Establishment and characterisation of a new cell line derived from Culex quinquefasciatus (Diptera: Culicidae)

Insect cell cultures are an important biotechnological tool for basic and applied studies. The objective of this work was to establish and characterise a new cell line from Culex quinquefasciatus embryonic tissues. Embryonated eggs were taken as a source of tissue to make explants that were seeded in L-15, Grace's, Grace's/L-15, MM/VP12, Schneider's and DMEM culture media with a pH range from 6.7-6.9 and incubated at 28oC. The morphological, cytogenetic, biochemical and molecular characteristics of the cell cultures were examined by observing the cell shapes, obtaining the karyotypes, using a cellulose-acetate electrophoretic system and performing random amplified polymorphic DNA-polymerase chain reaction analysis, respectively. The Grace's/L-15 medium provided the optimal nutritional conditions for cell adhesion and proliferation. Approximately 40-60 days following the explant procedure, a confluent monolayer was formed. Cellular morphology in the primary cultures and the subcultures was heterogeneous, but in the monolayer the epithelioid morphology type predominated. A karyotype with a diploid number of six chromosomes (2n = 6) was observed. Isoenzymatic and molecular patterns of the mosquito cell cultures matched those obtained from the immature and adult forms of the same species. Eighteen subcultures were generated. These cell cultures potentially constitute a useful tool for use in biomedical applications.     

Screening for compounds that induce apoptosis of cancer cells grown as multicellular spheroids

Screening and initial characterization of anticancer drugs are typically performed using monolayer cultures of tumor cells. It is well established that such monolayer cultures do not represent the characteristics of 3-dimensional solid tumors. The multicellular tumor spheroid model is of intermediate complexity between in vivo tumors and in vitro monolayer cultures and would be more suitable for drug screening. The authors describe a procedure in which multicellular spheroids are used to screen for compounds that induce tumor cell apoptosis. Multicellular spheroids were generated in 96-well plates, and apoptosis was determined using the M30-Apoptosense enzyme-linked immunosorbent assay method. A Z' factor of approximately 0.5 was observed for HCT116 colon carcinoma spheroids using staurosporine to induce apoptosis. This procedure is attractive for secondary screening of hits from larger cell-based screens.     

Micro Radiometric Method for Study of Acid-insoluble Materials in Monolayer Cell Cultures

A simple radiometric procedure for study of acid-insoluble products synthesized in monolayer cell cultures is described. Cell cultures were produced directly on the bottom surface of scintillation vials or on glass cover slips (8 X 30 mm). The cells were labeled and extracted; the radioactivity was determined while the cells remained affixed to the glass surface upon which they were grown. This procedure enabled rapid investigations of certain biosynthetic processes to be carried out by using many individual cell cultures. The method was applied to an investigation of (3)H-thymidine incorporation induced by vaccinia virus in a 5-bromodeoxyuridine-resistant cell line. (14)C-labeling was evaluated as an alternate procedure for cell quantitation.     

Multiparametric cell cycle analysis by automated microscopy

Cell cycle analysis using flow cytometry (FC) to measure cellular DNA content is a common procedure in drug mechanism of action studies. Although this technique lends itself readily to cell lines that grow in suspension, adherent cell cultures must be resuspended in a cumbersome and potentially invasive procedure that normally involves trypsinization and mechanical agitation of monolayer cultures. High-content analysis (HCA), an automated microscopy-based technology, is well suited to analysis of monolayer cell cultures but provides intrinsically less accurate determination of cellular DNA content than does FC and thus is not the method of choice for cell cycle analysis. Using Cellomics's ArrayScan reader, the authors have developed a 4-color multiparametric HCA approach for cell cycle analysis of adherent cells based on detection of DNA content (4,6-diamidino-2-phenylindole [DAPI] fluorescence), together with the known cell cycle markers bromo-2-deoxyuridine (BrdU) incorporation, cyclin B1 expression, and histone H3 (Ser28) phosphorylation within a single cell population. Considering all 4 markers together, a reliable and accurate quantification of cell cycle phases was possible, as compared with flow cytometric analysis. Using this assay, specific cell cycle blocks induced by treatment with thymidine, paclitaxel, or nocodazole as test drugs were easily monitored in adherent cultures of U-2 OS osteosarcoma cells.     

Potentiation of the destructive effect of heat (42°C) on synchronized cancer cells in culture by cell specific antiserum

1. 1. A kinetic study was made of the effects of hyperthermia and cell specific antibody on non-synchronized lag phase cultures and synchronized exponentially growing cultures of a malignant cell line. The effects on the degree of cell synchrony were also investigated.

