Loss of cancer drug activity in colon cancer HCT-116 cells during spheroid formation in a new 3-D spheroid cell culture system

Clinically relevant in vitro methods are needed to identify new cancer drugs for solid tumors. We report on a new 3-D spheroid cell culture system aimed to mimic the properties of solid tumors in vivo. The colon cancer cell lines HCT-116 wt and HCT-116 wt/GFP were grown as monolayers and for 3 or 6 days on 96-well NanoCulture® plates to form spheroids. Expression of surface markers, genes and hypoxia were assessed to characterize the spheroids and drug induced cytotoxicity was evaluated based on fluorescein diacetate (FDA) conversion by viable cells to fluorescent fluorescein or by direct measurement of fluorescence of GFP marked cells after a 72 h drug incubation. The cells reproducibly formed spheroids in the NanoCulture® plates with tight cell-attachment after 6 days. Cells in spheroids showed geno- and phenotypical properties reminiscent of hypoxic stem cells. Monolayer cultured cells were sensitive to standard and investigational drugs, whereas the spheroids gradually turned resistant. Similar results for cytotoxicity were observed using simplified direct measurement of fluorescence of GFP marked cells compared with FDA incubation. In conclusion, this new 3-D spheroid cell culture system provides a convenient and clinically relevant model for the identification and characterization of cancer drugs for solid tumors.     

Generation of Multicellular Tumor Spheroids with Microwell-Based Agarose Scaffolds for Drug Testing

Three dimensional multicellular aggregate, also referred to as cell spheroid or microtissue, is an indispensable tool for in vitro evaluating antitumor activity and drug efficacy. Compared with classical cellular monolayer, multicellular tumor spheroid (MCTS) offers a more rational platform to predict in vivo drug efficacy and toxicity. Nevertheless, traditional processing methods such as plastic dish culture with nonadhesive surfaces are regularly time-consuming, laborious and difficult to provide uniform-sized spheroids, thus causing poor reproducibility of experimental data and impeding high-throughput drug screening. In order to provide a robust and effective platform for in vitro drug evaluation, we present an agarose scaffold prepared with the template containing uniform-sized micro-wells in commercially available cell culture plates. The agarose scaffold allows for good adjustment of MCTS size and large-scale production of MCTS. Transparent agarose scaffold also allows for monitoring of spheroid formation under an optical microscopy. The formation of MCTS from MCF-7 cells was prepared using different-size-well templates and systematically investigated in terms of spheroid growth curve, circularity, and cell viability. The doxorubicin cytotoxicity against MCF-7 spheroid and MCF-7 monolayer cells was compared. The drug penetration behavior, cell cycle distribution, cell apoptosis, and gene expression were also evaluated in MCF-7 spheroid. The findings of this study indicate that, compared with cellular monolayer, MCTS provides a valuable platform for the assessment of therapeutic candidates in an in vivo-mimic microenvironment, and thus has great potential for use in drug discovery and tumor biology research.     

Imaging- and Flow Cytometry-based Analysis of Cell Position and the Cell Cycle in 3D Melanoma Spheroids

Three-dimensional (3D) tumor spheroids are utilized in cancer research as a more accurate model of the in vivo tumor microenvironment, compared to traditional two-dimensional (2D) cell culture. The spheroid model is able to mimic the effects of cell-cell interaction, hypoxia and nutrient deprivation, and drug penetration. One characteristic of this model is the development of a necrotic core, surrounded by a ring of G1 arrested cells, with proliferating cells on the outer layers of the spheroid. Of interest in the cancer field is how different regions of the spheroid respond to drug therapies as well as genetic or environmental manipulation. We describe here the use of the fluorescence ubiquitination cell cycle indicator (FUCCI) system along with cytometry and image analysis using commercial software to characterize the cell cycle status of cells with respect to their position inside melanoma spheroids. These methods may be used to track changes in cell cycle status, gene/protein expression or cell viability in different sub-regions of tumor spheroids over time and under different conditions.     

Importance of tyrosine phosphatases in the effects of cell-cell contact and microenvironments on EGF-stimulated tyrosine phosphorylation.

