Showing papers by "Mehmet Toner published in 1999"

Effect of cell–cell interactions in preservation of cellular phenotype: cocultivation of hepatocytes and nonparenchymal cells

Abstract: Heterotypic cell interaction between parenchymal cells and nonparenchymal neighbors has been reported to modulate cell growth, migration, and/or differentiation. In both the developing and adult liver, cell-cell interactions are imperative for coordinated organ function. In vitro, cocultivation of hepatocytes and nonparenchymal cells has been used to preserve and modulate the hepatocyte phenotype. We summarize previous studies in this area as well as recent advances in microfabrication that have allowed for more precise control over cell-cell interactions through 'cellular patterning' or 'micropatterning'. Although the precise mechanisms by which nonparenchymal cells modulate the hepatocyte phenotype remain unelucidated, some new insights on the modes of cell signaling, the extent of cell-cell interaction, and the ratio of cell populations are noted. Proposed clinical applications of hepatocyte cocultures, typically extracorporeal bioartificial liver support systems, are reviewed in the context of these new findings. Continued advances in microfabrication and cell culture will allow further study of the role of cell communication in physiological and pathophysiological processes as well as in the development of functional tissue constructs for medical applications.

Molding of Deep Polydimethylsiloxane Microstructures for Microfluidics and Biological Applications

Abstract: Here we demonstrate the microfabrication of deep (> 25 microns) polymeric microstructures created by replica-molding polydimethylsiloxane (PDMS) from microfabricated Si substrates. The use of PDMS structures in microfluidics and biological applications is discussed. We investigated the feasibility of two methods for the microfabrication of the Si molds: deep plasma etch of silicon-on-insulator (SOI) wafers and photolithographic patterning of a spin-coated photoplastic layer. Although the SOI wafers can be patterned at higher resolution, we found that the inexpensive photoplastic yields similar replication fidelity. The latter is mostly limited by the mechanical stability of the replicated PDMS structures. As an example, we demonstrate the selective delivery of different cell suspensions to specific locations of a tissue culture substrate resulting in micropatterns of attached cells.

Effects of cryoprotectants and ice-seeding temperature on intracellular freezing and survival of human oocytes

Abstract: The accurate determination of the freezing conditions that promote intracellular ice formation (IIF) is crucial for designing cryopreservation protocols for cells. In this paper, the range of temperatures at which IIF occurs in human oocytes was determined. Fresh oocytes with a germinal vesicle, failed-to-fertilize (metaphase I and metaphase II stages) and polyspermic eggs were used for this study. The occurrence of IIF was first visualized at a cooling rate of 120 degrees C/min using a programmable thermal microscope stage connected to a videomicroscope. Then, with a cooling rate of 0.2 degrees C/min, the seeding temperature of the extracellular ice was modified to decrease the incidence of IIF and increase the survival rate of frozen-thawed human oocytes. After adding different cryoprotectants, the median temperature of IIF (TMED) was decreased by approximately 23 degrees C in mouse and only by approximately 6.5 degrees C in human oocytes. Using 1.5 M propylene glycol and seeding temperatures of -8.0, -6.0 and -4.5 degrees C, the incidence of IIF was 22/28 (78%), 8/24 (33%) and 0/33 (0%) and the 24 h post-thaw survival rate was 10/31(32%), 19/34 (56%) and 52/56 (93%) respectively. The results show that IIF occurs more readily in human oocytes, and that ice seeding between -6 degrees C and -8 degrees C triggers IIF in a large number of human oocytes. Undesirable IIF can be prevented and survival rates maximized by raising the seeding temperature as close as possible to the melting point of the solution, which in our instrument was -4.5 degrees C.

Numerical Model of Fluid Flow and Oxygen Transport in a Radial-Flow Microchannel Containing Hepatocytes

Abstract: The incorporation of monolayers of cultured hepatocytes into an extracorporeal perfusion system has become a promising approach for the development of a tempo­ rary bioartificial liver (BAL) support system. In this paper we present a numerical investigation of the oxygen tension, shear stress, and pressure drop in a bioreactor for a BAL composed of plasma-perfused chambers containing monolayers of porcine hepatocytes. The chambers consist of microfabricat ed parallel disks with center-toedge radial flow. The oxygen uptake rate (OUR), measured in vitro for porcine hepatocytes, was curve-fitted using Michaelis-Menten kinetics for simulation of the oxygen concentration profile. The effect of different parameters that may influence the oxygen transport inside the chambers, such as the plasma flow rate, the chamber height, the initial oxygen tension in the perfused plasma, the OUR, and K^ was investigated. We found that both the plasma flow rate and the initial oxygen tension may have an important effect upon oxygen transport. Increasing the flow rate and/ or the inlet oxygen tension resulted in improved oxygen transport to cells in the radial-flow microchannels, and allowed significantly greater diameter reactor without oxygen limitation to the hepatocytes. In the range investigated in this paper (10 p,m < H < 100 jim), and for a constant plasma flow rate, the chamber height, H, had a negligible effect on the oxygen transport to hepatocytes. On the contrary, it strongly affected the mechanical stress on the cells that is also crucial for the successful design of the BAL reactors. A twofold decrease in chamber height from 50 to 25 jjm produced approximately a fivefold increase in maximal shear stress at the inlet of the reactor from 2 to 10 dyn/cm^. Further decrease in chamber height resulted in shear stress values that are physiologically unrealistic. Therefore, the channel height needs to be carefully chosen in a BAL design to avoid deleterious hydrodynamic effects on hepatocytes.

