen-coated dishes.     

Control of adhesion and detachment of parenchymal liver cells using lactose-carrying polystyrene as substratum.

Adhesion of hepatocytes on culture dishes whose surface was coated with a lactose-carrying styrene homopolymer (PVLA) was investigated. Hepatocytes maintained their round shape on PVLA substratum, which is in contrast to the usual spread shape characteristic of those cultured on collagen and fibronectin substrata. Calcium ion was indispensable for hepatocyte adhesion in PVLA substratum, and hence the hepatocytes on PVLA were easily detached when the culture was treated with ethylenediamine tetraacetic acid (EDTA). The recovered hepatocytes readheres to PVLA. The adhesion of hepatocytes to PVLA was not inhibited by cytochalasin B but by colchicine. Hepatocytes recognize the galactose moieties on the surface of asialoglycoproteins and removes these proteins from the blood stream by receptor mediated endocytosis. The mechanism of adhesion of hepatocytes on PVLA substratum which contains a high density of galactose residues was distinct from the attachment on collagen and fibronectin substrata, and showed great similarity to the receptor and ligand interactions which occurs in the clearance of asialoglycoproteins by hepatocytes.     

Stable immobilization of rat hepatocyte spheroids on galactosylated nanofiber scaffold

Primary rat hepatocytes self-assemble into multi-cellular spheroids and maintain differentiated functions when cultured on a two-dimensional (2-D) substrate conjugated with galactose ligand. The aim of this study is to investigate how a functional nanofiber scaffold with surface-galactose ligand influences the attachment, spheroid formation and functional maintenance of rat hepatocytes in culture, as compared with the functional 2-D substrate. Highly porous nanofiber scaffolds comprising of fibers with an average diameter of 760 nm were prepared by electrospinning of poly(epsilon-caprolactone-co-ethyl ethylene phosphate) (PCLEEP), a novel biodegradable copolymer. Galactose ligand with a density of 66 nmol/cm(2) was achieved on the nanofiber scaffold via covalent conjugation to a poly(acrylic acid) spacer UV-grafted onto the fiber surface. Hepatocytes cultured on the galactosylated PCLEEP nanofiber scaffold exhibited similar functional profiles in terms of cell attachment, ammonia metabolism, albumin secretion and cytochrome P450 enzymatic activity as those on the functional 2-D substrate, although their morphologies are different. Hepatocytes cultured on galactosylated PCLEEP film formed 50-300 microm spheroids that easily detached from surface upon agitation, whereas hepatocytes cultured on galactosylated nanofiber scaffold formed smaller aggregates of 20-100 microm that engulfed the functional nanofibers, resulting in an integrated spheroid-nanofiber construct.     

Control of adhesion and detachment of parenchymal liver cells using lactose-carrying polystyrene as substratum

Adhesion of hepatocytes on culture dishes whose surface was coated with a lactose-carrying styrene homopolymer (PVLA) was investigated. Hepatocytes maintained their round shape on PVLA substratum, which is in contrast to the usual spread shape characteristic of those cultured on collagen and fibronectin substrata. Calcium ion was indispensable for hepatocyte adhesion in PVLA substratum, and hence the hepatocytes on PVLA were easily detached when the culture was treated with ethylenediamine tetraacetic acid (EDTA). The recovered hepatocytes readheres to PVLA. The adhesion of hepatocytes to PVLA was not inhibited by cytochalasin B but by colchicine. Hepatocytes recognize the galactose moieties on the surface of asialoglycoproteins and removes these proteins from the blood stream by receptor mediated endocytosis. The mechanism of adhesion of hepatocytes on PVLA substratum which contains a high density of galactose residues was distinct from the attachment on collagen and fibronectin substrata, and showed great similarity to the receptor and ligand interactions which occurs in the clearance of asialoglycoproteins by hepatocytes.     

Adhesion contact dynamics of primary hepatocytes on poly(ethylene terephthalate) surface

