Migration and internalization of cells and polystyrene microspheres in tumor cell spheroids

The movement and internalization of ³H-labelled cells and of inert polystyrene microspheres within multicellular spheroids has been examined through histological sectioning and autoradiography. EMT6 and RIF-1 spheroids were cultured in spinner flasks for approx. 2.5 weeks. At this time, ³H-labelled cells and/or microspheres were allowed to adhere to the spheroid surface. Microspheres, ³H-labelled RIF-1 monolayer cells and ³H-labelled EMT6 monolayer cells were observed to move centripetally as a wave into EMT6 spheroids. In contrast, ³H-labelled trypsinized RIF-1 and EMT6 spheroid cells became mixed with the other non-labelled spheroid cells in homotypic RIF-1 and EMT6 spheroids, respectively. Reduction of spheroid growth by maintaining the spheroids at room temperature and by treatment with 2500 rads irradiation did not prohibit the internalization of ³H-labelled EMT6 cells and microspheres in EMT6 spheroids.     

Radiation Response of Connexin43-Transfected Cells in Relation to the “Contact Effect”

Some cell lines grown for only two cell doublings as multicell spheroids develop a form of resistance to killing by ionizing radiation that has been called the “contact” effect. While our previous results have implicated a role for higher order chromatin structure in the contact effect, another possible explanation is the presence of intercellular gap junctions that might facilitate communication between cells grown as spheroids and thereby enhance the ability of cells to resist or recover from radiation damage. To examine the role of gap junctions in the contact effect, rat glioma C6 and mouse EMT6 cell lines were transfected with a gene encoding the gap junctional protein connexin43. While C6 glioma cells are deficient in gap junctional communication, cells from spheroids were nonetheless more resistant than monolayers to killing by ionizing radiation, and the contact effect was present to a similar extent in the three transfected clones. For mouse EMT6 cells, radiosensitivity was similar whether cells were grown as monolayers or spheroids. Transfection of EMT6 cells with connexin43 increased gap junctional communication but did not promote development of a contact effect. Tumor volume doubling time in SCID mice increased significantly for one transfected clone; however, doubling timein vitrowas also increased relative to the EMT6 parent. We conclude that extensive gap junctional communication is not a requirement for the increased radiation resistance observed when some cell lines are grown as spheroids.     

Increased thermoresistance developed during growth of small multicellular spheroids

Mammalian cells growing as multicell spheroids, an in vitro model of tumor microregions, have been shown previously to be more resistant than single cells from monolayer cultures to killing by ionizing radiation, hyperthermia, ultrasound, and chemotherapeutic drugs. Although the mechanisms by which cells in spheroids acquire these increased resistances are unknown, available evidence has indicated that intercellular contact mediates the process for ionizing radiation. This investigation was undertaken to evaluate the role of intercellular contact produced during growth of small spheroids on the sensitivity of EMT6/Ro mouse mammary tumor cells to moderate hyperthermia. Increased thermoresistance developed in small spheroids (approximately 70 micron diameter, 25 cells/spheroid), as measured by colony formation, after exposures to different temperatures in the range of 37 to 45 degrees C for periods less than or equal to 2 hr and at 42.5 degrees C for less than or equal to 8 hr. Experiments were performed to determine the relative contributions to this increased thermoresistance of 1) the extent of intercellular contact in spheroids of different cellular multiplicities, 2) differences in membrane damage influenced by trypsin heat treatment sequence, and 3) physiological changes associated with growth of cells as spheroids in suspension compared to monolayer culture. Treatment with trypsin prior to heating sensitized cells to killing by hyperthermia but did not account for the differential thermoresistance between cells from spheroids and monolayers. Spheroid multiplicity in the range of 1.16 to 76.2 cells/spheroid had no significant effect on cell survival after hyperthermia. However, cells grown in spinner suspension culture were more thermoresistant than cells from monolayer cultures and nearly as thermoresistant as cells in spheroids. From these data we conclude that the greater thermoresistance of EMT/Ro cells in spheroids is the result of cellular physiological changes associated with growth in suspension and is not mediated by intercellular contact.     

Variations in tumor cell growth rates and metabolism with oxygen concentration, glucose concentration, and extracellular pH.

