A new continuous cell line from the mosquito Psorophora confinnis (Diptera: Culicidae) and its susceptibility to infections with some arboviruses

A new cell line, PC-0199-BR, was established from embryonated eggs of the mosquito Psorophora confinnis. To date (September 2000) it has had 62 continuous passages. This is the first report of a cell line of mosquitoes belonging to the genus Psorophora. Cell growth initially was achieved in the MM/VP12 medium, supplemented with 20% fetal bovine serum; however, the subcultures were later adapted to Grace's medium with 10% fetal bovine serum. Cell morphology in the primary cultures was heterogeneous; but later in the established cell line, the predominant cell type was epithelioid. Cultured cells were predominantly diploid (2n=6); however, chromosome abnormalities were observed in a small proportion of the cells in later passages. C and G band patterns were also determined in the karyotype. The cell line isozyme profiles coincided with pupae and adult samples of the species taken from the same colony. A preliminary arbovirus susceptibility study for the cell line was undertaken. No evidence was observed of contamination of the cell line with bacteria, fungi or mycoplasma.     

Growth limiting factors influencing high density culture of insect cells in Grace's medium

Summary Growth limiting factors influencing the high density cultivation of insect cells were studied. A stationary phase in Grace's medium was found to be due to the deprivation of the active form of FBS components. Various compounds were added in the early stationary phase to observe the recovery of cell growth. With the addition of yeastolate, final cell densities were 4-fold and 3.5-fold higher in monolayer and suspension cultures, respectively. Pluronic F-68 increases the specific growth rate and the growth yield of the cell as well as protects the cell from the shear damage.     

Culture of primary lung tumors using medium conditioned by a lung carcinoma cell line

Medium conditioned by the established lung tumor cell line A549 was used as a supplement to culture cells from primary solid lung tumors. Of 36 cases placed into culture, primary cells were obtained in 33 (91.7%). Of 29 cases in which subcultures were attempted, 18 (62.1%) were successful. Nine cell lines have been established by this technique to date. In growth assays, conditioned medium (CM) was found to stimulate both monolayer colony formation and growth in semi-solid medium of cells cultured from primary solid tumors. CM has been found to contain factors with the properties of both transforming growth factor alpha (TGF alpha) and insulin-like growth factor-I (IGF-I). The addition of a combination of these factors as purified peptides to basal medium at levels found in CM (0.1-0.5 ng/ml) stimulated colony formation of lung tumor cells by up to fourfold. These results indicate that secretion of growth factors may be important in tumor growth in vivo, and that use of CM may be a valuable tool for obtaining cultures from primary solid tumors.     

A comparison of bovine serum albumin and chicken ovalbumin as supplements for the serum-free growth of chinook salmon embryo cells,CHSE-214.

Chicken ovalbumin and bovine serum albumin (BSA) were compared as supplements to the basal medium, L-15, for the serum-free cultivation of the Chinook Salmon Embryo cell line, CHSE-214. Unlike L-15 alone, ovalbumin and some commercial BSA preparations allowed cell proliferation and development of confluent monolayer cultures. However, only a fatty acid-free BSA (2%) supported continuous proliferation for two years through approximately 15 subcultivations. For this, subcultivation was achieved with non enzymatic cell dissociation solutions. Also, new serum-free subcultivation techniques were developed that utilized avian egg white trypsin inhibitors to terminate the action of either bovine or cod trypsin. Finally, CHSE-214 were successfully cryopreserved in 2% BSA, allowing all cell cultivation steps to be performed in the absence of FBS.     

Secretagogue induction of cell differentiation in pancreatic acinar cells in vitro.