The design of bioartificial liver assist device requires an effective attachment of primary hepatocytes on polymeric biomaterials. A better understanding of this cell-surface interaction would aid the optimal choice of biomaterials. In this study, the adhesion contact dynamics of primary hepatocytes on poly(ethylene terephthalate) (PET) surface with grafted poly(acrylic acid) (PAA) and coated collagen is probed with confocal reflectance interference contrast microscopy (C-RICM) in conjunction with phase contrast microscopy. An increase of acrylic acid density from 0 to 12 nmole/cm2 raises both the root-mean-square surface roughness and amount of adsorbed collagen of PET surface. C-RICM demonstrates that hepatocytes form tight adhesion contacts upon seeding on both plain PET and PAA-grafted PET (both with collagen coating) despite the insignificant two-dimensional cell spreading. At two hours after cell seeding, the normalized contact area and adhesion energy of hepatocytes on 12 nmole/cm2 PAA-grafted-PET (with collagen coating) is 27% and 114% higher, respectively, than that on collagen coated plain PET. Interestingly, the growth kinetics of adhesion patch for hepatocyte on PAA-grafted PET with collagen coating is best fitted by R proportional to t0.5 and is significantly different from that on collagen coated plain PET, which is best fitted by R proportional to t0.25. Overall, this study demonstrates the modulation of biophysical response of adherent hepatocytes through the control of the biomaterial surface properties.     

Mixed-ligand modification of polyamidoamine dendrimers to develop an effective scaffold for maintenance of hepatocyte spheroids

Compared with a monolayer culture, hepatocyte spheroids are known to maintain liver function for long periods. We found that hepatocytes formed spheroids when cultured on polyamidoamine dendrimers modified with fructose. Because galactose is a ligand for the asialoglycoprotein receptor on the hepatocyte cytoplasmic membrane, it was chosen as another ligand for modification in order to maintain adhesion of spheroids for long periods. Simultaneous modification of dendrimers with fructose and galactose had a marked effect on the time length of spheroid adhesion. Suppression of apoptosis and necrosis was observed in hepatocyte spheroids cultured on a dendrimer modified with fructose and galactose (F/G dendrimer). Moreover, the hepatocyte spheroids cultured on the F/G dendrimer had higher activities of liver-specific functions, such as urea synthesis and albumin gene expression, than did those cultured on single-ligand-modified dendrimers. The expression of heat shock protein (HSP) genes was examined to evaluate the stress response of cells to scaffolds. The hepatocytes cultured on the F/G dendrimer had very low expression levels of both HSP60 and HSP70 mRNAs. Thus immobilization of mixed-ligand-modified dendrimers could generate a suitable surface for hepatocyte spheroid formation. These dendrimers could be a powerful tool for generating custom-made scaffolds for cells other than hepatocytes by selecting the ligands suitable for each cell type.     

Galactosylated poly(vinylidene difluoride) hollow fiber bioreactor for hepatocyte culture

To overcome the limitations of long-term expression of highly differentiated hepatocyte functions, we have developed a novel bioreactor in which hepatocytes are seeded in a ligand-immobilized hollow fiber cartridge. Galactosylated Pluronic polymer is immobilized on poly(vinylidene difluoride) (PVDF) hollow fiber surface through an adsorption scheme yielding a substrate with hepatocyte-specific ligand and a hydrophilic surface layer, which can resist nonspecific protein adsorption and facilitate cell binding to the galactose ligand. Interestingly, the galactosylated PVDF hollow fiber shows enhanced serum albumin diffusion across the membrane. Freshly isolated rat hepatocytes were seeded and cultured in the extralumenal space of the hollow fiber cartridge for 18 days in a continuously circulated system. Albumin secretion function of the seeded hepatocytes was monitored by analyzing circulating medium by enzyme-linked immunosorbent assay. Urea synthesis and P-450 function (7-ethoxycoumarin dealkylase activity) were measured periodically by doping the circulating medium with NH4Cl and 7-ethoxycoumarin, respectively. Hepatocytes cultured on galactosylated PVDF hollow fibers maintained better albumin secretion and P-450 functions than on unmodified and serum-coated PVDF hollow fibers when cultured in serum-containing medium. Morphological examination by scanning electron microscopy showed that hepatocytes cultured on galactosylated PVDF hollow fibers developed significant aggregation, in contrast to those cultured on unmodified PVDF fibers or on serum-coated PVDF fibers. Transmission electron microscopy images revealed that tight junctions and canaliculus-like structures formed in these aggregates. These results suggest the potential application of this galactosylated PVDF hollow fiber cartridge for the design of a bioartificial liver assist device.     

Plasma-induced graft polymerization of acrylic acid onto poly(ethylene terephthalate) films: characterization and human smooth muscle cell growth on grafted films.

