Review-progress in concept and practice of growing anchorage-dependent mammalian cells in three dimension

Tissue culture has played a major role in the rapid advances made in medical science in the past 50 yr. The full potential of the technique, however, is limited by the fact that growth of cells is usually restricted to a monolayer accompanied by major decreases in many of their tissue-specific functions. This has been shown to be due, in large part, to the inadequate oxygenation of cells growing in tissue culture dishes. Studies that show that the high charge density and rigidity of the plastic and glass surfaces used for culture are also major factors limiting growth of cells to a monolayer, are reviewed. A new culture system has been developed in which cells are grown on substrata made using perfluorocarbons (PFCs) coated with collagen type 1 and other adhesive factors. Perfluorocarbons have a much higher solubility for oxygen than water and have been used as oxygen delivery systems to protect cells from hypoxia. These new PFC-based substrata can provide both the optimal level of oxygen cells need to maintain differentiated functions and the flexible and weaker type of adhesion that allows cells to round up, interact with each other, and when provided with adequate nutritional support, to grow in three dimension.     

EFFECTS OF INCREASED PARTIAL PRESSURES OF OXYGEN, NITROGEN AND HELIUM ON CELLS IN CULTURE

The effects of gas-phase oxygen, nitrogen and helium at superatmospheric as well as at atmospheric partial pressures have been studied, using both normal and malignant cells. The ability of certain types of cells in monolayer cultures to proliferate when exposed to high or low oxygen tensions, and to recover when cultured further under more optimal conditions, has been assessed. The presence of an inert gas at high partial pressure permits survival of various types of cells in culture at much higher oxygen tensions than would otherwise be possible. A tentative explanation for this phenomenon is provided and its significance discussed.     

New perfluorocarbon system for multilayer growth of anchorage-dependent mammalian cells.

A novel tissue culture system has been developed that supports the multilayer growth of Hep G2 cells. The system consists of growing cells on collagen-coated perfluorocarbon substrata in the wells of a multi-well plate designed so that, even at very high densities, the oxygen in the cultures is replenished as rapidly as it is consumed. Hep G2 cells, which are typically contact inhibited, grow to form more than 10 layers of cells that continue to secrete albumin. Both multilayer growth and high rates of albumin depend on using a very enriched nutrient medium, compared to media usually used for monolayer culture of Hep G2 cells. The role played by increased oxygenation, enriched media, and the unique properties of the perfluorocarbon substrata for the 3-D growth of anchorage-dependent cells is discussed.     

Proliferation-associated oxygen consumption and morphology of tumor cells in monolayer and spheroid culture.

The oxygen consumption rate, proliferative activity, and morphology of EMT6/Ro mouse mammary sarcoma cells in monolayer and multicellular spheroid culture have been investigated in a comparative study. During the transition of monolayer cells from the exponential into the plateau growth phase, there is a distinct decrease in the cellular volume that is associated with a corresponding decrease in the proliferative and respiratory activity of the cells. The decline in cell volume is mainly due to a decrease in the content of cytoplasm, whereas the size of the nucleus is only slightly reduced. A concomitant decrease in the number of mitochondria per cell obviously accounts for the reduction in cellular oxygen uptake. Despite a continuous decrease of cell proliferation from the surface to interior regions of EMT6 spheroids reflected by a gradient in tritiated thymidine labeling, volume-related oxygen consumption is rather uniform in viable regions of these aggregates. The finding can be explained by the results of the morphometric evaluation showing a uniform volume density of mitochondria, i.e., of oxygen-consuming sites within these spheroids.     

Tumorsphere assay provides more accurate prediction of in vivo responses to chemotherapeutics

Although the sphere culture system has been widely used in stem cell biology, its application for drug screening is limited due to lack of standardized, rapid analytical tools. To optimize sphere cultures for in vitro screening of drugs, we evaluated the properties of primary tumor cells growing as tumorspheres and compared their chemosensitivity to those of cells growing in monolayer. Most cells in tumorsphere cultures were quiescent whereas cells in monolayer culture had a high mitotic index. Moreover, doxorubicin showed better cytotoxicity than paclitaxel in the sphere cultures, but their efficacy was reversed in the monolayer cultures. Importantly, the response of cytotoxic outcomes for suspension cultures matched the in vivo response better than monolayer cultures, providing support for the use of short term suspension cultures of primary cells as a model for drug testing.     

Surface glycosaminoglycans as a differentiation cofactor in neuroblastoma cell cultures.