The effect of two different types of secretagogues on rat pancreatic acinar cells cultured onto a reconstituted basement membrane was studied. Cells cultured without any secretagogue were able to reaggregate but did not form monolayer patches. Most of them lost their differentiated ultrastructural characteristics but regained their polarity. In contrast, when CCK, caerulein, or carbamylcholine was added to the culture medium cells developed both acini-like structures and cell monolayer patches. The cells retained the differentiated ultrastructural appearance and polarity resembling their in situ morphology. Furthermore, secretagogue-conditioned cells presented higher amylase contents. The use of secretagogue antagonists such as L-364,718 and L-365,260 for caerulein, or atropine and mecamylamine for carbamylcholine, did not profoundly modify the cultures and the morphological effects triggered by the secretagogues alone. However, both CCK antagonists and cholinergic antagonists inhibited to a certain degree the secretory stimulation. Our data support the theory that a major role is played by secretagogues in conjunction with the basement membrane for the maintenance of differentiation in pancreatic acinar cells in vitro which appears to be independent from their secretory effect.     

Development of transformed characteristics by sheep thyroid cells irradiated as differentiated primary cultures

A method has been developed which allows four stages of transformation to be detected in subcultured populations of sheep thyroid cells exposed to gamma irradiation as differentiated primary cultures. The cell populations initially irradiated and the subcultures deriving from these retain a number of differentiated properties of thyroid cells, including iodine uptake, follicle formation and T₄ production. The stages of transformation which develop in the subcultures are nonsenescence, focus development on a confluent monolayer, i.e. loss of contact inhibition, colony formation in soft agar, i.e. anchorage independence, and reduced serum dependence.The technique offers a method for detecting the development of transformation in primary differentiated epithelial cultures.     

Cell cultures from the symbiotic soft coral Sinularia flexibilis

The symbiotic octocoral Sinularia flexibilis is a producer of potential pharmaceuticals. Sustainable mass production of these corals as a source of such compounds demands innovative approaches, including coral cell culture. We studied various cell dissociation methodologies and the feasibility of cultivation of S. flexibilis cells on different media and cell dissociation methodologies. Mechanical dissociation of coral tissue always yielded the highest number of cells and allowed subsequent cellular growth in all treatments. The best results from chemical dissociation reagents were found with trypsin-ethylene diamine tetraacetic acid. Coral cells obtained from spontaneous dissociation did not grow. Light intensity was found to be important for coral cell culture showing an enduring symbiosis between the cultured cells and their intracellular algae. The Grace's insect medium and Grace's modified insect medium were found to be superior substrates. To confirm the similarity of the cultured cells and those in the coral tissue, a molecular test with Internal Transcribed Spacer primers was performed. Thereby, the presence of similar cells of both the coral cells and zooxanthella in different culture media was confirmed.     

A Cell Line (HEW) from Embryos of Haddock (Melanogrammus aeglefinius) and Its Capacity to Tolerate Environmental Extremes

Cell lines can be useful experimental tools for studying marine fish, which are often difficult to routinely obtain and maintain in the laboratory. As few cell lines are available from coldwater marine fish, cultures were initiated from late gastrula embryos of haddock (Melanogrammus aeglefinus) in Leibovitz's L-15 with fetal bovine serum (FBS). From one culture, a cell line (HEW) emerged that has been grown for close to 100 population doublings, was heteroploid, and expressed telomerase activity, all of which suggest HEW is immortal. Growth occurred only if FBS was present and was optimal at 12 to 18°C. Usually most cells had an epithelial-like morphology, but under some conditions, cells drew up into round central bodies from which radiated cytoplasmic extensions with multiple branches. These neural-like cells appeared within a few hours of cultures being placed at 28°C or being switch to a simple salt solution (SSS). At 28°C, cells died within 24 h. In SSS, HEW cells survived as a monolayer for at least 7 days. The sensitivity of HEW cells to morphological change and their capacity to withstand starvation should make them useful for investigating cellular responses to environmental stresses.     

