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M L Yarmush

Publications and source records attributed to M L Yarmush.

At least 73 records · Page 4Linked to original sources
PubMed1996

Culture matrix configuration and composition in the maintenance of hepatocyte polarity and function.

Several extracellular matrix (ECM) configurations involving type I collagen and Matrigel were examined for their ability to support differentiated function and polarity of cultured adult rat hepatocytes. Collagen sandwich- and Matrigel-based cultures yielded superior and comparable albumin secretion for at least 2 weeks. In collagen sandwich, hepatocytes were polygonal, and formed multicellular arrays. Collagen sandwich was also found to promote in vivo-like polarization of F-actin, cell adhesion molecules (E-cadherin), and lateral (Na+, K(+)-ATPase, glucose transporter) and apical (dipeptidyl peptidase, aminopeptidase) membrane polarity markers, but not the expression of the gap junction protein connexin 32 and the epidermal growth factor (EGF) receptor. In contrast, hepatocytes cultured in or on Matrigel were more rounded and formed aggregates. Matrigel-based cultures also elicited detectable levels of connexin and EGF receptor and an altered distribution of F-actin, E-cadherin, and apical and lateral membrane proteins. Composite sandwich configurations containing collagen I and Matrigel restored markers lacking in the collagen sandwich, and showed a variable morphology and membrane polarity. Hepatocyte polarity could thus be manipulated by the overall ECM composition. Furthermore, in composite sandwich cultures, these manipulations can be effected largely independent of changes in hepatocyte morphology and albumin secretion.

Actins↗
PubMed1996

An analysis of transport resistances in the operation of BIAcore; implications for kinetic studies of biospecific interactions.

The mass-transfer processes that affect kinetic measurements of biospecific interactions between one species in a flowing solution and another species immobilized in a thin hydrogel instrument were analysed by means of convection-diffusion reaction models. The specific purpose was to identify experimental design considerations for kinetic measurements using the BIAcore instrument. Numerical solutions identified three different regimes of operation. A kinetic regime exists at low values of Damkohler number (Da), when the intrinsic kinetics are slow and the diffusion is relatively fast. This allows for the accurate determination of kinetic constants. A limiting value of Da, above which mass-transfer limitations appear, is presented as a function of Peclet number, Pe. At higher Da values, the reaction occurs in the mass-transfer-controlled regime where the reaction-rate is independent of the intrinsic kinetics. It was observed that, frequently, the reaction occured in an intermediate regime where, although the mass-transfer rate was not strictly limiting, substantial concentration gradients were present. Analysing the data in this regime by direct application of kinetic equations underestimates the association rate constant. Even when the reaction was not limited by mass-transfer in the flow channel, it may have been affected by steric hindrance to transport in the hydrogel, if a large concentration of capturing antibody or ligand was immobilized. The primary effect of the hindrance was to lower the soluble-species (analyte) concentration in the hydrogel when compared to the bulk solution. Non-uniformity of conditions within the hydrogel in the presence of steric hindrance had a significant effect on the observed reaction. The effect was most prominent at higher analyte concentration, when the rate constant showed an apparent reduction as the reaction progressed.

Antibodies, Monoclonal↗
PubMed1996

Effect of extracellular matrix topology on cell structure, function, and physiological responsiveness: hepatocytes cultured in a sandwich configuration.

Extracellular matrix (ECM) geometry is an important modulator of cell polarity and function. For example, 3-dimensional matrices often more effectively induce differentiated cell function than traditional 2-dimensional substrates. The effect of ECM topology can be investigated in a controlled fashion using a technique whereby cells cultured on a single surface are overlaid with a second layer of ECM, thereby creating a "sandwich" configuration. Confluent monolayers of epithelial or endothelial cells overlaid in this fashion often reorganize into structures that are reminiscent of their native tissue. In the case of hepatocytes, the overlay causes a dramatic reorganization of the cytoskeleton, adoption of in vivo-like morphology and polarity, and expression of a wide array of liver-specific functions. In this short review, we use the sandwiched hepatocyte culture system to illustrate the effect of ECM geometry on cellular function. Pertinent studies are summarized in the context of defining the parallels, strengths, and limitations of this culture system as an in vitro model to study the physiology and morphogenesis of liver tissue. We also explore some of its potential uses as a model to study liver pharmacology and toxicology, and for the development of liver preservation techniques and liver-assist devices.

Animals↗
PubMed1996

Targeted expression of insulin-like growth factor to human keratinocytes: modification of the autocrine control of keratinocyte proliferation.