Graft polymerization of acrylic acid onto plasma treated poly(ethylene terephthalate) (PET) films was carried out to develop surfaces for protein immobilization and smooth muscle cell seeding. Films with various graft densities were characterized by contact angle measurements, attenuated total reflectance infrared spectroscopy, X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM). The contact angle was observed to decrease from 72.9 degrees for the virgin PET films to between 26 degrees and 33 degrees depending on the graft density. Storage of grafted films led to an increase in the contact angle, suggesting molecular rearrangement at the surface. However, films with the lowest graft levels showed maximum enhancement in the contact angle up on storage. XPS confirmed the presence of the polyacrylic acid grafts at the film surface and AFM showed a marked increase in the wavelength of the surface roughness as the graft density increased. The amount of collagen immobilized at the surface of the grafted films also increased as the graft density increased. The collagen immobilized films provided an excellent substrate for the growth of human smooth muscle cells.     

Priming of hepatocytes for cell culture by partial hepatectomy prior to cell isolation

The combination of ex vivo gene transfer and a sufficient transplant model for hepatocytes may permit treatment of single enzyme-based metabolic liver diseases. Induction of replicative potential (priming) in hepatocyte cultures may enhance the efficiency of gene transfer under stable in vitro conditions. It is known that hepatocyte replication is increased in vivo after partial hepatectomy. We investigated the effect of partial hepatectomy prior to cell isolation on hepatocytes in vitro. Male Lewis rats served as donors. Hepatocytes were isolated by collagenase digestion from either intact livers or from livers 48 h after 70% hepatectomy (PH). Cells were seeded on collagen-coated culture dishes with hormone-supplemented culture media. Hepatocyte morphology, number, albumin secretion rate, and mono-ethyl-glycin-xylidid (MEGX)-biotransformation capacity were assessed on days 1, 3, and 5 in culture. PH significantly increased hepatocyte number and albumin secretion of cultured hepatocytes over the whole observation period. In contrast, MEGX-biotransformation capacity was significantly decreased. Morphology of cultured hepatocytes was not affected by PH prior to hepatocyte isolation. These results suggest a prolonged and complex response of hepatocytes to PH in vitro. Hepatocyte priming by PH is a promising approach toward stable cultures of proliferating hepatocytes and may provide a model for in vitro studies of hepatic regeneration mechanisms. Further research on hepatocyte priming toward an application in ex vivo gene transfer and hepatic tissue engineering seems justified.     

3D hepatocyte monolayer on hybrid RGD/galactose substratum

Hepatocyte-based applications such as xenobiotics metabolism and toxicity studies usually require hepatocytes anchoring onto flat substrata that support their functional maintenance. Conventional cell culture plates coated with natural matrices or synthetic ligands allow hepatocytes to adhere tightly as two-dimensional (2D) monolayer but these tightly anchored hepatocytes rapidly lose their differentiated functions. On galactosylated substrata, hepatocytes adhere loosely; and readily form three-dimensional (3D) spheroids that can maintain high levels of cellular functions. These spheroids detach easily from the substrata and exhibit poor mass transport properties unsuitable for many applications. Here, we have developed a hybrid RGD/galactose substratum based on polyethylene terephthalate film conjugated with both RGD peptide and galactose ligand to enhance cell adhesion and functions synergistically. Primary hepatocytes adhere effectively onto the transparent hybrid substratum in 96-well plates as monolayer while exhibiting high levels of liver-specific functions, morphology and cell-cell interactions typically seen in the 3D hepatocyte spheroids. The hepatocytes cultured onto the hybrid substratum also exhibit high levels of sensitivity to a model drug acetaminophen similar to the 3D hepatocyte spheroids. The monolayer of hepatocytes exhibiting the 3D cell behaviors on this flat hybrid substratum can be useful for various applications requiring both effective mass transfer and cellular support.     

Adhesion contact dynamics of HepG2 cells on galactose-immobilized substrates

The specific recognition between asialoglycoprotein receptor and galactose ligand at cell-substrate interfaces has been shown to mediate hepatocyte adhesion and maintain liver specific functions of hepatocytes. Conventionally, the success of hepatocyte attachment on engineered tissue scaffold is inferred from the degree of two-dimensional cell spreading that is measured by transmitted light microscopy. However, the actual contact mechanics and adhesion strength of hepatocytes during two-dimensional cell spreading has not been elucidated due to lack of biophysical probe. In this study, a novel biophysical technique known as confocal reflectance interference contrast microscopy (C-RICM) in conjunction with phase contrast microscopy is utilized to probe the adhesion dynamics, contact mechanics and two-dimensional spreading kinetics of HepG2 cells on galactose immobilized and collagen gel coated substrates. C-RICM demonstrates that HepG2 cells form strong adhesion contacts with both galactose-immobilized surfaces and collagen gel coated substrates. Moreover, HepG2 cells maintain their compact shapes in the presence of asialoglycoprotein receptor-mediated recognition while they become exceedingly spread under integrin-mediated adhesion on collagen gel coated substrate. The initial rate of adhesion contact formation and the steady-state adhesion energy of HepG2 cell population are highest on substrate conjugated with galactose ligand via a longer spacer. The adhesion dynamics and final adhesion energy of HepG2 cells depends both on the type of ligand-receptor interaction and the length of spacer between the ligand and substrate. Most importantly, new biophysical insights into the initial hepatocyte attachment that are critical for hepatocyte culture are provided through the decomposition of two-dimensional spreading and adhesion contact formation on bio-functional substrates.     