Plasma-derived serum as a selective agent to obtain endothelial cultures from swine aorta

Endothelial cell and smooth muscle cell cultures from artery wall provide a potential model system for studying cellular processes involved in atherogenesis. To prepare serial subcultures of swine arterial endothelial cells that are free of smooth muscle cells without either selecting a small population or subjecting the cells to cytotoxic conditions, we used swine plasma-derived serum (SPDS) to establish conditions in which endothelial cells have a growth advantage. Endothelial cells were collected by collagenase digestion and smooth muscle cell cultures were prepared by outgrowth from explants of arterial medial segments. Growth rates were compared when each cell type was maintained on SPDS, or fetal bovine serum (FBS), or swine whole serum (SWS). When 20% FBS or SWS were used the doubling times were less than 30 h for both endothelial cells and smooth muscle cells. On 20% SPDS the doubling time for endothelial cells was 32 h, but for smooth muscle cells it was at least 168 h. Using SPDS, we prepare endothelial subcultures from swine aorta that express principally polygonal morphology at confluence. Endothelial cell cultures grown on SPDS have higher angiotensin-converting enzyme than those grown on FBS.     

Anionic glycoconjugates from differentiated and dedifferentiated cultures of bovine articular chondrocytes: Modulation by TGF-β

Summary Primary, high density bovine articular chondrocyte (BAC) cultures, stimulated with transforming growth factor-β-1, elaborated a high molecular weight anionic glycoconjugate, kDa 540, which does not contain glycosaminoglycan chains (Chan and Anastassiades, 1996). The effect of exogenously added transforming growth factor-β-1 on the elaboration of the high molecular weight glycoconjugate and of proteoglycans was studied during dedifferentiation of the chondrocytes, utilizing a serial subculture technique under anchorage-dependent conditions, up to four subcultures. The high molecular weight glycoconjugate was detected in the media of all growth-factor-stimulated chondrocyte subcultures, as well as stimulated primary cultures, but not in unstimulated primary cultures or subcultures. By contrast, a large proteoglycan, was only secreted by primary cultures and first subcultures, whether treated with transforming growth factor-β-1 or untreated. This proteoglycan contained mostly chondroitin sulfate chains, whose hydrodynamic size was increased by the addition of transforming growth factor-β-1. Further, the pattern of the proteoglycans appearing in the media of subcultures 2–4 was influenced by the addition of transforming growth factor-β-1, so that while these control subcultures elaborated both the large and small chondroitin sulfate proteoglycans, the equivalent stimulated subcultures elaborated only intermediate sized chondroitin sulfate proteoglycan(s). These results suggest that while dedifferentiation of articular chondrocytes, achieved by subculturing, strongly modulates the effect of exogenously added transforming growth factor-β-1 on the type of proteoglycan elaborated, the process of dedifferentiation does not influence the transforming-growth-factor-β-dependent synthesis of the high molecular weight anionic glycoconjugate.     

Cell morphology in long-term cultures of normal and abnormal amniotic fluids

Summary The cell morphology of long-term cultures of amniotic fluid cells from 10 fetuses with a neural tube defect (NTD) and three with omphalocele was examined and compared to 30 long-term cultures of normal amniotic fluids as well as a long-term culture of human fetal brain. Cultures from the amniotic fluids of the fetuses with NTD and omphalocele showed cells with the same general characteristics as normal amniotic fluid cells. However, the cultures of amniotic fluid cells from NTD pregnancies had an additional cell type also seen in fetal brain culture. This was a neuroblast-like cell, with small rounded refractile morphology and long branching processes forming clusters of varying sizes which lay on top of large flat cells. These neuroblast-like cells diminished in number with time in culture and were not present in subcultures. Their possible neuronal origin is discussed.     

Rapid Growth of Acanthamoeba In Defined Media; Induction of Encystment By Glucose-Acetate Starvation

Defined media are described that support 14-20 h generation times for Acanthamoeba castellanii and A. rhysodes in monolayer cultures. the media differ in minor ways from previously described media, but the growth rates are greatly improved over previously reported values. Maximum growth rates were observed for A, castellanii in a complex medium containing 21 amino acids, but near-maximum rates could be achieved in relatively simple media containing 9 amino acids. Growth occurred with 6 amino acids, as reported by others, but generation times exceeded 30 h. Amitosis was a common problem during early subcultures in defined media, but became infrequent after repeated transfers. Synchronous encystment resulting in 70-80% cyst formation could be induced in the defined media by glucose and acetate starvation. the rate of encystment varied with cell density at the time of starvation and was optimal at initial densities of 400-800 amebae/mm2.     