Somatomedin C/insulin-like growth factor-I (IGF-I) is required for the proliferation of keratinocytes in vitro. In skin, the cells known to synthesize IGF-I are melanocytes and fibroblasts of the dermis. To investigate the role of IGF-I as a mediator of keratinocyte proliferation, we have used retroviral-mediated gene transfer to introduce the gene encoding human IGF-I into diploid human keratinocytes, thus causing these cells to produce a growth factor they normally do not express. Modified cells synthesized and secreted significant levels of IGF-I (560 ng/10(7) cells/24 h) in vitro. Cells expressing IGF-I were no longer dependent on exogenously added IGF-I or insulin for their sustained growth in vitro under serum-free conditions. The growth of these cells did require added epidermal growth factor (EGF) and bovine pituitary extract. The addition of an antibody that neutralizes IGF-I inhibited cell growth, suggesting that IGF-I must be secreted by the cells to promote cell proliferation. To investigate the role of IGF-I in vivo, we grafted modified keratinocytes expressing IGF-I onto athymic mice. Grafts of epithelial sheets of modified cells formed a stratified epithelium comparable to control grafts of unmodified cells. When analyzed for keratin 16 expression and by quantitative staining for the nuclear proliferation antigen Ki-67, however, modified epithelia showed an increase in these markers of proliferation when compared with grafts of unmodified cells. This study demonstrates that genetic modification can be used to modify the autocrine control of keratinocyte proliferation. The de novo synthesis of IGF-I by keratinocytes could sustain keratinocytes growth in vitro and stimulate proliferation in vivo without significantly altering epidermal differentiation. These data further support the role of IGF-I as a paracrine mediator of epidermal proliferation and as a potential signal of mesenchymal-epithelial interactions.

Animals↗
PubMed1996

Evaluation of human skin reconstituted from composite grafts of cultured keratinocytes and human acellular dermis transplanted to athymic mice.

This study evaluates the use of composite grafts of cultured human keratinocytes and de-epidermalized, acellular human dermis to close full-thickness wounds in athymic mice. Grafts were transplanted onto athymic mice and studied up to 8 wk. Graft take was excellent, with no instances of infection or graft loss. By 1 wk, the human keratinocytes had formed a stratified epidermis that was fused with mouse epithelium, and by 8 wk the grafts resembled human skin and could be freely moved over the mouse dorsum. Immunostaining for keratins 10 and 16 and for involucrin revealed an initial pattern of epithelial immaturity, which by 8 wk had normalized to that of mature unwounded epithelium. Mouse fibroblasts began to infiltrate the acellular dermis as early as 1 wk. By 8 wk fibroblasts had completely repopulated the dermis, and blood vessels were evident in the most superficial papillary projections. Dermal elements, such as rete ridges and elastin fibers, which were present in the starting dermis, persisted for the duration of the experiment. Grafts using keratinocytes from dark-skinned donors as opposed to light-skin donors had foci of pigmentation as early as 1 wk that progressed to homogenous pigmentation of the graft by 6 wk. These results indicate that melanocytes that persist in vitro are able to resume normal function in vivo. Our study demonstrates that composite grafts of cultured keratinocytes combined with acellular dermis are a useful approach for the closure of full-thickness wounds.

Animals↗
PubMed1996

Proteoglycans secreted by packaging cell lines inhibit retrovirus infection.

Using a model recombinant retrovirus encoding the Escherichia coli lacZ gene, we have found that medium conditioned with NIH 3T3 cells and packaging cell lines derived from NIH 3T3 cells inhibits infection. Most of the inhibitory activity was greater than 100 kDa and was sensitive to chondroitinase ABC digestion, which is consistent with the inhibitor being a chondroitin sulfate proteoglycan. Proteoglycans secreted by NIH 3T3 cells and purified by anion-exchange chromatography inhibited amphotropic retrovirus infection. Pretreatment of amphotropic retrovirus stocks with chondroitinase ABC boosted the level of transduction efficiency by more than twofold. The implications of these findings with respect to retrovirus-cell interactions and the production of high-titer retroviral stocks are discussed.

3T3 Cells↗
PubMed1995

Effects of hypothermia on the function, membrane integrity, and cytoskeletal structure of hepatocytes.