Xyloglucan as a synthetic extracellular matrix for hepatocyte attachment

The possibility of employing naturally derived xyloglucan (XG) having galactose moieties in the side chain for the development of synthetic extracellular matrix in tissue engineering was studied. Hepatocyte adhesion to the XG-coated polystyrene (PS) dish was 73.9% after 30 min incubation, whereas that to the PS dish as a negative control was 59.1%. The hepatocyte adhesion to the XG-coated surface was dependent on the presence of Ca2+ ions, whereas that to the XG-coated surface could not be induced by Mg2+ ions alone, indicating specific interaction between galactose moieties of XG and asialoglycoprotein receptors of hepatocytes. From the results of fluorescence, confocal laser micrographs and flow cytometry, it was suggested that XG was internalized by hepatocytes through a receptor-mediated mechanism. The DNA synthesis of hepatocytes attached to the XG-coated surface was decreased with an increase of the coating concentration of XG and in the presence of epidermal growth factor (EGF). The spreading shapes of the hepatocytes attached to the surface in the presence of EGF at low concentration of XG (1 microg/ml) were enhanced. The hepatocytes attached to the surface at a high concentration of XG (200 microg/ml) showed round shapes with spheroids after 16 h in the presence of EGF.     

Evaluation of a galactose-carrying gelatin sponge for hepatocytes culture and transplantation

This study proposes a new three-dimensional culture of mouse hepatocytes in a porous galactose-carrying modified gelatin sponge matrix. The modification of gelatin using galactose residues significantly increased the attachment of hepatocytes on the substrate. A modified gelatin sponge with lactobionic acid (MGLA) was prepared to increase the specific interaction between the hepatocytes and the matrix. Hepatocytes cultured in a three-dimensional MGLA sponge released much less lactate dehydrogenase than those cultured on a collagen Type I-coated monolayer. Moreover, the survival rate of hepatocytes cultured on an MGLA sponge was longer than the survival rate of hepatocytes cultured on a collagen Type I-coated monolayer. Hepatic specific metabolic functions, namely, the secretion of serum albumin and the synthesis of urea, were well maintained and promoted by spheroidal hepatocytes formed in the MGLA sponge.     

Adhesion behaviours of hepatocytes cultured onto biodegradable polymer surface modified by alkali hydrolysis process

Rat hepatocytes were cultured onto the surface of various amorphous biodegradable polymers composed of lactic acid and glycolic acid which were partially surface hydrolyzed by treating with sodium hydroxide. The polymer surface progressively became more hydrophilic with increasing NaOH treatment time, which was confirmed by measuring water contact angles and XPS results. The number of hepatocytes attached onto the NaOH treated hydrophilic surfaces was greater than that of the non-treated control surface. The extent of hepatocytes adhesion onto the surface-modified poly(D,L-Iactic-co-glycolic acid, 85/15) depended on the NaOH treatment time. Under the optimal conditions of lactic/glycolic composition in the polymer and the surface hydrolysis time, the adhesion of hepatocytes were comparable or even better than the collagen-coated biodegradable polymer surface used as a control.     

Adhesion behaviours of hepatocytes cultured onto biodegradable polymer surface modified by alkali hydrolysis process

Rat hepatocytes were cultured onto the surface of various amorphous biodegradable polymers composed of lactic acid and glycolic acid which were partially surface hydrolyzed by treating with sodium hydroxide. The polymer surface progressively became more hydrophilic with increasing NaOH treatment time, which was confirmed by measuring water contact angles and XPS results. The number of hepatocytes attached onto the NaOH treated hydrophilic surfaces was greater than that of the non-treated control surface. The extent of hepatocytes adhesion onto the surface-modified poly(D,L-lactic-co-glycolic acid, 85/15) depended on the NaOH treatment time. Under the optimal conditions of lactic/glycolic composition in the polymer and the surface hydrolysis time, the adhesion of hepatocytes were comparable or even better than the collagen-coated biodegradable polymer surface used as a control.     