Tumors and multicellular tumor spheroids can develop gradients in oxygen concentration, glucose concentration, and extracellular pH as they grow. In order to calculate these gradients and assess their impact on tumor growth, it is necessary to quantify the effect of these variables on tumor cell metabolism and growth. In this work, the oxygen consumption rates, glucose consumption rates, and growth rates of EMT6/Ro mouse mammary tumor cells were measured at a variety of oxygen concentrations, glucose concentrations, and extracellular pH levels. At an extracellular pH of 7.25, the oxygen consumption rate of EMT6/Ro cells increased by nearly a factor of 2 as the glucose concentration was decreased from 5.5 mM to 0.4 mM. This effect of glucose concentration on oxygen consumption rate, however, was slight at an extracellular pH of 6.95 and disappeared completely at an extracellular pH of 6.60. The glucose consumption rate of EMT6/Ro cells increased by roughly 40% when the oxygen concentration was reduced from 0.21 mM to 0.023 mM and decreased by roughly 60% when the extracellular pH was decreased from 7.25 to 6.95. The growth rate of EMT6/Ro cells decreased with decreasing oxygen concentration and extracellular pH; however, severe conditions were required to stop cell growth (0.0082 mM oxygen and an extracellular pH of 6.60). Empirical correlations were developed from these data to express EMT6/Ro cell growth rates, oxygen consumption rates, and glucose consumption rates, as functions of oxygen concentration, glucose concentration, and extracellular pH. These empirical correlations make it possible to mathematically model the gradients in oxygen concentration, glucose concentration, and extracellular pH in EMT6/Ro multicellular spheroids by solution of the diffusion/reaction equations. Computations such as these, along with oxygen and pH microelectrode measurements in EMT6/Ro multicellular spheroids, indicated that nutrient concentration and pH levels in the inner regions of spheroids were low enough to cause significant changes in nutrient consumption rates and cell growth rates. However, pH and oxygen concentrations measured or calculated in EMT6/Ro spheroids where quiescent cells have been observed were not low enough to cause the cessation of cell growth, indicating that the observed quiescence must have been due to factors other than acidic pH, oxygen depletion, or glucose depletion.     

ATP concentrations in multicellular tumor spheroids assessed by single photon imaging and quantitative bioluminescence

To evaluate the interrelationship among the cellular energy status and the development of necrosis in tumor microregions, local ATP concentrations and the extent of necrosis were determined in multicellular tumor spheroids, i.e., in spherical tumor cell aggregates. The spheroids were grown in rotated suspension cultures using EMT6 cells that were derived from a murine mammary sarcoma. The distribution of viable and necrotic cell areas was assessed by histological investigations. The regional distribution of ATP concentrations was measured with a novel technique using quantitative bioluminescence and single photon imaging. This method makes it possible to determine ATP concentrations in absolute terms with a spatial resolution at the level of a single cell. The results show that ATP concentrations in the center of EMT6 spheroids decrease from values of 1.0 to 1.5 mM in small spheroids with 300 microns in diameter to values close to or at the background level in 750 microns spheroids. Necrosis was detectable in spheroids larger than 300 microns, and virtually no spheroid without necrosis was found at sizes larger than 600 microns. Since the emergence of central necrosis precedes the drop in ATP to undetectably low values, the data suggest that energy metabolism is not or not directly involved in the development of necrosis in tumor spheroids under the growth conditions investigated.     

Culturing of human mesenchymal stem cells as three-dimensional aggregates induces functional expression of CXCR4 that regulates adhesion to endothelial cells

Culture-expanded human mesenchymal stem cells (hMSCs) are increasingly used in a variety of preclinical and clinical studies. However, these cells have a low rate of engraftment to bone marrow or damaged tissues. Several laboratories have shown that during isolation and subculturing mesenchymal stem cells quickly lose the expression of CXCR4, the key receptor responsible for lymphocytes and hematopoietic stem cell homing. Here we show that culturing of hMSCs as three-dimensional aggregates (hMSC spheroids) restores CXCR4 functional expression. Expression of CXCR4 inversely correlates with the secretion of SDF-1 by hMSCs. Cells from hMSC spheroids up-regulate expression of CD49b, the alpha2 integrin subunit, and suppress the expression of CD49d, the alpha4 integrin subunit. Transfer of cells from the spheroids back to a monolayer suppresses the expression of CXCR4 and CD49b and restores the expression of CD49d. Treatment of cells from the spheroids with SDF-1 leads to CXCR4 internalization and activation of ERK-1,2. Adhesion of hMSCs to human umbilical vein endothelial cells (HUVECs) was investigated. SDF-1, AMD-3100, or exposure of HUVECs to hypoxia did not affect adhesion of hMSCs from a monolayer to HUVECs. Adhesion of cells from hMSC spheroids to HUVECs was stimulated by SDF-1, AMD-3100, or by exposure of HUVECs to hypoxia. Stimulatory effects of hypoxia and addition of SDF-1 or AMD-3100 were not additive. Overall, our data indicate that the expression of CXCR4 by hMSCs regulates hMSC adhesion to endothelial cells.     