We have compared the EGF responses of A431 cells when grown as monolayers at a variety of cell densities or as multicellular spheroids in order to investigate the effects of cell contact and 3-dimensional structure on signal transduction. Proliferation of the A431 squamous carcinoma cell line grown in our laboratory was unaffected by EGF when grown in monolayer culture. As 3-dimensional, multicellular spheroids, however, growth was stimulated by EGF. The maximum volume attainable in the presence of EGF was more than 30 times that in its absence. EGF-dependent tyrosine phosphorylation was compared under these conditions by immunohistochemistry and Western blotting. In initial experiments using published procedures, tyrosine phosphorylation was density-dependent in monolayers and undetectable in spheroids. However, the density-dependence was abolished by the addition of high concentrations of protein tyrosine phosphatase inhibitors (1 mM Zn++ and VO4(3)-). The density dependence of EGF-stimulated tyrosine phosphorylation in monolayers was, therefore, largely the result of changes in phosphatase activity rather than kinase. Using high concentrations of phosphatase inhibitors, phosphotyrosine was clearly visible by immunohistochemistry in the outermost cells of spheroids, but it was still not visible in the spheroid center. The lack of response within the spheroid was not related to the presence of EGF receptor nor diffusion of EGF. In companion experiments, we showed that staining for EGF receptor was present homogeneously throughout the spheroid and that EGF penetrated to its center under the conditions of the experiment. Thus, although an increase in tyrosine phosphatase activity was a major factor affecting tyrosine phosphorylation in the outer cells, other factors were important in the inner cells. We concluded that an increase of tyrosine phosphatase activity was the most important component of the adaptation of the EGF signal transduction system to high cell density in monolayer cultures. In spheroids, tyrosine phosphatases are also enhanced, but other factors, such as autocrine synthesis of TGF-alpha and possibly the cellular distribution of EGF receptors and cell shape, play a role.     

Rapid generation of single-tumor spheroids for high-throughput cell function and toxicity analysis

Spheroids are widely used in biology because they provide an in vitro 3-dimensional (3D) model to study proliferation, cell death, differentiation, and metabolism of cells in tumors and the response of tumors to radiotherapy and chemotherapy. The methods of generating spheroids are limited by size heterogeneity, long cultivation time, or mechanical accessibility for higher throughput fashion. The authors present a rapid method to generate single spheroids in suspension culture in individual wells. A defined number of cells ranging from 1000 to 20,000 were seeded into wells of poly-HEMA-coated, 96-well, round-or conical-bottom plates in standard medium and centrifuged for 10 min at 1000 g. This procedure generates single spheroids in each well within a 24-h culture time with homogeneous sizes, morphologies, and stratification of proliferating cells in the rim and dying cells in the core region. Because a large number of tumor cell lines form only loose aggregates when cultured in 3D, the authors also performed a screen for medium additives to achieve a switch from aggregate to spheroid morphology. Small quantities of the basement membrane extract Matrigel, added to the culture medium prior to centrifugation, most effectively induced compact spheroid formation. The compact spheroid morphology is evident as early as 24 h after centrifugation in a true suspension culture. Twenty tumor cell lines of different lineages have been used to successfully generate compact, single spheroids with homogenous size in 96-well plates and are easily accessible for subsequent functional analysis.     

Multicellular tumour spheroids derived from human brain tumours

Multicellular tumour spheroids were prepared from a total of 46 human brain tumour biopsies by collagenase digestion and plating into agar coated flasks. Both primary malignant and secondary tumours formed spheroids with some correlation between the malignancy of tumour and the ability to undergo spheroid formation. The spheroids were capable of progressive growth, the rate of which was dependent, to some extent, on environmental conditions and was reflected by an increase in cell number within the spheroids. Spheroids prepared in this way may prove to be useful models for in vitro chemosensitivity and the general biology of brain tumours.     

Cell Fusion Studies to Examine the Mechanism for Etoposide Resistance in Chinese Hamster V79 Spheroids

