Assessment of virus production and chloramphenicol acetyl transferase expression by insect cells in serum-free and serum-supplemented media using a temperature-sensitive baculovirus

Spodoptera frugiperda insect cells were grown in Sf-900 serum-free medium and two kinds of serum-supplemented media (IPL -41 and Grace's). The specific growth rates of uninfected cells were found to be 0.024, 0.35, and 0.034 h(-1) respectively, at 33 degrees C. The IPL -41 medium supported to highest maximum cell density (10.6 x 10(6) cells/mL) compared to 3.5 x 10(6) and 8.7 x 10(6) cells/mL with the Grace's and serum-free media, respectively. In temperature shifdown experiments with a temperature-sensitive baculo-virus (acts10YM1CAT), virus titer and chloramphenicol acetyl transferase (CAT) expression were highest in the IPL -41 (5.1 x 10(7) PFU/mL and 20000 U/mL). Use of Grace's medium gave higher virus titers than the serum-free medium (4.4 x 10(6) vs 4.1 x 10(5) PFU/mL) as well as higher CAT titers (7050 vs 1980 U/mL). Interestingly, in the three media used, the highest virus and CAT titers were obtained at MOI (multiplicity of infection) of 0.02 At MOI of 2.0 virtually no increase in virus of CAT titer was observed. This result is contrary to those obtained at constant-temperature (27 degrees C) infection and cell culture, in which higher virus titers and recombinant protein expression and obtained at higher MOI.     

Recombinant protein production in insect cell cultures infected with a temperature-sensitive baculovirus

Spodoptera frugiperda (IPLB-SF-21) insect cells were grown in shake-flasks and infected with a temperature-sensitive baculovirus to express the gene of chloramphenicol acetyl transferase (CAT) in serum-free medium (SF-900) and two serum-supplemented media (IPL-41 and Grace's). In temperature-shift experiments (cell growth at 33°C followed by virus replication at 27°C 3–4 days later), virus and CAT production were much poorer in the serum-free medium than in serum-supplemented media, though cell growth was virtually the same in the different media tested. In all the three media, highest virus and CAT titers were obtained at the lowest MOI (multiplicity of infection 0.02). This result is contrary to that obtained in constant-temperature culture (27°C for both cell growth and virus replication). Virus and CAT production was greatly improved when the entire culture was run at constant temperature. It appeared that infected cells were severely damaged at 33°C (6°C above the optimal 27°C), resulting in little or no virus and protein production. As a result of these temperature-shift experiments, a larger-scale (141 air-lift bioreactor) serum-free culture of Sf-9 insect cells was conducted at constant temperature (27°C) to produce recombinant protein (β-galactosidase). A cell density as high as 1×10⁷ cells.ml⁻¹, and a β-gal concentration of up to 104,000 unit.ml⁻¹ were achieved.     

Fed-batch culture of insect cells with exponential feeding of amino acid and yeastolate solution

Amino acids rather than sugars are the primary limiting substrates for the culture of insect cells in a Grace's medium. When cultures are supplemented with amino acids, the yeastolate components other than the amino acids become the secondary limiting substrates. For the fed-batch culture of insect cells, a solution containing concentrated amino acids and yeastolate was supplied using an exponential feed flow rate calculated from mass balance equations. During the batch period the specific growth rate was 0.02 h^у, whereas during the fed-batch period it was measured as 0.007 and 0.012 h^у on the basis of the cell numbers and the dry cell weight, respectively. This difference in the specific growth rates in the fed-batch period is caused by an increase in the cell size during this period. Furthermore, in fed-batch cultures, dissolved oxygen was found to be a limiting factor for high cell-density cultures.     

Physiological functions of hemocytes newly emerged from the cultured hematopoietic organs in the silkworm, Bombyx mori

Abstract  Cellular immunity is a very important part of insect innate immunity. It is not clear if hemocytes entering the hemolymph require a maturation process to become competent. The establishment of a tissue culture system for the insect hematopoietic organs would enable physiological function assays with hemocytes newly emerged from hematopoietic organs. To this end, we established a hematopoietic organ culture system for the purebred silkworm pnd pS and then studied the physiological functions of the newly emerged hemocytes. We found that Grace's medium supplemented with 10% heated silkworm larval plasma was better for culturing the hematopoietic organs of pnd pS . Newly emerged hemocytes phagocytosed propidium iodide-labeled bacteria and encapsulated the Iml-2 coated nickel beads as well as pupal tissue debris. This culture system is therefore capable of generating physiologically functional hemocytes. These hemocytes can be used to study the mechanisms of the hemocyte immune response among others.     

Trypanosoma theileri: In vitro cultivation in tsetse fly and vertebrate cell culture systems

Epimastigote forms of Trypanosoma theileri were grown at 25°C in insect cell culture media and in Glossina tissue cultures for more than 6 months. Doubling times of 10–14 h during exponential growth were observed. In cell cultures which had been derived from pupal tsetse flies growth rates were higher than in cell free media; in a larval cell line, however, growth of T. theileri was inhibited. Ecdysteroids and juvenile hormone I reduced multiplication of T. theileri in cell free media. When T. theileri was incubated in different sera only fetal calf serum (FCS) supported growth. Epimastigote forms transformed into trypomastigote bloodforms when cultured at 37°C in FCS, vertebrate cell cultures, and Eagle's medium, but not in insect media or Glossina cell cultures. Oxygen uptake of epimastigotes could be inhibited by rotenone antimycin A and cyanide; trypomastigotes were not affected by these inhibitors.     