The possible role of surface glycosaminoglycans (GAGs) in neuronal maturation occuring in neuroblastoma cultures has been investigated. GAGs of neuroblastoma cells, grown in suspension and monolayer, were labelled with 3H-glucosamine and 35S-sulfate. Neuron maturation, following cell adhesion to culture dishes, is accompanied by an increased ability of the cells to retain heparan sulfate (HS) on their surface, which is otherwise lost into the culture medium. The role of surface HS as a cofactor of cellular differentiation is discussed.     

The expression of differentiation by chick embryo thyroid in cell culture. I. Functional and fine structural stability in mass and clonal culture

The stability of glandular epithelial cells has been investigated utilizing the techniques of cell culture. Embryonic chick thyroid was chosen as a representative cell type and methods were developed for the successful clonal culture of thyroid follicular cells. Thyroid cells were found to be morphologically and functionally stable while undergoing rapid division in both dense (monolayer) and dilute (clonal) cell culture. Differentiated features were retained for a minimum of 32 days of primary clonal culture (approximately 17 generations under clonal conditions). During the culture period, the cells retained their epithelial morphology, retained their cytoplasmic rough endoplasmic reticulum, and continued to produce chromatographically detectable thyroid hormones. Hormone production in culture was a specific thyroid characteristic since control cultures of embryonic heart and liver did not contain the hormones.     

PARENCHYMAL CELLS FROM ADULT RAT LIVER IN NONPROLIFERATING MONOLAYER CULTURE : I. Functional Studies

Parenchymal cells from adult rat liver have been established in primary monolayer culture. Donor animals are subjected to a partial hepatectomy and, 4 days later, cells are prepared by collagenase perfusion of the regenerated liver. The hepatic parenchymal cells, separated from nonparenchymal material and suspended in serum-free medium, are placed in plastic tissue culture dishes, where they form a monolayer within 24 h. The monolayer cells exhibit minimal mitotic activity and demonstrate several major metabolic functions characteristic of liver in vivo; these include albumin synthesis and secretion, gluconeogenesis from 3-carbon precursors, responsiveness to insulin and glucagon, glycogen synthesis, and activity of two microsomal enzymes. These functions are present in the monolayer cells for several days at activities similar to those observed in the liver in vivo. The findings indicate that hepatic parenchymal cells in this monolayer system are viable and behave in many respects like normal adult rat liver.     

Thyrotropin effects on thyroid cells in culture. Effects of trypsin on the thyrotropin receptor and on thyrotropin-mediated cyclic 3':5'-AMP changes.

Dog, human, and bovine thyroid cells in culture have been shown to develop follicle-like structures when cells are cultured in conditions of confluency and when cells are incubated in the presence of bovine thyrotropin or N6,O2'-dibutyryl cyclic adenosine 3':5'-monophosphate during the first 24 to 48 hours after trypsinization. If thyrotropin is added 48 hours after trypsinization, these cells do not form follicle-like structures but remain as a monolayer culture. Although thyroid cells which grow as a monolayer have a thyrotropin receptor on their plasma membranes with the same in vitro binding properties as the thyrotropin receptor on the plasma membranes of the follicle-forming thyroid cells, there is a 1- to 2-fold greater number of receptors per mg of membrane protein when follicle-forming and monolayer cultures are compared...     

Differentiation of human stromal bone marrow cell precursors in monolayer culture

The colonies of human bone marrow fibroblasts in monolayer culture have been studied. It has been shown that there are two types of colonies in the cultures: monolayer and multilayer ones, both having alkaline phosphatase-positive cells. In monolayer colonies one can observe calcium deposition indicative of osteogenic differentiation of human bone marrow stromal cells.     

Studies of intercellular invasion in vitro using rabbit peritoneal neutrophil granulocytes (PMNS). I. Role of contact inhibition of locomotion

Intercellular invasion is the active migration of cells on one type into the interiors of tissues composed of cells of dissimilar cell types. Contact paralysis of locomotion is the cessation of forward extension of the pseudopods of a cell as a result of its collision with another cell. One hypothesis to account for intercellular invasion proposes that a necessary condition for a cell type to be invasive to a given host tissue is that it lack contact paralysis of locomotion during collision with cells of that host tissue. The hypothesis has been tested using rabbit peritoneal neutrophil granulocytes (PMNs) as the invasive cell type and chick embryo fibroblasts as the host tissue. In organ culture, PMNs rapidly invade aggregates of fibroblasts. The behavior of the pseudopods of PMNs during collision with fibroblasts was analyzed for contact paralysis by a study of time-lapse films of cells in mixed monolayer culture. In monolayer culture, PMNs show little sign of paralysis of the pseudopods upon collision with fibroblasts and thus conform in their behavior to that predicted by the hypothesis.     