2. 2. Hyperthermic treatment of synchronized SDB monolayer cell populations with maximum replication rate sensitized the cells to subsequent destruction by cell specific antibody.

3. 3. Hyperthermic treatment of SDB monolayer cultures with low replication rate and varying degrees of metabolic activity produced no such sensitization.

4. 4. Hyperthermia was disruptive to a synchrony procedure involving a blockade of DNA synthesis by excess thymidine.

Protein analysis of mammalian cells in monolayer culture using the bicinchoninic assay

We have applied the bicinchoninic acid (BCA) protein assay to rat brain primary astrocyte monolayer cultures growing in multiwell culture plates. The BCA method provides a more rapid and sensitive procedure with greater stability of color than is obtained using the Lowry method. Also, large numbers of samples can be read rapidly at the available wavelengths on an enzyme-linked immunosorbent assay microtiter plate reader. We found, however, artifactually high readings when using isotonic buffered sucrose to wash the cultures followed by sodium hydroxide to solubilize the cell protein. Such a procedure is commonly used for washing monolayer cell cultures in transport and binding studies. This effect was found to be due to hydrolysis of sucrose to the reducing sugar glucose. Use of Triton X-100 eliminated this problem, but this agent only solubilized about 80% of the protein that could be solubilized with sodium hydroxide. Furthermore, the high viscosity of Triton X-100 makes it more difficult to use. We found that washing the cells with isotonic mannitol solution followed by solubilization with sodium hydroxide gave reliable results. The sensitivity and speed of this method makes it suitable for multiple protein determinations in experiments using large numbers of cell culture samples.     

Comparison of endocrine secretion by monolayer cultures derived by different procedures from neonatal hamster and rat pancreas.

Monolayer cultures derived from neonatal hamster or rat pancreas by two different epithelioid cell-enriching gravity sedimentation procedures varied in ability to maintain uniform levels of insulin secretion with increased culture age. Rat pancreatic cultures were superior in this respect to identically derived hamster preparations, depending on the preparative procedure employed. Quantitative differences in the temporal pattern of insulin secretion by different rat pancreatic culture preparations were ascribed to plating cell density and consequent terminal cell density as a function of preparative procedure such that reduced densities favored sustained secretory levels. These findings suggest the importance of tissue species and preparative procedure in deriving pancreatic monolayer cultures capable of sustained levels of insulin secretion with age.     

The immunohistochemical characterisation of an SV40-immortalised human corneal epithelial cell line

Alternatives to the Draize rabbit eye irritation test are currently being investigated. Because of morphological and biochemical differences between the rabbit and the human eye, continuous human cell lines have been proposed for use in ocular toxicology studies. Single cell-type monolayer cultures in culture medium have been used extensively in ocular toxicology. In the present study, an SV40-immortalised human corneal epithelial (HCE) cell line was characterised immunohistochemically, by using 13 different monoclonal antibodies to cytokeratins (CKs), ranging from CK3 to CK20. The results from the monolayer HCE cell cultures were compared with those from the corneal epithelium of human corneal cryostat sections. Previous studies have shown that the morphology of the HCE cell is similar to that of primary cultured human corneal epithelial cells, and that the cells express the cornea-specific CK3. In the study reported here, we show that the cell line also expresses CKs 7, 8, 18 and 19. These CKs are typically expressed by simple epithelial cells, and are not found in the human cornea in vivo. Therefore, the monolayer HCE cell line grown in culture medium does not express the CK pattern that is typical of human corneal epithelium. This should be taken into consideration when using HCE cell cultures in similar single cell-type experiments for ocular toxicology.     

A "hanging" coverslip method for cell surface iodination of monolayer cultures

A procedure is described by which the use of a 1,3,4,6-tetrachloro-3 alpha,6 alpha-diphenyl glycoluril (iodogen)-coated coverslip to iodinate the cell surface proteins of monolayer cultures has been improved by "hanging" the coverslip at a defined distance from the cells. This method allows gentle manipulation of the cell culture, resulting in retention of high cell viability and in recovery of the cell monolayer with a minimum of mechanical damage. In addition, it allows the safe disposal of the radioactive coverslip upon completion of the reaction. Finally, the labeling is surface specific. The application of this procedure to 3T3 fibroblasts results in labeling of proteins comparable to lactoperoxidase-catalyzed iodinations.     