Actin and tubulin synthesis in monolayer and suspension cultures of murine L cells

The rates of total protein, actin and tubulin synthesis were studied for monolayer (L-929) and suspension (LS) cultures of mouse L cell. Data on pulse 34S-label incorporation into the cellular protein pool show that LS characterized by a short cell cycle have, comparatively to L-929, higher rates of protein synthesis and phosphorylation. According to PAGE data, the level of actin and tubulin synthesis in suspension line exceeds that in monolayer one. The correlation between growth conditions, biosynthetic parameters and dynamics of cytoskeleton is discussed.     

Release into culture medium of membrane-associated, tumor-specific antigen by B-16 melanoma cells.

Serum-free supernatant fluids from monolayer cultures of B-16 mouse melanoma cells were found to contain a soluble membrane associated tumor-specific antigen. The 100,000 g supernatant of the culture fluid induced an antibody response to the B-16 cells both in rabbits and in the mouse strain of origin (C57Bl/6J). Similar supernatant fluids derived from an unrelated cell line (L-929) or from normal C57Bl/6 fibroblasts did not contain the B-16 specific material. Preliminary results indicate that the B-16 specific material is a protein of low molecular weight which is released into the culture fluid chiefly by living cells and, to a lesser extent, by autolysing cells.     

Transformation of avian myogenic cultures with myelocytomatosis virus strain 29

Summary Primary cultures of proliferating chick presumptive myoblasts were exposed of either to two RNA tumor viruses and shortly thereafter treated with 5-bromodeoxyuridine (BUdR) to suppress differentiation. The effect of a Rous sarcoma virus which was temperature-sensitive for transformation (tsRSV) has been characterized previously and was used as a reference for evaluating the effect of a myelocytomatosis virus (MC29) and its helper. Two subcultures following exposure, both infected cultures were extensively transformed as indicated by cell morphology. Relaxation of the BUdR block at this time resulted in cultures which still appeared transformed and did not contain myoblast or myotube-like cells or two of their molecular markers. In contrast, uninfected controls and tsRSV-infected cultures which were shifted-up to the nonpermissive temperature produced numerous spontaneously contracting myotubes. The results confirm previous evidence that infection of presumptive myoblasts by tsRSV at the premissive temperature preserves the extant state of differentiation of presumptive myoblasts and suggest, by analogy, that MC29-infection renders a similar effect.     

Use of Refrigeration as a Practical means to Preserve Viability of in Vitro-Cultured IDE8 Tick Cells

In vitro cultivation of the IDE8 cell line, derived from embryonic Ixodes scapularis ticks, constitutes an important system for the study of tick-borne pathogens, as these cells support growth of rickettsial species which are not normally transmitted by this tick. However, since cryopreservation of IDE8 cells is not always successful, there is a need to develop alternative ways to preserve these cells. In the present study, a suspension of IDE8 cells in culture medium was kept under refrigeration at 4°C for up to 60 days. Every 15 days, the suspension was mixed and aliquots were re-cultured in 2-ml tubes, under standardized conditions. In addition, three techniques for cryopreservation, using two different cryoprotectants (DMSO and glycerol), were evaluated. Medium changes were carried out every week and subculturing every 2 weeks. The development of cultures and their respective subcultures, after returning to standard culture temperature, was evaluated by percentage viability and by cellular morphology evaluated in Giemsa-stained cytocentrifuge smears. All cultures and subcultures appeared healthy, showing growth rates comparable to cultures that had not been kept under refrigeration. The results demonstrated that storage under refrigeration at 4°C is an efficient method for preservation of IDE8 cells for up to 60 days and that refrigeration may be preferable to cryopreservation for short-term preservation of IDE8 cells.     