Development of rat mast cells in vitro. I. Differentiation of mast cells from thymus cells.

Mast cells were differentiated by long-term culture of rat thymus cells on rat embryonic fibroblasts monolayers. Mature mast cells obtained in the culture were morphologically similar to normal peritoneal and thoracic mast cells and possessed specific receptors for IgE on their surface. In culture, blast cells appeared on the monolayer several days after seeding of thymus cells. These cells developed into young mast cells in the monolayer and became free in the culture medium with maturation. Receptors for IgE were detected on the surface of mastoblasts which contained a small amount of metachromatic granules. Evidence was obtained which suggested that the number and/or affinity of the receptors for IgE increases with maturation of mast cells. It was found that some mast cells differentiated from monolayers of embryo cells without seeding thymus cells. The present experiments, however, clearly showed that mast cells can be differentiated from thymus cell culture without monolayer. It appears that both thymus and embryo tissues contain precursors of mast cells.     

Immune response to a syngeneic mammary adenocarcinoma. II. In vitro generation of cytotoxic lymphocytes.

A method is described for the consistent in vitro generation cytotoxic cells by incubating Fischer 344 rat spleen cells on monolayers of a syngeneic mammary adenocarcinoma. Significant cytotoxicity by in vitro culture is generated as early as 3 days after initiation and effector cells are cytolytic only toward target cells of the sensitizing monolayer. Reciprocal sensitization with allogeneic fibroblasts as the immunizing monolayer yielded effector cells cytolytic for the fibroblasts but without effect on the mammary tumor. The consistency in the generation of cytotoxic cells by in vitro culture should permit its standardized use in following other related immune phenomena such as blocking by serologic factors and suppression, recritment of memory for cytotoxic function.     

The increase in radioresistance of Chinese hamster cells cultured as spheroids is correlated to changes in nuclear morphology

Chinese hamster V79 cells grown as spheroids in roller culture are more radioresistant than those grown as monolayers. The supercoiled structure of chromatin, as salt-extracted nucleoids, has been examined using flow cytometry. Irradiated viable cells from spheroid culture contain restraints to supercoil relaxation that are absent in monolayer cells. Further analysis of the chromatin organization from each growth form shows that the radioresistant spheroid cells contain a DNA-protein matrix that is more resistant to detergent-induced degradation. The increase in structural integrity may be due to the retention of a 55-60 kDa protein that is apparent in the nucleoids of spheroid, but not monolayer cells. The increase in structural integrity of the spheroid cells may explain their greater radioresistance by providing a more stable platform for high-fidelity DNA damage repair.     

The K(m) for Radiosensitization of Human Tumor Cells by Oxygen is Much Greater than 3 mmHg and is Further Increased by Elevated Levels of Cysteine

We studied the role of cysteine as an intracellular radiation protector under conditions in which both oxygen and thiols were monitored at 37 degrees C. In HCT-116 human colon cancer cells, the intracellular cysteine content affects the radiation survival dramatically at intermediate oxygen levels, but not at zero or high oxygen levels. Using a spin-through-oil method with a dual radioactive label detection system, we measured intracellular cysteine and glutathione (GSH) levels for cells in suspension culture. A comparison of the cysteine levels of monolayer cells lysed in situ and of trypsinized monolayer cells in suspension (Horan et al., Cytometry 29, 76-82, 1997) revealed that, upon trypsinization from monolayer culture and transfer to a spinner apparatus at 37 degrees C, HCT-116 cells lose most of their intracellular cysteine. Over the 60-min time course of control experiments, these cells do not recover intracellular cysteine despite the availability of cystine (the disulfide of cysteine) in the medium. When cells in spinner culture are provided with exogenous cysteine, they initially concentrate it to 10-fold the extracellular concentration, with the concentration factor decreasing to about 5-fold over the course of an hour. The intracellular GSH concentration changes little throughout this period, regardless of the changes in cysteine levels. The same apparatus was used to assess the survival of HCT-116 cells irradiated at 37 degrees C under conditions of constant pO(2) monitoring. For cells without added cysteine, the oxygen concentration for half-maximal radiation sensitivity was about 7.5 mmHg (intermediate hypoxia), more than twice the commonly accepted value (3 mmHg). At 7.5 mmHg, cells with added cysteine (intracellular concentration 3.5 mM) were almost as radioresistant as severely hypoxic cells (approximately 0.005% oxygen). Cells in parallel experiments in which the cells were grown in monolayers on glass Petri dishes had intermediate cysteine values and also intermediate radiosensitivity. We conclude that the radiation response of cells at intermediate oxygen levels is controlled predominantly by intracellular cysteine levels and that the cysteine levels commonly found in tumors may increase the K(m) for radiosensitivity to values much higher than suggested previously.     