Studies using an in vitro model show evidence of involvement of epithelial-mesenchymal transition of human endometrial epithelial cells in human embryo implantation

Human embryo implantation is a critical multistep process consisting of embryo apposition/adhesion, followed by penetration and invasion. Through embryo penetration, the endometrial epithelial cell barrier is disrupted and remodeled by an unknown mechanism. We have previously developed an in vitro model for human embryo implantation employing the human choriocarcinoma cell line JAR and the human endometrial adenocarcinoma cell line Ishikawa. Using this model we have shown that stimulation with ovarian steroid hormones (17β-estradiol and progesterone, E2P4) and suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor, enhances the attachment and adhesion of JAR spheroids to Ishikawa. In the present study we showed that the attachment and adhesion of JAR spheroids and treatment with E2P4 or SAHA individually induce the epithelial-mesenchymal transition (EMT) in Ishikawa cells. This was evident by up-regulation of N-cadherin and vimentin, a mesenchymal cell marker, and concomitant down-regulation of E-cadherin in Ishikawa cells. Stimulation with E2P4 or SAHA accelerated Ishikawa cell motility, increased JAR spheroid outgrowth, and enhanced the unique redistribution of N-cadherin, which was most prominent in proximity to the adhered spheroids. Moreover, an N-cadherin functional blocking antibody attenuated all events but not JAR spheroid adhesion. These results collectively provide evidence suggesting that E2P4- and implanting embryo-induced EMT of endometrial epithelial cells may play a pivotal role in the subsequent processes of human embryo implantation with functional control of N-cadherin.     

Evaluation of various types of new hypoxic cell sensitizers using the EMT6 single cell-spheroid-solid tumour system

Eleven new hypoxic cell sensitizers representative of those developed in Japan between 1980 and 1985 were evaluated in vitro and in vivo in comparison with misonidazole (MISO), SR-2508, Ro 03-8799, and ANT (2-amino-5-nitrothiazole). The new compounds included 2-nitroimidazole nucleoside analogues, nitrotriazoles and other nitroaromatics, non-nitro compounds, and electron-affinic compounds that readily intercalate DNA. The sensitizing activity in the EMT6 single cells correlated not only with the reduction potential but, for some compounds, also with the reactivity with non-protein sulphydryls. The sensitizers were also tested using the EMT6 spheroids and solid tumours. The patterns of changes in sensitizer enhancement ratios (SERs) for single cells, spheroids, and solid tumours were classified into two types: (1) SERs for the three testing systems were similar; and (2) SERs decreased in the order of: single cells, spheroids, and solid tumours. Only nitroimidazole and nitrotriazole derivatives belonged to the former type. RK-28 and RK-29, 2-nitroimidazoles with sugar analogue components, had in vivo effects almost equal to those of MISO. Also 3- and 4-nitrotriazole derivatives had definite in vivo effects.     

Hypoxic fraction and binding of misonidazole in EMT6/Ed multicellular tumor spheroids

Misonidazole has been shown to bind selectively to hypoxic cells in tissue culture and to cells which are presumed to be chronically hypoxic in EMT6 spheroids and tumors. Thus it has considerable potential as a marker of hypoxic cells in vivo. To further evaluate this potential EMT6/Ed spheroids were used to quantitate misonidazole binding under conditions which resulted in hypoxic fractions between 0 and 1. Hypoxic fractions were quantitated using radiation survival curves. A doubling of the oxygen in the gas phase to 40% was required to fully oxygenate all chronically hypoxic cells. The patterns of binding of 14C-labeled misonidazole determined by autoradiography were consistent with the regions of radiobiological hypoxia as predicted by oxygen diffusion theory. The overall uptake of 3H-labeled misonidazole by spheroids correlated well with the hypoxic fraction, although binding to aerobic cells and necrotic tissue contributed appreciably to the total label in the spheroids. It is concluded that misonidazole is an excellent marker of hypoxia in EMT6/Ed spheroids at the microscopic level, and the total amount bound per spheroid provides a potentially useful measure of the hypoxic fraction.     