Albumin and urea production by hepatocytes cultured on extracellular matrix proteins-conjugated poly(vinyl alcohol) membranes

Production of albumin and urea by mouse hepatocytes on poly(vinylalcohol-co-ethylamine) (PVA-EA) membranes containing immobilized extracellular matrix (ECM) proteins was investigated for 7 days. The amount of ECM proteins (collagen, vitronectin and laminin) immobilized on PVA-EA and PVA-ECM membranes was determined to range from 1.09 microg/cm2 to 1.60 microg/cm2. Hepatocytes cultured on PVA-ECM membranes in serum-free media showed higher albumin production than those cultured on PVA-EA membranes after a 7-day incubation under the conditions in this study. Urea production by hepatocytes on PVA-ECM membranes was also determined to be higher than that on PVA-EA membranes up until day 5 of incubation in serum-free media, whereas no difference of urea production by hepatocytes on different PVA-ECM membranes and PVA-EA membranes was observed at 7 days of incubation. The effect of ECM proteins in PVA-ECM membranes on hepatocyte function (such as albumin and urea production) was observed in hepatocytes cultured in serum-free media up to day 5 of incubation. The ECM proteins immobilized on the PVA-ECM membranes contributed not only to the long-term stable production of albumin and urea by hepatocytes, but also the improved surviVal (viability) of hepatocytes on PVA-ECM membranes.     

A dual-functional fibrous scaffold enhances P450 activity of cultured primary rat hepatocytes

We have designed a novel dual-functional electrospun fibrous scaffold comprising two fiber mesh layers that were modified differently to induce two separate biological responses from hepatocytes. The first fiber layer was galactosylated on the surface to mediate hepatocyte attachment, while the second layer was loaded with 3-methylcholanthrene (3-Mc) to enhance cytochrome P450 activity of hepatocytes. Primary rat hepatocytes cultured on the galactosylated fibrous scaffolds loaded with different concentrations of 3-Mc were compared for their cell attachment efficiency, albumin secretion activity and cytochrome P450-dependent 7-ethoxycoumarin O-deethylase activity. This hybrid fibrous scaffold mediated hepatocyte attachment with slightly lower efficiency (76+/-2.3%) than a single-layer galactosylated fibrous scaffold (84+/-3.5%). More importantly, the cytochrome P450 activity of the hepatocytes cultured on the hybrid scaffold correlated well with the 3-Mc loading level. The results also showed that transfer of 3-Mc to hepatocytes through direct cell-fiber contact was the dominant transport route, with the induced cytochrome P450 activity being 1.9- to 4.8-fold higher than that of transfer of 3-Mc to hepatocytes via dissolution from fibers to medium. This study demonstrates the feasibility of creating multi-functional fibrous scaffolds that serve both as an adhesive substrate and as a delivery vehicle for bioactive molecules.     

The effect of surface roughness of microporous membranes on the kinetics of oxygen consumption and ammonia elimination by adherent hepatocytes.

In membrane hybrid liver support devices (HLSDs) using isolated hepatocytes where oxygen is transported only by diffusion to the cells, about 15-40% of the cell mass is likely to be in direct contact with the semipermeable membranes used as immunoselective barriers: quantitative effects of membrane surface properties on the kinetics of hepatocyte metabolic reactions may also affect HLSD performance. In this paper, we report our investigation of the effects of surface morphology of two microporous commercial membranes on the kinetics of oxygen consumption and ammonia elimination by primary hepatocytes in adhesion culture. Isolated rat hepatocytes were cultured on polypropylene microporous membranes with different surface roughness and pore size in a continuous-flow bioreactor whose fluid dynamics was optimized for the kinetic characterization of liver cell metabolic reactions. Collagen-coated membranes were used as the reference substratum. Hepatocyte adhesion was not significantly affected by membrane surface morphology. The rates of the investigated reactions increased with ammonia concentration according to saturation kinetics: the values of kinetic parameters Vmax and K(M) increased as cells were cultured on the membrane with the greatest membrane surface roughness and pore size. For the reaction of oxygen consumption, Vmax increased from 0.066 to 0.1 pmol h(-1) per cell as surface roughness increased from 70 to 370 nm. For the kinetics of ammonia elimination. K(M) increased from 0.23 to 0.32 mM and Vmax increased from 1.49 to 1.79 pmol h(-1) per cell with membrane surface roughness increasing from 70 to 370 nm. Cells cultured on collagen-coated membranes consistently yielded the highest reaction rates. The Vmax values of 0.18 and 2.84 pmol h(-1) per cell for oxygen consumption and ammonia elimination, respectively, suggest that cell functions are also affected by the chemical nature of the substratum.