Controlled reversible poration for preservation of biological materials

Abstract: A preservation method for biological material having cell membranes includes reversibly porating the cell membranes; loading a bio-protective agent having bio-preservation properties to a predetermined intracellular concentration; preparing the bio-protective agent loaded biological material for storage; storing the biological material; recovering the stored biological material from storage; and reversing the cell membrane poration. H5 α-toxin, a genetically engineered mutant of Staphylococcus aureus α-hemolysin, may be used as a porating agent. Non-permeating sugars such as trehalose and sucrose may be used as the bio-protective agent.

Posttranslational modifications of cardiac and skeletal muscle proteins by reactive oxygen species after burn injury in the rat.

Abstract: OBJECTIVE: To determine the involvement of oxidative damage in muscle wasting after burn injury. SUMMARY BACKGROUND DATA: Burn injury damages tissue at the site of the burn and also affects peripheral tissue. There is evidence to suggest that reactive oxygen species may be generated in increased amounts after burn, and these may contribute to wound healing and to posttranslational modifications of tissue constituents distant from the wound site. METHODS: The oxidation of muscle proteins was assessed, using the dinitrophenylhydrazine assay for carbonyl content, in muscles of rats after a full-thickness skin scald burn covering 20% of the total body surface area, over a 6-week period. In this model, rats failed to incur normal body weight or muscle weight gain. RESULTS: Soleus, extensor digitorum longus, diaphragm, and heart ventricle proteins were oxidatively damaged after injury. The extent of tissue protein oxidation, however, differed depending on the time points studied. In general, higher levels of protein carbonyl group formation, an indicator of oxidative damage, were found to occur within 1 to 5 days after injury, and the oxidized protein content of the various tissues decreased during the later stages. Both sarcoplasmic and myofibrillar carbonyl-containing proteins accumulated in diaphragm 3 days after burn injury and were rapidly removed from the tissue during a 2-hour in vitro incubation. This coincided with increased proteolytic activity in diaphragm. CONCLUSIONS: These observations suggest that the loss of proteins modified by reactive oxygen species may contribute to the burn-induced protein wasting in respiratory and other muscles by a proteolytically driven mechanism.

Micropatterning cells in tissue engineering.

Abstract: Recent advances in tissue engineering have been facilitated by the ability to control the environment of cells in vitro. Modulation of cell-extracellular matrix, cell-substrate, and cell-cytokine interactions have proven to be useful in organotypic cultures of many kinds. In some cases, such as skin, bone marrow, and liver, the recovery of physiologic tissue function is enhanced by co-cultivation of two or more cell types together. The in vitro dependence of tissue function on cell-cell interactions is reminiscent of cellular cues during embryogenesis, or adult interactions between parenchymal cells and supporting stroma.

Cryopreservation of rat hepatocytes in a three-dimensional culture configuration using a controlled-rate freezing device.

Abstract: From engineered tissues to transfected cell lines, the long term storage of living biologicals is desirable for a variety of medical, scientific, economic, and regulatory concerns, including transport, the expense of development, repeatability issues, and the point of use. Currently, the best option is cryogenic storage, placing the biomaterials in suspended animation at very low temperatures (-196°C), halting all chemical reactions, limiting genetic drift, and ensuring the maintenance of cell viability and function upon thawing (1). Obtaining such an advantageous state, however, can be a difficult achievement. This problem becomes further complicated as we move toward next generation multicomponent products such as engineered skin and cartilage substitutes, composed of multiple cell types oriented in complicated three-dimensional geometries within an extracellular matrix scaffold (2-4).

Microfabrication of basal lamina analogs with complex topographic features

Abstract: A microfabrication approach was used to produce novel analogs of the basal lamina with complex topographic features. A test pattern of ridges and channels with length scales (40 /spl mu/m to 310 /spl mu/m) similar to invaginations in native basal lamina was laser machined into a polyimide master chip. Negative replicates of the chip were used as templates to produce thin (/spl sim/21 /spl mu/m) collagen or gelatin membranes that recapitulated the complex topographic features of the master chip. To demonstrate their utility, membranes were incorporated into dermal analogs and their surfaces seeded with cultured human epidermal keratinocytes to form skin equivalents. The keratinocytes formed a differentiated and stratified epidermis that conformed to the features of the microfabricated membrane. Interestingly, the topography of the membrane influenced the differentiation of the keratinocytes as stratification was enhanced in the deeper channels. Membrane topography also controlled the gross surface features of the skin equivalent; infolds of the epidermis increased with channel depth. These microfabricated basal lamina analogs will help to elucidate the influence of topography on epithelial cell proliferation and differentiation and should have applications in the tissue engineering of skin equivalents as well as other basal lamina containing tissues.