The role of actin filaments and microtubules in hepatocyte spheroid self-assembly

Cultured rat hepatocytes self-assemble into three-dimensional structures or spheroids that exhibit ultrastructural characteristics of native hepatic tissue and enhanced liver-specific functions. The spheroid formation process involves cell translocation and changes in cell shape, indicative of the reorganization of the cytoskeletal elements. To elucidate the function of the cytoskeleton, hepatocytes undergoing spheroid formation were treated with drugs that disrupt the different cytoskeletal components. Cytochalasin D, which targets the actin filaments, caused inhibition of spheroid formation. The role of microtubules in this process was assessed by incubating the cells with taxol or nocodazole. Perturbation of microtubules had minimal effects on spheroid assembly. Scanning electron micrographs showed no morphological differences between spheroids formed in control cultures and those formed in the presence of taxol or nocodazole. In addition, the effects of those agents on hepatocyte functions were investigated. Albumin secretion and cytochrome P450 2B1/2 activities of hepatocytes were comparable in spheroids formed in the presence of taxol or nocodazole to those formed in control cultures. The levels of these liver-specific activities were lower in cytochalasin D--treated cultures where only dispersed cells or cell clumps were found but spheroids had not found. Thus, hepatocytes require an intact actin network to self-assemble efficiently into functional tissue-like structures. Perturbation of the microtubule lattice does not impair the formation process. Events that transpire during hepatocyte spheroid self-assembly exhibit striking similarities to processes commonly observed in tissue morphogenesis. The results provide insight into the mechanisms that cells employ to organize into tissues and can contribute to our understanding of how to control the cellular assembly in tissue engineering and clinical applications.     

Mathematical modelling of microenvironment and growth in EMT6/Ro multicellular tumour spheroids

In order to determine the role of micromilieu in tumour spheroid growth, a mathematical model was developed to predict EMT6/Ro spheroid growth and microenvironment based upon numerical solution of the diffusion/reaction equation for oxygen, glucose, lactate ion, carbon dioxide, bicarbonate ion, chlorine ion and hydrogen ion along with the equation of electroneutrality. This model takes into account the effects of oxygen concentration, glucose concentration and extracellular pH on cell growth and metabolism. Since independent measurements of EMT6/Ro single cell growth and metabolic rates, spheroid diffusion constants, and spinner flask mass transfer coefficients are available, model predictions using these parameters were compared with published data on EMT6/Ro spheroid growth and micro-environment. The model predictions of reduced spheroid growth due to reduced cell growth rates and cell shedding fit experimental spheroid growth data below 700 microns, but overestimated the spheroid growth rate at larger diameters. Predicted viable rim thicknesses based on predicted near zero glucose concentrations fit published viable rim thickness data for 1000 microns spheroids grown at medium glucose concentrations of 5.5 mM or less. However, the model did not accurately predict the onset of necrosis. Moreover, the model could not predict the observed decreases in oxygen and glucose metabolism seen in spheroids with time, nor could it predict the observed growth plateau. This suggests that other unknown factors, such as inhibitors or cell-cell contact effects, must also be important in affecting spheroid growth and cellular metabolism.     

Extracellular matrix proteins modulate endocytosis of the insulin receptor

Internalization of the insulin receptor (IR) is a highly regulated multi-step process whose underlying molecular basis is not fully understood. Here we undertook to study the role of extracellular matrix (ECM) proteins in the modulation of IR internalization. Employing Chinese hamster ovary cells that overexpress IR (CHO-T cells), our results indicate that IR internalization proceeds unaffected even when Tyr phosphorylation of IR substrates, such as IRS-1, is impaired (e.g. in CHO-T cells overexpressing IRS-1 whose pleckstrin-homology domain has been deleted or in CHO-T cells that overexpress the PH/PTB domain of IRS-1). In contrast, IR internalization is affected by the context of the ECM proteins to which the cells adhere. Hence, IR internalization was inhibited 40-60% in CHO-T cells adherent onto galectin-8 (an ECM protein and an integrin ligand of the galectin family) when compared with cells adherent onto fibronectin, collagen, or laminin. Cells adherent to galectin-8 manifested a unique cytoskeletal organization, which involved formation of cortical actin and generation of F-actin microspikes that contrasted with the prominent stress-fibers formed when cells adhered to fibronectin. To better establish a role for actin filament organization in IR endocytosis, this process was assayed in CHO-T cells (adherent onto fibronectin), whose actin filaments were disrupted upon treatment with latrunculin B. Latrunculin B did not affect insulin-induced Tyr phosphorylation of IR or its ability to phosphorylate its substrates; still, a 30-50% reduction in the rate of IR internalization was observed in cells treated with latrunculin B. Treatment of cells with nocodazole, which disrupts formation of microtubules, did not affect IR internalization. These results indicate that proper actin, but not microtubular, organization is a critical requirement for IR internalization and suggest that integrin-mediated signaling pathways emitted upon cell adhesion to different extracellular matrices and the altered cytoskeletal organizations generated thereof affect the itinerary of the insulin receptor.     