To increase the storage time of livers for transplantation, a better understanding of hypothermia-induced hepatocyte damage is necessary. To this end, we have characterized the effects of hypothermia on long-term function and cytoskeletal organization of hepatocytes cultured in the collagen sandwich configuration, which maintains the expression of liver-specific functions for several weeks. In these studies, cultured hepatocytes (maintained at 37 degrees C for 7 days) were exposed to 4 degrees C in Leibovitz-15 (L15), University of Wisconsin (UW) solution, or L15 supplemented with 2.5 g% polyethylene glycol (PEG) for various time periods followed by a return to normothermia. When L15 medium was used, the long-term albumin secretion rate of cultured hepatocytes was decreased by 50% after 4 h, and by 95% after 24 h of exposure to 4 degrees C. Amorphous precipitates of F-actin and fragmented short microtubules were also observed after 4 and 12 h of hypothermia, respectively. Similar results were obtained when hepatocytes were stored in UW solution. However, in L15 supplemented with PEG, no significant reduction in long-term albumin secretion rates and intact actin and microtubule morphology was observed even after 24 h of exposure to 4 degrees C. The membrane integrity and long-term albumin secretion of hepatocytes stored in the presence of PEG were decreased to approximately 50% only after 48 h of exposure to 4 degrees C. Thus, PEG may be a useful additive in preservation solutions for hepatocytes in hepatocyte-based liver support systems and for intact tissue as well.

Actins↗
PubMed1995

Dynamics of photoinduced cell plasma membrane injury.

We have developed a video microscopy system designed for real-time measurement of single cell damage during photolysis under well defined physicochemical and photophysical conditions. Melanoma cells cultured in vitro were treated with the photosensitizer (PS), tin chlorin e6 (SnCe6) or immunoconjugate (SnCe6 conjugated to a anti-ICAM monoclonal antibody), and illuminated with a 10 mW He/Ne laser at a 630 nm wavelength. Cell membrane integrity was assessed using the vital dye calcein-AM. In experiments in which the laser power density and PS concentration were varied, it was determined that the time lag before cell rupture was inversely proportional to the estimated singlet oxygen flux to the cell surface. Microscopic examination of the lytic event indicated that photo-induced lysis was caused by a point rupture of the plasma membrane. The on-line nature of this microscopy system offers an opportunity to monitor the dynamics of the cell damage process and to gain insights into the mechanism governing photolytic cell injury processes.

Cell Line↗
PubMed1995

Dynamics of cell membrane permeability changes at supraphysiological temperatures.

A quantitative fluorescent microscopy system was developed to characterize, in real time, the effects of supraphysiological temperatures between 37 degrees and 70 degrees C on the plasma membrane of mouse 3T3 fibroblasts and isolated rat skeletal muscle cells. Membrane permeability was assessed by monitoring the leakage as a function of time of the fluorescent membrane integrity probe calcein. The kinetics of dye leakage increased with increasing temperature in both the 3T3 fibroblasts and the skeletal muscle cells. Analytical solutions derived from a two-compartment transport model showed that, for both cell types, a time-dependent permeability assumption provided a statistically better fit of the model predictions to the data than a constant permeability assumption. This finding suggests that the plasma membrane integrity is continuously being compromised while cells are subjected to supraphysiological temperatures.

3T3 Cells↗
PubMed1995

Genetically modified human epidermis overexpressing PDGF-A directs the development of a cellular and vascular connective tissue stroma when transplanted to athymic mice--implications for the use of genetically modified keratinocytes to modulate dermal regeneration.

We investigated the hypothesis that keratinocyte-produced platelet-derived growth factor-AA (PDGF-AA) is involved in epidermal-dermal interactions and that PDGF-AA is an important mediator of the temporal and spatial events of tissue repair. Retroviral-mediated gene transfer was used to introduce the gene encoding human PDGF-A into cultures of human diploid keratinocytes. Genetic modification boosted the endogenous in vitro level of PDGF-AA secretion by over 300 fold. When PDGF-secreting cells were transplanted as epithelial sheets to athymic mice, modified keratinocytes underwent terminal differentiation and generated a stratified epithelium comparable to unmodified cells. Seven days after grafting the newly synthesized connective tissue layer subjacent to the PDGF-A-modified grafts was significantly thicker, was rich in mononuclear cells and fibroblasts, and had increased numbers of blood vessels when compared to control grafts of unmodified cells. These results suggest that PDGF-AA secreted by the epidermis is an important mediator of epithelial-mesenchymal interactions and helps to promote growth and vascularization of the underlying dermal tissue. Further, these data demonstrate the feasibility of using genetically modified cells to modulate tissue regeneration.

Animals↗
PubMed1995

Retrovirus infection: effect of time and target cell number.