The matrix-forming phenotype of cultured human meniscus cells is enhanced after culture with fibroblast growth factor 2 and is further stimulated by hypoxia

Human meniscus cells have a predominantly fibrogenic pattern of gene expression, but like chondrocytes they proliferate in monolayer culture and lose the expression of type II collagen. We have investigated the potential of human meniscus cells, which were expanded with or without fibroblast growth factor 2 (FGF2), to produce matrix in three-dimensional cell aggregate cultures with a chondrogenic medium at low (5%) and normal (20%) oxygen tension. The presence of FGF2 during the expansion of meniscus cells enhanced the re-expression of type II collagen 200-fold in subsequent three-dimensional cell aggregate cultures. This was increased further (400-fold) by culture in 5% oxygen. Cell aggregates of FGF2-expanded meniscus cells accumulated more proteoglycan (total glycosaminoglycan) over 14 days and deposited a collagen II-rich matrix. The gene expression of matrix-associated proteoglycans (biglycan and fibromodulin) was also increased by FGF2 and hypoxia. Meniscus cells after expansion in monolayer can therefore respond to chondrogenic signals, and this is enhanced by FGF2 during expansion and low oxygen tension during aggregate cultures.     

Microencapsulated multicellular tumor spheroids as a novel in vitro model for drug screening

To generate multicellular tumor spheroids (MTS) based on human breast adenocarcinoma MCF-7 cells and to study them as a novel in vitro model for anticancer drug screening, a technique for cell microencapsulation in biocompatible alginate-chitosan microcapsules has been used in this study. Using the MTS based on the MCF-7 cells methotrexate (MTX) cytotoxicity has been investigated. A set of MTS with an average size of 150, 200 and 300 μm was prepared as a function of cultivation time. Cell viability was evaluated after MTS incubation in cultivation medium containing various MTX concentrations (1, 2, 10, 50 and 100 nM) for 48 h. MTS were shown to be markedly more resistant to MTX than the monolayer culture. The increase of the spheroid size was in correlation with the enhanced MTS resistance to MTX. Thus, at 100 nM MTX a number of viable cells in MTS with the size of 300 μm was 2.5-fold higher than that in the monolayer culture. It is suggested that the cells microencapsulated into MTS can better mimic cell behavior in small solid tumors compared to the monolayer culture. In the future MTS could be proposed as a novel in vitro model for anticancer drug screening.     

Dispersed mammary gland epithelial cells. I. Isolation and separation procedures

The mammary gland from midpregnant rabbits has been dissociated into individual cells by enzymatic digestion, divalent cation chelation, and gentle shearing. A heterogeneous cell population is obtained, comprising approximately 60% parenchymal cells, approximately 10% myoepithelial cells, and approximately 30% connective tissue cells, including fibroblasts, plasma cells, and microphages. The epithelial cells are characterized by the presence of fat droplets, which in 65% of the cells form large supranuclear vacuoles. Their buoyant density is less than 1.045, allowing their separation from myoepithelial cells and connective tissue cells by isopycnic centrifugation in a density gradient. The homogeneity of the epithelial cell fraction has been assessed by light and electron microscopy. The cells are viable and functionally active as indicated by their ability to exclude vital dyes, incorporate labeled precursors, consume oxygen, maintain intracellular Na+ and K+ concentrations, and retain their structural integrity. In addition, when cultured in Petri dishes, the cells grow as a monolayer, reestablish junctional complexes and retain cell polarity.     

Kinetics of gas diffusion in mammalian cell culture systems. II. Theory

Mathematical models have been constructed which relate the depth of the culture fluid overlay to the oxygen available to mammalian cells cultured under static conditions. These models suggest that the maintenance of a given rate of oxygen utilization by some culture systems may be critically depended on this fluid depth and on the solubility and rate of diffusion of oxygen in the culture fluid. The importance of these concepts as applied to the isolation and growth of differentiated cells representative of the tissue of origin are noted.