Initiation and characterization of a diploid cell line from larval tissues of Aedes dorsalis (Meigen).

Mosquito cell cultures were initiated from the minced tissues of newly hatched Aedes dorsalis (Meigen) larvae. Continuous cell division occurred only after an adaptive period of approximately 6 months. Optimal growth of the cells required a relatively low pH of 6.5. Karyological studies showed that the cells have remained diploid (2n = 6) for 60 serial passages and that the cultures are free of contaminating cells. The cultures also were shown to be free of bacteria (including Mycoplasma), fungi and virions. Subpopulations (strains) of the original parental cultures have been selected and characterized on the basis of morphology, karyology, growth rate and monolayer formation.     

In vitro expression of a 38,000 dalton heparin-binding glycoprotein by morphologically differentiated smooth muscle cells

In vitro, high density monolayer cultures of vascular smooth muscle cells can be induced to form multicellular nodules. The nodular cells appear to be morphologically differentiated smooth muscle cells. Sodium dodecyl sulfate (NaDodSO4)-polyacrylamide gel electrophoresis was used to compare the proteins synthesized and secreted by monolayer and nodular cultures of smooth muscle cells. Although most proteins appeared to be similar, the nodular cultures contained a unique heparin binding protein of Mr = 38,000 (38kD protein) (Millis, A.J.T., Hoyle, M., Reich, E., and Mann, D.M., 1985, J. Biol. Chem., 260:3754-3761). The 38kD protein was glycosylated and its apparent molecular weight was shifted to Mr = 32,500 after synthesis in the presence of tunicamycin or digestion with endoglycosidase F. The production of 38kD protein by nodular cell cultures did not appear to result from the degradation of a high molecular weight precursor in nodular conditioned medium. Further, it was not detected in monolayer cell conditioned medium that had been incubated with nodular cells. Finally, its synthesis was not induced in monolayer cell cultures that had been labeled in nodular cell conditioned medium. The 38kD protein appears to be uniquely associated with nodular cultures of smooth muscle cells.     

Modifications of proteoglycans produced by human skin fibroblast cultures during replicative senescence

The properties of proteoglycans (PGs) produced by normal human skin fibroblast were investigated with increasing passage. The increase of subculture number was associated with a constant increase in PG molecular size, which was particularly evident in cell layer extracts. In the cell layer, the ratio of DS-PGs/HS-PGs was markedly higher in early passage cultures. Moreover, the cell layer from young cells contained lower amounts of radioactivity incorporated into the most hydrophobic PG populations, suggesting that the PG core protein might also undergo significant modification with increasing subcultures. There was no significant difference in energy charge value between early and late passage cultures, whereas the NAD/NADH ratio was found to decrease markedly in senescent cells.     

Culture conditions affect induction of vitellogenin synthesis by estradiol-17 beta in primary cultures of tilapia hepatocytes

In vitro synthesis of vitellogenin (VTG), a female-specific protein, after estradiol-17 beta (E(2)) treatment was compared among different culture conditions using the hepatocytes of tilapia, Oreochromis mossambicus. VTG was measured by enzyme-linked immunosorbent assay. Comparison of Leibovitz's L-15 medium (L-15), Williams' medium E (WE) and Medium 199 (M199), which have been used for hepatocyte cultures in certain teleost fishes, showed that monolayer formation of the hepatocytes on the plate in WE and M199 was faster than in L-15 at the beginning of the culture. Morphological differences in the hepatocytes among the culture media were not evident by 96 h after culture. VTG synthesis in L-15 after E(2) treatment was higher than in WE and M199. A concentration of NaHCO(3) at 5 mM in L-15 resulted in faster monolayer formation of the cells and higher VTG synthesis than at 0 and 23 mM. Primary culture of the tilapia hepatocytes at 28 degrees C showed higher synthesis of VTG than at 23 and 33 degrees C. These results suggest that nutritional requirements are vitally different among species, and there are optimal ranges in the pH and the temperature in cultured hepatocytes.