Using a model amphotropic recombinant retrovirus encoding the Escherichia coli lacZ gene and quantitative assays to measure virus infection, we have determined the effects of time and target cell number on infectivity. Infection of various numbers of NIH 3T3 fibroblasts showed that the extent of lacZ virus infection was dependent on virus concentration and independent of target cell number. These results demonstrate that multiplicity of infection is not an accurate predictor of the efficiency of retroviral infection. Varying the time of viral infection revealed that maximal infection occurred after greater than 24 h of exposure of the cells to the lacZ virus. Half-maximal infection occurred after 5 h of exposure. After 2 h of adsorption at 37 degrees C, the majority of infectious virus was not adsorbed to cells but was unbound and able to infect other cells. These results are discussed in terms of both their relevance to the fundamental biology of retrovirus infection and the use of recombinant retroviruses for retrovirus-mediated gene transfer with purposes of gene therapy.

3T3 Cells↗
PubMed1994

Selective adhesion of hepatocytes on patterned surfaces.

Successful development of cell-biased bioartificial liver devices necessitates the establishment of techniques and designs for long-term, stable hepatocellular function and efficient transport of nutrients and wastes within the device. Given the relatively large cell mass that one must consider, one possible solution involves the use of micropatterning technology to sandwich hepatocytes aligned in rows between two micropatterned surfaces. Rows of cells would alternate with hepatocyte-free areas, creating efficient transport channels for fluid flow and nutrient exchange. Ultimately, this type of device could also be used as a three-dimensional construct for investigating a variety of cell-surface, cell-extracellular matrix, and cell-cell interactions. To achieve this goal, one must develop techniques for selectively adhering hepatocytes to solid substrates. In this study, reproducible, selective adhesion of hepatocytes on a glass substrate with large regions of adhesive (AS) and nonadhesive (NAS) surfaces was obtained. The AS had hydrophilic characteristics, enhancing deposition of collagen molecules from an aqueous solution, and subsequent hepatocyte adhesion, whereas the NAS had hydrophobic properties and remained collagen-free and hepatocyte-free. In addition, a reproducible processing technique for obtaining patterns of hepatocytes was developed and optimized, using a surface with a single AS band as a first approximation to a micropatterned device. This was achieved by spincoating an aqueous collagen type I solution (0.1 mg/mL) on a banded surface at 500 rpm for 25 seconds. The morphology and long-term function of the hepatocytes attached to AS in nonbanded and banded surface configurations was assessed by mimicking sandwich culture and was shown to be similar to stable, differentiated sandwich cultures. Mathematical modeling was used to determine critical design criteria for the hypothetical micropatterned device. The oxygen distribution and viscous pressure drop were modeled along a typical microchannel and limited to in vivo values. An optimal channel length of 0.6 cm and a flow rate of 2.0 x 10(-6) mL/s were obtained for a channel of 100 microns in width and 10 microns in height. These values were reasonable in terms of practical implementation.

Albumins↗
PubMed1994

Compartmental distribution of tumor-specific monoclonal antibodies in human melanoma xenografts.

Monoclonal antibodies (MAb) are attractive for tumor therapy because of their exquisite specificity. Although a majority of tumor cells in small (< or = 20 mg) solid tumors can be labeled following systemic administration of antitumor cell MAbs, little quantitative information is available as to the distribution of these MAbs within the several compartments that comprise solid tumors. Our goal was to provide such data in a well-characterized melanoma xenograft system. In accord with earlier work, i.v.-injected, melanoma-specific MAbs 436 and IND1, directed, respectively, against the 125 kD and HMW-melanoma-associated antigens, accumulated in M21 and SK-MEL-2 tumor xenografts in amounts of approximately 20% of injected dose/g. However, only 20-24% of the MAbs present in tumor xenografts was bound to tumor cells; the great majority (76-80%) was in the tumor extracellular fluid (ECF) and collagenous residue fractions. These results could not be accounted for by MAb degradation or release of MAbs from tumor cells during xenograft dissociation. Rather, they reflected in large part interactions of MAbs with antigens which tumors had shed into the ECF. Thus, 48 h after i.v. injection of 20 micrograms of melanoma-specific, biotin-tagged MAb, 46-66% of that present in the tumor ECF was complexed with melanoma-associated antigens. Overall, 61-73% of the MAbs recovered from tumor xenografts were bound to tumor antigens (either to tumor cells themselves or to tumor-shed antigens). In contrast, only approximately 4% of a melanoma-nonspecific MAb (B72.3) accumulated per g tumor after i.v. injection and nearly all of this was free in the ECF. Consistent with these data, fluorescence microscopy revealed that i.v.-injected, fluorescein-tagged MAbs achieved highest concentrations in tumor stroma, particularly at the tumor-host interface. Flow cytometry of dissociated solid tumors revealed that both the fraction of MAb-labeled tumor cells and the amount of MAb/tumor cell could be increased by increasing the administered i.v. dose of melanoma-specific MAb. Nonetheless, even at the highest i.v. injected dose (300 micrograms), 15-37% of tumor cells lacked detectable MAb labeling. Taken together, the data indicate that delivery of tumor cell-specific MAbs to solid tumors cannot be equated with their delivery to tumor cells. This distinction is important for immunotherapeutic approaches that require MAb contact with tumor cells.