When exposed to etoposide, the outer cells from Chinese hamster V79 spheroids are about 10 times more resistant to DNA strand breaks and cell killing than V79 cells grown as monolayers. Previous results have shown that the outer cells of both spheroids and monolayers grow at the same rate and contain the same amount and activity of the target enzyme, topoisomerase II. In order to examine possible mechanisms for this resistance, cell fusion studies were conducted with fluorescent dye-tagged monolayer and spheroid cells. Fused cells were exposed for 30 min to 1.2 μg/ml etoposide and then separated using fluorescence-activated cell sorting into binucleate cells consisting of two monolayer cells, two spheroid cells, or a mixed doublet consisting of one cell of each type. Individual sorted cell doublets were examined for the presence of etoposide-induced DNA strand breaks using the alkaline comet assay. As expected, doublets of monolayer cells were sensitive to etoposide and doublets of spheroid cells were resistant. However, mixed doublets were as resistant to DNA damage by etoposide as spheroid doublets. In comparison, when etoposide- or adriamycin-resistant V79 monolayer cells were fused to the parent monolayer cells, the expected intermediate sensitivity to etoposide was observed for the mixed doublets. We conclude that etoposide resistance associated with the outer cells of spheroids can be “transferred” to produce resistance in monolayer cells. Rapid changes in phosphorylation that can affect topoisomerase II activity or localization, or that can alter chromatin structure, are suggested as possible mechanisms of resistance. In support of this hypothesis, topo IIα phosphorylation was at least 10 times greater in monolayers than in the outer cell layer of spheroids.     

Three-dimensional perfused tumour spheroid model for anti-cancer drug screening

Objective

To build an in vitro-perfused, three-dimensional (3D) spheroid model based on the TissueFlex system for anti-cancer drug efficacy testing in order to mimic avascular micro-tissues with inherent O₂, nutrient and metabolite gradients, and to provide a more accurate prediction of drug toxicity and efficacy than traditional in vitro tumour models in conventional static culture well plates.

Results

The perfused cancer spheroid model showed higher cell viability and increased diameter of spheroids over a relatively long culture period (17 days). Three anti-cancer drugs with different cytotoxic mechanisms were tested. In perfusion, lower cytotoxicity was observed for traditional cytotoxic drug 5-fluorouracil and microtubule-interfering, paclitaxel, showed greater interruption of spheroid integrity. For the hypoxic-dependent drug, tirapazamine, there was no significant difference observed between static and perfusion cultures.

Conclusion

The perfusion culture provides a better homeostasis for cancer cell growth in a more controllable working platform for long-term drug testing.

     

A reliable tool to determine cell viability in complex 3-d culture: the acid phosphatase assay

Cell-based assays are more complex than cell-free test systems but still reflect a highly artificial cellular environment. Incorporation of organotypic 3-dimensional (3-D) culture systems into mainstream drug development processes is increasingly discussed but severely limited by complex methodological requirements. The objective of this study was to explore a panel of standard assays to provide an easy-handling, standardized protocol for rapid routine analysis of cell survival in multicellular tumor spheroid-based antitumor drug testing. Spheroids of 2 colon carcinoma cell lines were characterized for evaluation. One of the assay systems tested could reliably be used to determine cell viability in spheroids. The authors verified that the acid phosphatase assay (APH) is applicable for single spheroids in 96-well plates, does not require spheroid dissociation, and is linear and highly sensitive for HT29 and HCT-116 spheroids up to diameters of 650 microm and 900 microm, consisting of 40,000 and 80,000 cells, respectively. Treatment of HT29 and HCT-116 cells with 5-fluorouracil, Irinotecan, and C-1311 revealed critically reduced drug efficacies in 3-D versus monolayer culture, which is discussed in light of literature data. The experimental protocol presented herein is a small but substantial contribution to the establishment of sophisticated 3-D in vitro systems in the antitumor drug screening scenario.     

Multicellular Tumor Spheroids for Evaluation of Cytotoxicity and Tumor Growth Inhibitory Effects of Nanomedicines In Vitro: A Comparison of Docetaxel-Loaded Block Copolymer Micelles and Taxotere?

While 3-D tissue models have received increasing attention over the past several decades in the development of traditional anti-cancer therapies, their potential application for the evaluation of advanced drug delivery systems such as nanomedicines has been largely overlooked. In particular, new insight into drug resistance associated with the 3-D tumor microenvironment has called into question the validity of 2-D models for prediction of in vivo anti-tumor activity. In this work, a series of complementary assays was established for evaluating the in vitro efficacy of docetaxel (DTX) -loaded block copolymer micelles (BCM+DTX) and Taxotere® in 3-D multicellular tumor spheroid (MCTS) cultures. Spheroids were found to be significantly more resistant to treatment than monolayer cultures in a cell line dependent manner. Limitations in treatment efficacy were attributed to mechanisms of resistance associated with properties of the spheroid microenvironment. DTX-loaded micelles demonstrated greater therapeutic effect in both monolayer and spheroid cultures in comparison to Taxotere®. Overall, this work demonstrates the use of spheroids as a viable platform for the evaluation of nanomedicines in conditions which more closely reflect the in vivo tumor microenvironment relative to traditional monolayer cultures. By adaptation of traditional cell-based assays, spheroids have the potential to serve as intermediaries between traditional in vitro and in vivo models for high-throughput assessment of therapeutic candidates.     