Microinjected fluorescent polystyrene beads exhibit saltatory motion in tissue culture cells

Microinjected 0.26-micron fluorescent, carboxylated microspheres were found to display classical saltatory motion in tissue culture cells. The movement of a given particle was characterized by a discontinuous velocity distribution and was unaffected by the activity of adjacent particles. The microspheres were translocated at velocities of up to 4.7 micron/s and sometimes exhibited path lengths greater than 20 micron for a single saltation . The number of beads injected into a cell could range from a few to over 500 with no effect on the cell's ability to transport them. Neither covalent cross-linking nor preincubation of the polystyrene beads with various proteins inhibited the saltatory motion of the injected particles. The motion of the injected beads in cultured cells was reversibly inhibited by the microtubule poison nocodazole, under conditions in which actin-rich, nitrobenzoxadiazol - phallacidin -staining structures remain intact. Whole-cell high voltage electron microscopy of microinjected cells that were known to be moving the fluorescent microspheres revealed that the beads were embedded in the cytoplasmic matrix and did not appear to be membrane bound. The enhanced detectability of the fluorescent particles over endogenous organelles and the ability to modify the surfaces of the beads before injection may enable more detailed studies on the mechanism of saltatory particle motion.     

Serum, trypsin, and cell shape but not cell-to-cell contact influence the X-ray sensitivity of Chinese hamster V79 cells in monolayers and in spheroids

Nutrient concentration in the growth medium and trypsin affect cellular radiosensitivity in a manner that is related to cell shape (Reddy, Stevenson, and Lange, Int. J. Radiat. Biol. 55, 105-117 (1989); Reddy and Lange, Radiat. Res. 119, 338-347 (1989]. Hence we hypothesized that the concentration of serum in the medium could influence the X-ray sensitivity of cells and that the spread cells in monolayers and round cells in spheroids may differ in their response to the radiosensitizing effect of trypsin. We compared the X-ray sensitivity of monolayer and spheroid cells grown for 19 +/- 1 h in MEM supplemented with 5 or 15% serum. Cells were trypsinized and plated either immediately before, or 2.5 +/- 0.5 h after, irradiation and incubation for repair in situ. Survival of cells in monolayers and in spheroids was higher in MEM with 5% serum than with 15% serum. Trypsin treatment affected the shape and radiosensitivity of cells in monolayers but not in spheroids. When all cells were grown in the same serum concentration and a 2.5-h postirradiation incubation was allowed prior to trypsinization, the X-ray sensitivity of cells in spheroids was greater than that of cells in monolayers. The survival of cells in spheroids became equal to that of monolayer cells when cells in spheroids were converted to monolayers by placing them in 25-cm2 flasks and allowing them 3 h to attach and spread. Cell cycle distributions were nearly the same in monolayers and spheroids cultured in MEM with 5 or 15% serum. We conclude that: (1) serum concentration in the growth medium and trypsin do appear to contribute to the differences in the radiosensitivity of spheroids and monolayer V79 cells; (2) these differences are associated with changes in cell morphology.     

The multicellular tumor spheroid model: I. Characterization of the allograft response in sensitized mice

Intraperitoneal implants of multicellular spheroids of EMT6 mammary sarcoma cells into specifically sensitized allogeneic mice have been used as a model to study in situ immunity to solid tumor allografts. Morphological examination of the spheroids recovered from sensitized mice revealed massive infiltration by host cells. There was an early but apparently nonspecific infiltration by large numbers of polymorphonuclear leukocytes followed by the infiltration of large numbers of macrophages and smaller numbers of lymphocytes. The later infiltration correlated with tumor cell killing as determined by a cloning assay for surviving EMT6 cells, and with the presence of cells with cytotoxic activity in in vitro assays. The kinetics of tumor cell killing indicated that tumor cell survival decreased within a few hours of spheroid implantation and less than 1% of the tumor cells survived after 48 h. Purification of the infiltrating cells by nylon wool fractionation and by treatment with anti-T-cell serum and complement indicated that the primary cytotoxic cell is a T lymphocyte.     