Mechanisms of protein degradation in growing and non-growing L-cell cultures.

L-cells prelabelled with [14C]leucine and [3H]thymidine were placed in either fresh growth medium (minimal essential medium with 10% serum) or stepdown medium (minimal essential medium) for 3 days. The 14C/3H ratio remained constant in the growing cultures and decreased in the stationary-phase cultures, indicating no protein turnover in growing cultures and a degradative rate of 0.6%/h in the stationary-phase cultures. Media analysis, however, indicated that 14C-labelled proteins were being degraded at approx. 1.2%/h in growing cultures and 1.7%/h in stationary-phase cultures. Additional studies indicated that a subpopulation of L-cells in the monolayer, comprising approx. 20--30% of the total, were lost in the original processing procedure. Experiments in which recoveries approached 100% by fixation of the monolayer in situ indicated that a protein-degrading subpopulation accounted for all the observed proteolysis in the growing cultures. Proteolysis in these cultures was only partially inhibited with NH4Cl, indicating that only a small part of the protein degradation was occurring in an activated lysosomal-autophagic system. NaF produced a more effective inhibition of proteolysis, but we were not able to distinguish whether this effect was on an ATP-requiring basal-turnover mechanism or a direct effect on unregulated activity of proteinases in the cell hyaloplasm. However, NH4Cl inhibited the proteolysis induced when cells were placed in stepdown medium, suggesting that the induced proteolysis was occurring via the autophagic system. We conclude that L-cells exist in at least two states with respect to protein degradation: (a) a subpopulation that is actively replicating and does not degrade cellular proteins, and (b) a second subpopulation of cells, derived from the preceding one, which degraded most of their labelled proteins, are not capable of further replication, and are not sedimented in an iso-osmotic EDTA buffer solution. In addition, proliferating L-cells, when placed in stepdown medium, begin to degrade cell protein through a mechanism involving autophagolysosomes.     

In vitro evaluation of alginate encapsulated adipose-tissue stromal cells for use as injectable bone graft substitute

This study aims to investigate the survival and osteogenic behavior of murine-derived adipose-tissue stromal cells (ATSCs) encapsulated in alginate microcapsules thereby instigating further studies in this cell delivery strategy for in vivo osteogenesis. Cell viability was quantified using a tetrazolium-based assay and osteogenic differentiation was evaluated by both alkaline-phosphatase (ALP) histochemistry and osteocalcin mRNA analysis. Following microencapsulation, cell numbers increased from 3.9 x 10(3) on day 1 to 7.8 x 10(3) on day 7 and maintained excellent viability in the course of 21-day culture. ALP was 6.9, 5.5, and 3.2 times higher than monolayer cultures on days 7, 14, and 21, respectively. In addition, osteocalcin mRNA was detectable in encapsulated cultures earlier (day 14) than monolayer cultures. We conclude that alginate microcapsules can act as three-dimensional matrix for ATSC proliferation and has potential for use as injectable, biodegradable scaffold in bone tissue engineering.     

Modulation of proteoglycan metabolism by human fibroblasts maintained in an endogenous three-dimensional matrix.

This report describes synthesis and degradation of proteoglycans by human gingival fibroblasts growing in an endogenous three-dimensional matrix. Cells grown in the matrix cultures demonstrated a high rate of proteoglycan synthesis, varying between 2 and 4 times that of cells maintained in monolayer cultures. In addition, the relative amount deposited into the cell layer was increased in the matrix cultures, constituting 70% to 90% of the synthesized material during the first 24 h. Comparable levels for the monolayer cultures were 30% to 60%. The majority of the 35S-sulfate-labeled material in both matrix (80%) and monolayer (62%) cultures was susceptible to chondroitin ABC-lyase digestion. The major product was a low Mr (120,000) proteoglycan which could be immunoprecipitated by an antibody against PGII (decorin). In addition, the cells synthesized two chondroitin ABC-lyase-sensitive proteoglycans, one with Mr greater than 400,000, one with an apparent Mr of 250,000, as well as two heparan sulfate proteoglycans with Mr greater than 250,000. The low Mr dermatan sulfate, decorin, was also the major component deposited in the three-dimensional matrix, constituting about 60% of the total sulfate incorporation. In contrast, fibroblasts in monolayer cultures deposited only a small amount (13%) of decorin (PGII) in the cell layer, and the major proteoglycan in this compartment was heparin sulfate. The rate of release of the newly deposited proteoglycans was the same in the two culture conditions, although material released from the three-dimensional matrix cultures contained small Mr components indicating a higher degree of degradation. These studies show differences in proteoglycan metabolism by gingival fibroblasts grown in an endogenous matrix and in monolayer cultures.(ABSTRACT TRUNCATED AT 250 WORDS)     