6-Shogaol Inhibits Breast Cancer Cells and Stem Cell-Like Spheroids by Modulation of Notch Signaling Pathway and Induction of Autophagic Cell Death

Cancer stem cells (CSCs) pose a serious obstacle to cancer therapy as they can be responsible for poor prognosis and tumour relapse. In this study, we have investigated inhibitory activity of the ginger-derived compound 6-shogaol against breast cancer cells both in monolayer and in cancer-stem cell-like spheroid culture. The spheroids were generated from adherent breast cancer cells. 6-shogaol was effective in killing both breast cancer monolayer cells and spheroids at doses that were not toxic to noncancerous cells. The percentages of CD44⁺CD24^-/^low cells and the secondary sphere content were reduced drastically upon treatment with 6-shogaol confirming its action on CSCs. Treatment with 6-shogaol caused cytoplasmic vacuole formation and cleavage of microtubule associated protein Light Chain3 (LC3) in both monolayer and spheroid culture indicating that it induced autophagy. Kinetic analysis of the LC3 expression and a combination treatment with chloroquine revealed that the autophagic flux instigated cell death in 6-shogaol treated breast cancer cells in contrast to the autophagy inhibitor chloroquine. Furthermore, 6-shogaol-induced cell death got suppressed in the presence of chloroquine and a very low level of apoptosis was exhibited even after prolonged treatment of the compound, suggesting that autophagy is the major mode of cell death induced by 6-shogaol in breast cancer cells. 6-shogaol reduced the expression levels of Cleaved Notch1 and its target proteins Hes1 and Cyclin D1 in spheroids, and the reduction was further pronounced in the presence of a γ-secretase inhibitor. Secondary sphere formation in the presence of the inhibitor was also further reduced by 6-shogaol. Together, these results indicate that the inhibitory action of 6-shogaol on spheroid growth and sustainability is conferred through γ-secretase mediated down-regulation of Notch signaling. The efficacy of 6-shogaol in monolayer and cancer stem cell-like spheroids raise hope for its therapeutic benefit in breast cancer treatment.     

High-throughput Image Analysis of Tumor Spheroids: A User-friendly Software Application to Measure the Size of Spheroids Automatically and Accurately

The increasing number of applications of three-dimensional (3D) tumor spheroids as an in vitro model for drug discovery requires their adaptation to large-scale screening formats in every step of a drug screen, including large-scale image analysis. Currently there is no ready-to-use and free image analysis software to meet this large-scale format. Most existing methods involve manually drawing the length and width of the imaged 3D spheroids, which is a tedious and time-consuming process. This study presents a high-throughput image analysis software application – SpheroidSizer, which measures the major and minor axial length of the imaged 3D tumor spheroids automatically and accurately; calculates the volume of each individual 3D tumor spheroid; then outputs the results in two different forms in spreadsheets for easy manipulations in the subsequent data analysis. The main advantage of this software is its powerful image analysis application that is adapted for large numbers of images. It provides high-throughput computation and quality-control workflow. The estimated time to process 1,000 images is about 15 min on a minimally configured laptop, or around 1 min on a multi-core performance workstation. The graphical user interface (GUI) is also designed for easy quality control, and users can manually override the computer results. The key method used in this software is adapted from the active contour algorithm, also known as Snakes, which is especially suitable for images with uneven illumination and noisy background that often plagues automated imaging processing in high-throughput screens. The complimentary “Manual Initialize” and “Hand Draw” tools provide the flexibility to SpheroidSizer in dealing with various types of spheroids and diverse quality images. This high-throughput image analysis software remarkably reduces labor and speeds up the analysis process. Implementing this software is beneficial for 3D tumor spheroids to become a routine in vitro model for drug screens in industry and academia.     