Lipid recycling between the plasma membrane and intracellular compartments: transport and metabolism of fluorescent sphingomyelin analogues in cultured fibroblasts

We examined the metabolism and intracellular transport of the D-erythro and L-threo stereoisomers of a fluorescent analogue of sphingomyelin, N-(N-[6-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino] caproyl])-sphingosylphosphorylcholine (C6-NBD-SM), in Chinese hamster ovary (CHO-K1) fibroblast monolayers. C6-NBD-SM was integrated into the plasma membrane bilayer by transfer of C6-NBD-SM monomers from liposomes to cells at 7 degrees C. The cells were washed, and within 10-15 min of being warmed to 37 degrees C, C6-NBD-SM was internalized from the plasma membrane to a perinuclear location that colocalized with the centriole and was distinct from the lysosomes and the Golgi apparatus. This perinuclear region was also labeled by internalized rhodamine-conjugated transferrin. C6-NBD-SM endocytosis was not inhibited when the microtubules were disrupted with nocodazole; rather, the fluorescent lipid was distributed in vesicles throughout the cell periphery instead of being internalized to the perinuclear region of the cell. The metabolism of C6-NBD-SM to other fluorescent sphingolipids at 37 degrees C and its effect on C6-NBD-SM transport was also examined. To study plasma membrane lipid recycling, C6-NBD-SM was first inserted into the plasma membrane of CHO-K1 cells and then allowed to be internalized by the cells at 37 degrees C. Any C6-NBD-SM remaining at the plasma membrane was then removed by incubation with nonfluorescent liposomes at 7 degrees C, leaving cells containing only internalized fluorescent lipid. The return of C6-NBD-SM to the plasma membrane from intracellular compartments upon further 37 degrees C incubation was then observed. The half-time for a complete round C6-NBD-SM recycling between the plasma membrane and intracellular compartments was approximately 40 min. Pretreatment of cells with either monensin or nocodazole did not inhibit C6-NBD-SM recycling.     

The radiation response of hypoxic cells in EMT6 spheroids in suspension culture does model data from EMT6 tumors

Radiation survival curves of EMT6/Ed spheroids have been obtained under conditions which eliminate changes in oxygen concentration between growth and irradiation. These curves show a high-dose, resistant component which is nearly parallel to the curves obtained when spheroids were irradiated under nitrogen. Thus EMT6 spheroids appear to model accurately the radiation responses of EMT6 tumors. In contrast, when spheroids were grown to relatively high density (300-400 spheroids per 250-ml spinner flask), then separated into several flasks for irradiation, an increase in oxygen concentration in the medium occurred which fully oxygenated the previously hypoxic cells. The two causes for the oxygen depletion in sealed growth flasks were quantitated. Depletion of total oxygen in the flask occurred, and, more importantly, oxygen consumption kept the growth medium well below equilibrium with the oxygen in the gas phase. Smaller but similar effects on oxygen concentration were found in flasks containing V79 spheroids.     

Biochemical and functional changes of rat liver spheroids during spheroid formation and maintenance in culture: I. morphological maturation and kinetic changes of energy metabolism, albumin synthesis, and activities of some enzymes

In the process of isolated single liver cells coming together to form three-dimensional spheroids, cells undergo dramatic environmental changes. How liver cells respond to these changes has not been well studied before. This study characterized the functional and biochemical changes during liver spheroid formation and maintenance. Spheroids were prepared in 6-well plates from freshly isolated liver cells from male Sprague rats by a gyrotatory-mediated method. Morphological formation, and functional and biochemical parameters of liver spheroids were evaluated over a period of 21 days in culture. Liver spheroid formation was divided into two stages, immature (1-5 days) and mature (>5 days), according to their size and shape, and changes in their functionality. Galactose and pyruvate consumption was maintained at a relatively stable level throughout the period of observation. However, glucose secretion and cellular GPT and GOT activities were higher in immature spheroids, decreased upto day 5 and remained stable thereafter. Cellular gamma-glutamyltransferase (gamma-GT) and lactate dehydrogenase (LDH) activities were initially undetectable or low and increased as spheroids matured. Albumin secretion decreased rapidly within the first 2 days and increased as spheroids matured. It is concluded that cells undergo functional and biochemical changes during spheroid formation following isolation of liver cells from intact tissue. Functionality and biochemical properties recovered and were maintained in mature spheroids. A relatively stable period (6-15 days) of functionality in mature spheroids was identified and is recommended for applications of the model.