Growth of Respiratory Syncytial Virus in Suspension Cell Culture

Respiratory syncytial virus, Burnett strain, adsorbed efficiently and grew to high titers in suspension cultures of HEp-2 and MA-160 cells. Our results compared favorably with previous experience with the growth of respiratory syncytial virus in monolayer cell cultures. The use of suspension cell cultures provides a convenient and simple procedure for producing high-titering respiratory syncytial virus pools.     

Changes in protein content per cell during growth of mouse L cells

The relationship between cell density and protein content per cell was examined in monolayer and suspension cultures of mouse L cells. In monolayer cultures, the protein content per cell reached a maximum at 6 h after plating and retained this level for 18 h. Thereafter, the protein content per cell declined gradually during the exponential growth phase and finally returned to the initial level at the stationary phase. The changes were neither due to the effect of trypsinization nor to the exhaustion of the medium. The protein content per cell in a sparse culture was always greater than that in a dense culture for monolayer culture of L cells. In suspension culture the increase of protein content per cell during the lag phase was similar to that found in monolayer cultures. However, the gradual decline of protein content per cell observed during the exponential phase of monolayer cultures was not detected during that of a suspension culture. The results suggest that the decrease of protein content per cell in monolayer cultures may be related to some function of cell plasma membrane which could be inhibited by a cell-to-cell contact.     

Epidermal growth factor induces cell cycle arrest and apoptosis of squamous carcinoma cells through reduction of cell adhesion

Most squamous epithelial cells are strictly anchorage-dependent cell types. We observed that epidermal growth factor (EGF) promoted the growth of A431 squamous carcinoma cells in suspension cultures but suppressed cell growth and induced apoptosis in monolayer cultures, suggesting that loss of adhesion is responsible for the effects observed in monolayer culture, before cell death. Consistent with this finding, we demonstrated that EGF reduced cell attachment, cell-cell interaction, and cell spreading. Treatment with EGF increased cell adhesion-regulated expression of p21 but suppressed expressions of cyclin A, D1, cdk2, and retinoblastoma protein (pRb), leading to cell cycle arrest and adhesion-regulated programmed cell death. To test directly whether promoting cell adhesion could reduce the effects of EGF, we grew cultures on plates coated with type II collagen. On these plates, cell adhesion was enhanced and EGF treatment had little effect on cell adhesion and apoptosis when cells were attached to the collagen. The collagen effects were dose dependent, and cell cycle and cell cycle-associated proteins were altered accordingly. Finally, when cultures were plated on bacterial Petri dishes, which completely disrupted cell attachment to substratum, the level of apoptosis was greatly higher and cell cycle was arrested as compared with monolayer cultures. Taken together, our results strongly suggest that the EGF-induced cell cycle arrest and apoptosis in monolayer cultures was the result of a decline in cell adhesion.     

Differences in the X-ray sensitivity of cells in different regions of the sandwich, a diffusion-limited system for cell growth

The sandwich system was recently developed as a tumor analog; like spheroids, sandwiches are diffusion-limited multicellular systems which exhibit a necrotic center and a viable cell border. Using sandwiches of the 9L cell line, we compared the X-ray sensitivity of cells in the inner half of the viable border, adjacent to the necrotic center, with that of cells in the outer half of the viable border, adjacent ot the medium. No cells were hypoxic at the time of irradiation. The cells in the inner half of the viable border exhibited an increased radioresistance over cells in the outer half. The effect was dose multiplying with a multiplying factor of 1.5. Besides the sandwich studies, the X-ray sensitivity of 9L plateau monolayer cultures (induced by starvation) was compared to exponentially growing monolayer cultures. The plateau cultures exhibited an increased radioresistance over the exponentially growing cultures. The effect was also dose multiplying with a multiplying factor of 1.5.     

Changes in surface relief of suspended cells are morphological signs of the initial stage of neoplastic transformation in fibroblastic monolayer cultures.