Mathematical modelling of drug transport in tumour multicell spheroids and monolayer cultures

In this paper we adapt an avascular tumour growth model to compare the effects of drug application on multicell spheroids and on monolayer cultures. The model for the tumour is based on nutrient driven growth of a continuum of live cells, whose birth and death generates volume changes described by a velocity field. The drug is modelled as an externally applied, diffusible material capable of killing cells, both linear and Michaelis-Menten kinetics for drug action on cells being studied. Numerical solutions of the resulting system of partial differential equations for the multicell spheroid case are compared with closed form solutions of the monolayer case, particularly with respect to the effects on the cell kill of the drug dosage and of the duration of its application. The results show an enhanced survival rate in multicell spheroids compared to monolayer cultures, consistent with experimental observations, and indicate that the key factor determining this is drug penetration. An analysis of the large time tumour spheroid response to a continuously applied drug at fixed concentration reveals up to three stable large time solutions, namely the trivial solution (i.e. a dead tumour), a travelling wave (continuously growing tumour) and a sublinear growth case in which cells reach a pseudo-steady-state in the core. Each of these possibilities is formulated and studied, with the bifurcations between them being discussed. Numerical solutions reveal that the pseudo-steady-state solutions persist to a significantly higher drug dose than travelling wave solutions.     

Multicell spheroids as a model for cell kinetic studies

Cells growing in tissue culture as three-dimensional, multicellular aggregates called 'spheroids' typically show a decreasing growth fraction and development of quiescent subpopulations as the spheroids enlarge. Kinetic studies in a number of spheroid systems have indicated that the primary reason for the tumour-like growth is a progressive decrease in growth fraction, with only a modest elongation of cell cycle time in larger spheroids. In this paper, the cellular growth kinetics for spheroids of V79 Chinese hamster lung cells are reviewed, and the regrowth kinetics of cells resuming growth after recovery from quiescent regions of the spheroids are described. Further, the role of regrowth/repopulation in determining the spheroid response to anti-tumour cytotoxics is explored, with particular emphasis on treatment with cisplatin and etoposide. By separating the effects of cytotoxicity and regrowth in the overall spheroid response to anti-neoplastic drugs, it is suggested that 'drug resistance' in tumours can be a kinetic as well as a genetic problem.     

Invited review Multicell spheroids as a model for cell kinetic studies

Abstract. Cells growing in tissue culture as three-dimensional, multicellular aggregates called 'spheroids' typically show a decreasing growth fraction and development of quiescent subpopulations as the spheroids enlarge. Kinetic studies in a number of spheroid systems have indicated that the primary reason for the tumour-like growth is a progressive decrease in growth fraction, with only a modest elongation of cell cycle time in larger spheroids. In this paper, the cellular growth kinetics for spheroids of V79 Chinese hamster lung cells are reviewed, and the regrowth kinetics of cells resuming growth after recovery from quiescent regions of the spheroids are described. Further, the role of regrowth/repopulation in determining the spheroid response to anti-tumour cytotoxics is explored, with particular emphasis on treatment with cisplatin and etoposide. By separating the effects of cytotoxicity and regrowth in the overall spheroid response to anti-neoplastic drugs, it is suggested that 'drug resistance' in tumours can be a kinetic as well as a genetic problem.     

Prolonged lidocaine metabolizing activity of primary hepatocytes with spheroid culture using polyurethane foam as a culture substratum

Primary rat hepatocytes formed spheroids in the pores of polyurethane foam (PUF) used as a culture substratum. The hepatocytes in monolayer and spheroid stationary culture converted lidocaine to monoethylglycinexylidide (MEGX) which was N-deethylation of lidocaine. The metabolic activity of the hepatocytes/spheroid stationary culture system was 1.5∼2.0-fold higher than that of monolayer culture for 10 days. The activity of albumin production and cell survival of hepatocytes in monolayer and spheroid cultures decrease due to lidocaine treatment dependend on the lidocaine concentration, but the activity and cell survival in PUF/spheroid stationary culture were maintained at a higher level than that in monolayer culture under the lidocaine treatment. We developed a device for an in vitro liver model, drug metabolism simulator (DMS), using a PUF/spheroid packed-bed module including 4.00 ± 0.68 × 10⁷ hepatocytes and analyzed pharmacokinetics of lidocaine in a one-compartment model. Lidocaine clearance and extraction ratio of hepatocytes in the DMS corresponded to 1.354 ± 0.318 ml/min/g-liver and 0.677 ± 0.0159/g-liver, respectively (N=4). These values were comparable with in vivo values, 1.930 ml/min g-liver and 0.965/g-liver reported by Nyberg (1977). Consequently, PUF/spheroid culture maintained high lidocaine metabolizing activity over a long term and seems to provide a promising culture system as a drug metabolism simulator which will be used for drug screening, cytotoxicity tests and prediction of pharmacokinetics. This revised version was published online in July 2006 with corrections to the Cover Date.     