Animals↗
PubMed1994

Antibody-targeted photolysis of bacteria in vivo.

We have evaluated the efficacy of antibody-targeted photolysis to kill bacteria in vivo using specific antibacterial photosensitizer (PS) immunconjugates. After infecting the dorsal skin in mice with Pseudomonas aeruginosa, both specific and nonspecific tin (IV) chlorin e6-monoclonal antibody conjugates were injected at the infection site. After a 15 min incubation period, the site was exposed to 630 nm light with a power density of 100 mW/cm2 for 1600 seconds. Irradiation resulted in a greater then 75% decrease in the number of viable bacteria at sites treated with a specific conjugate, whereas normal bacterial growth was observed in animals that were untreated or treated with a nonspecific conjugate. Antibody-targeted photolysis may be a selective and versatile tool for treating a variety of infections.

Animals↗
PubMed1994

Transport of fluorescent dextrans across the rat ileum after cutaneous thermal injury.

OBJECTIVE: To determine the time course and spatial distribution of uptake of macromolecules in the small intestine of rats subjected to cutaneous thermal injury. DESIGN: Prospective, controlled animal study. SUBJECTS: Fifty-five female Sprague-Dawley (CD) rats subjected to scald burn injury covering 20% (small injury; n = 29) and 40% (large injury; n = 6) of the total body surface area between 3 and 72 hrs after injury. Animals subjected to sham injury (n = 20) were used as controls. INTERVENTIONS: The intestine was cannulated near the distal ileum and incised 7 cm upstream. After perfusion with physiologic buffer, this intestinal loop was filled with the same buffer containing fluorescent-labeled dextrans (3 and 70 kilodaltons molecular weight) and ligated 4 cm from the injection point. After a 2-hr incubation period, the tissues were fixed with paraformaldehyde and cryosections were examined by laser confocal microscopy. The mesentery was also observed by laser confocal microscopy during incubation with the permeability probes. The disappearance of fluorescence was studied after washing the dextran probes from the gut lumen. MEASUREMENTS AND MAIN RESULTS: In small injuries, there was a transient uptake of the 3-kilodalton dextran by the epithelium in focal regions of the ileum with the effects seen between 7 and 21 hrs after injury. In large injuries, epithelial staining was visible within 3 hrs, and the marker was seen to translocate both to the lymphatics and the blood vessels of the mesentery. In comparison, the 70-kilodalton dextran was visible within the intercellular spaces. Little or no epithelial staining was seen in sham-injured animals. CONCLUSIONS: These results suggest that a transcellular pathway for the translocation of small macromolecules from the lumen to the mesentery can be activated after burn injury. The novel techniques described here will be useful to examine intestinal transport in various pathologic situations.

Animals↗
PubMed1994

An extracorporeal microscopy perfusion chamber for on-line studies of environmental effects on cultured hepatocytes.

The development of bioartificial-hybrid organ support systems is hampered by the lack of knowledge on the effects of different (in vivo) environments on cells during extracorporeal perfusion. In the present study, a perfusion chamber was designed for continuous monitoring of cultured cells during perfusion with media, as well as during plasma perfusion in an extracorporeal circuit. Chamber characterization showed satisfactory thermal and perfusion profiles and no major pH fluctuations. Further testing was performed with hepatocytes that were cultured in between two collagen layers, a configuration which was previously shown to preserve hepatocyte morphology and function for over six weeks of culture. Perfusion of the hepatocytes with culture media did not adversely affect cell morphology and function, provided the perfusion time was < or = 48 hours. Perfusion of the cultures during connection of the chamber to an extracorporeal circuit involving normal rats for six hours resulted in reversible cytoplasmic changes, unaltered cell shapes indices, and a 40 percent increase in albumin secretion rate during the first post-perfusion day, followed by a return to stable control levels. We expect that this chamber will be a valuable tool for on-line studies of cells under (extracorporeal) perfusion conditions and could be used for a large variety of studies on regeneration, reperfusion damage, and detoxification.

Albumins↗