The percentages of cells with different types of cell surface relief were determined in cell suspensions derived from monolayer cultures. Primary cultures of rat embryo fibroblasts (REF) and cell lines REF (LT) and REF-1, immortalized cells of which preserved normal phenotypic characteristics of the initial primary culture REF, as well as morphologically transformed tumorigenic lines REF (LT) ras and REF-2EJ were studied. In REF suspensions the cells with the blebbed type of surface relief were shown to be predominant as compared with those with microvillus relief whereas cell suspensions derived from both immortalized and fully transformed cultures display the reverse ratio of cells with those types of surface relief. Therefore, the pattern of cell surface relief in cell suspensions derived from fibroblastic monolayer cultures may serve as a morphological marker of the initial stage of neoplastic transformation-immortalization when typical morphological signs of cell transformation are not yet manifested in monolayer cultures.     

Fatty acid and glucose oxidation by cultured rat heart cells

Monolayer cultures of fetal rat myocardial cells can be utilized to examine substrate preferences and interactions. The specific activity of glucose oxidation by myocardial cell cultures was high in sparse cultures but decreased with increased cell density. In contrast, palmitate oxidation was independent of initial cell density. Palmitate inhibited glucose oxidation by 50% in rat heart cultures. Glucose had only a slight sparing effect on palmitate oxidation. This suggests that fetal and newborn rat myocardial cells in culture preferentially oxidize palmitate similar to adult heart. The sparing effect of palmitate on glucose oxidation is accounted for by inhibition of the glycolytic-aerobic pathway and not by inhibition of the pentose phosphate pathway. Data on oxidation of ¹⁴C-pyruvate specifically labelled suggest that palmitate or a product of its oxidation such as acetyl-CoA may be acting directly to inhibit the pyruvate dehydrogenase complex. Palmitate oxidation per mg of cell protein was constant from 15 days gestational age to 2 days postnatal age. The observed differences between cultured cells and the intact heart may relate to decreased aerobic metabolism in monolayer cell culture and suggest that the increase in fatty acid oxidation observed in vivo is controlled by the oxygen environment of the cell. These studies show that heart cells in monolayer culture can be utilized to obtain metabolic information similar to an adult organ perfusion model.     

An immunocytotoxicity assay for neural cells in monolayer cultures

The immunological analysis of cell surface constituents which may characterize neuronal and glial populations, though still in its infancy, will greatly facilitate the investigation of several important problems in neurobiology. One critical component of such analyses is the way by which a given antiserum can be shown to be active on, and possibly selective for neurons and glial cells from normal neural tissues. This report describes the use of monolayer cultures of normal neural cells for recognition and quantitative titration of antisera directed against them. Sera were collected from rabbits immunized with chick embryo spinal cord cell susptnsions, and found to be reactive to the same cells in the initial cell dissociate as well as in subsequent monolayer cultures of different in vitro ages. A monolayer assay procedure was developed, which (i) uses small numbers of cells and small volumes of immune reagents, with the possibility of further scaling down; (ii) applies equally to cultures using different substrata; (iii) permits differential counts of morphologically different cultured cells; (iv) allows to recognize cytological damage imposed by the immune serum in the presence, though not in the absence, of complement; and (v) quantitatively titrates the immune activity with 10- to 20-fold higher sensitivity than other titration procedures. While the study was not intended to investigate the possible specificities of the new antisera, it provided the unexpected observation that non-neuronal cells in these spinal cell cultures were considerably less sensitive than neurons to the complement-dependent action of the antisera.     

Effect of cyclophosphamide in vitro and on vaccinia virus replication in tissue culture.

The effect of cyclophosphamide on the growth of Vero, BSC-1, and HeLa cells in monolayer cultures was studied. By using hemocytometer counts and tritiated thymidine uptake as indicators of growth, it was found that cyclophosphamide significantly interfered with the metabolism of Vero and BSC-1 cells when sustained in Leibovitz medium. Vero cells and HeLa cells grown in Eagle medium were not affected by exposure to cyclophosphamide. Vaccinia virus replication in Vero cell monolayer cultures incubated with cyclophosphamide was markedly augmented, and this enhanced growth was reflected by virus quantitation techniques and metabolic studies using tritiated thymidine uptake. No difference in the distribution of infectious particles was found when cyclophosphamide-treated and control infected cultures were compared. Pathways other than through hepatic enzymes appear available to activate cyclophosphamide in vitro. These effects are dependent on both the cell type and the medium in which the cells are grown. Cyclophosphamide can facilitate vaccinia virus replication in vitro through metabolic interactions at the cellular level. The precise mechanisms underlying this effect require further study.