Human Cardiac Progenitor Spheroids Exhibit Enhanced Engraftment Potential

A major obstacle to an effective myocardium stem cell therapy has always been the delivery and survival of implanted stem cells in the heart. Better engraftment can be achieved if cells are administered as cell aggregates, which maintain their extra-cellular matrix (ECM). We have generated spheroid aggregates in less than 24 h by seeding human cardiac progenitor cells (hCPCs) onto methylcellulose hydrogel-coated microwells. Cells within spheroids maintained the expression of stemness/mesenchymal and ECM markers, growth factors and their cognate receptors, cardiac commitment factors, and metalloproteases, as detected by immunofluorescence, q-RT-PCR and immunoarray, and expressed a higher, but regulated, telomerase activity. Compared to cells in monolayers, 3D spheroids secreted also bFGF and showed MMP2 activity. When spheroids were seeded on culture plates, the cells quickly migrated, displaying an increased wound healing ability with or without pharmacological modulation, and reached confluence at a higher rate than cells from conventional monolayers. When spheroids were injected in the heart wall of healthy mice, some cells migrated from the spheroids, engrafted, and remained detectable for at least 1 week after transplantation, while, when the same amount of cells was injected as suspension, no cells were detectable three days after injection. Cells from spheroids displayed the same engraftment capability when they were injected in cardiotoxin-injured myocardium. Our study shows that spherical in vivo ready-to-implant scaffold-less aggregates of hCPCs able to engraft also in the hostile environment of an injured myocardium can be produced with an economic, easy and fast protocol.     

Assessment of the number of local cytotoxic T lymphocytes required for degradation of micrometer-size tumor spheroids

Adoptive immunotherapy with human cytotoxic T lymphocytes (CTL) is a promising cancer treatment. Previously we showed that human CTLs against various types of tumors can be efficiently produced by coculturing peripheral blood cells with target cells. The aims of this study were to simulate the interaction of CTLs and micrometer-size tumor tissues in vitro and to assess the required number of CTLs at local tumor sites for degradation of a tumor. Allogeneic CTLs against a human transitional cell carcinoma cell line and autologous CTLs against a renal cell carcinoma cell derived from a surgical specimen were generated. The cytotoxic activities of CTLs against tumor cells in monolayer culture and tumor spheroids formed in U-bottom 96-well culture plates were assessed. Both allogeneic and autologous CTLs showed greater destructive activity than lymphokine activated killer (LAK) cells against target tumor spheroids. CTLs inoculated at E/T ratios of 0.1 to 1 coexisted with the tumor spheroid for 5 to 6 days and then increased in number with apparently lethal activity against the tumor spheroid. In contrast to CTLs, the increase in LAK cell numbers was scarcely observed, and the proliferated LAK cells did not show cytotoxicity against the tumor spheroid. These observations suggest that, when a small number of CTLs reach a local tumor site, they can destroy micrometer-size tumors after considerable local proliferation. This revised version was published online in August 2006 with corrections to the Cover Date.     

Formation of multicellular spheroids composed of adult rat hepatocytes in dishes with positively charged surfaces and under other nonadherent environments

Adult rat hepatocytes formed floating multicellular spheroids in primary culture in an uncoated plastic dish with a positively charged surface. Cells in the spheroids formed in such a simple way were similar to those formed in dishes coated with proteoglycan fraction isolated from rat liver reticulin fibers; in both cases, cells maintained high ability to produce albumin and poor ability to proliferate in response to epidermal growth factor. Coating dishes with albumin was also helpful in spheroid formation; coating with 2-hydroxymethyl methacrylate resulted in formation of incomplete spheroids. Elimination of serum factors was essential for the formation of spheroids; when cells were washed with serum-containing medium before seeding or if the medium was replaced with a serum-containing medium, spheroid formation was completely inhibited. Collagens, fibronectin, and laminin, all of which promote the adhesion and spreading of hepatocytes on substrates, inhibited spheroid formation. Furthermore, collagens disintegrated spheroids, and cells in the monolayer initiated proliferation. Thus, two distinct, mutually exclusive features of primary culture of adult hepatocytes apparently exist; monolayer culture with proliferative activity in an adherent environment and spheroid culture with poor proliferative activity and high albumin-producing ability in a nonadherent environment.