Isolation and primary culture of viable multicellular endothelial isolates from hard corals

Summary Conditions for the primary culture of branching scleractinian coral (Acropora micropthalma and Pocillopora damicornis) cells were established with a calcium-free seawater cell dissociation method. Cells were isolated and cultured in supple-mented Dulbecco’s modified Eagle media with heat-inactivated fetal bovine serum, antibiotics, and sterile seawater. Among the isolated cell types, large (60–100 μm) multicellular endothelial isolates (MEIs) were seen in high numbers. These isolates were observed to continually spin for up to 300 h without media change. The following parameters were optimized: media, serum, light, trace elements, and growth factor supplements. Rotations per minute were calculated to determine MEI motility in relation to size. Finally, analyses of external and internal structures were conducted with scanning electron microscopy, transmission electron microscopy, and fluorescence microscopy. Additional coral species, Montipora digitata, Stylophora pistillata, Seriatopora hystrix and Porites sp. were also cultured to determine the applicability of isolation techniques. The relatively long survival time of MEIs in primary culture makes them ideal candidates for in vitro studies examining coral disease processes (e.g., mode of infection and intracellular effects of disease-causing agents) as well as aspects of general coral growth and health (e.g., trace element requirements and transfer of products between host cell and zooxanthellae).     

Growth of enteric neurones from isolated myenteric ganglia in dissociated cell culture

Summary Ganglia of the myenteric plexus from the newborn guinea-pig, isolated by microdissection, were dissociated by a combination of enzymatic and mechanical methods. The neurones and glial cells in the resulting cell suspension were cultured for up to 21 days in vitro. The growth of the enteric ganglion cells in serum-free, hormone-supplemented (N1) medium and in serum-supplemented medium containing a mitotic inhibitor was compared over a period of 14 days in vitro. Enteric neurones were outnumbered by glia in both culture media, although glial cell proliferation was inhibited in both media compared with that in serum-supplemented medium without mitotic inhibitors. Glial cell numbers appeared to decline in serum-free medium after the first week in vitro. Neurites tended to be more varicose in the serum-free medium, and the morphology of the enteric glial cells also differed markedly in the two media. This is the first report of the dissociation and subsequent culture of myenteric ganglia that had previously been completely isolated from the remainder of the gut wall.     

Optimization of a feed medium for fed-batch culture of insect cells using a genetic algorithm

Insect cells have been cultured for over 30 years, but their application is still hampered by low cell densities in batch fermentations and expensive culture media. With respect to the culture method, the fed-batch culture mode is often found to give the best yields. However, optimization of the feed composition is usually a laborious task. In this report, the successful use of genetic algorithms (GAs) to optimize the growth of insect cells is described. A feed was developed from 11 different medium components, each used at a wide range of concentrations. The feed was optimized within four sets of 20 experiments. The optimized feed was tested in bioreactors and the addition scheme was further improved. The viable-cell density of HzAm1 (Helicoverpa zea) insect cells improved 550% to 19.5 x 10(6) cells/mL compared to a control fermentation in an optimized commercial medium. No accumulation of waste products was found, and none of the amino acids was depleted. Glucose was depleted, which suggests that even further improvement is possible. We show that GAs are a successful method to optimize a complex fermentation in a relatively short time frame and without the need of detailed information concerning the cellular physiology or metabolism.     

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.     

Maintenance of the primary symbiote of the pea aphid Acyrthosiphon pisum in liquid media

The primary symbiote of the pea aphid was maintained in liquid culture media for 12 hr for the purpose of optimizing certain culture parameters. The optimum temperature for protein and nucleic acid synthesis was found to be 28–30°C. There was no dependence of this microorganism on fetal calf serum added to the growth medium. Grace's insect tissue culture medium and Grace's insect tissue culture medium (modified by Tokumitsu and Maramorosch) were found to be best suited for the maintenance of this organism.The suitability of the growth medium for the microorganism was directly related to the age of the culture medium.     

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.     

Effects of medium alterations on in vitro development of Brugia malayi larvae in cultured mosquito thoraces.

A recent study showed that 1-day-old, intracellularly lodged larvae of Brugia species develop in vitro to the infective third-stage larvae (L3) in excised thoraces of susceptible mosquitoes in the diphasic insect tissue culture medium containing a nutrient agar base overlaid with a 1:1 mixture of Schneider's Drosophila medium and Grace's insect cell culture medium supplemented with 20% fetal bovine serum (FBS) and antimicrobial agents. In the present investigation, the diphasic culture medium was used to evaluate the effects of medium alterations on the development of 1-day-old, intracellularly lodged larvae of subperiodic Brugia malayi in excised thoraces of Aedes aegypti to the L3. One-day-old larvae developed to the L3 in medium without nutrient agar base, at pH 7.0 and pH 7.5, in Hanks' balanced salt solution (HBSS) and in HBSS supplemented with bovine albumin fraction-V (BAF-V). These larvae also developed in the absence of FBS in the overlay medium, in overlay medium containing 5-20% FBS, in medium components obtained from different sources, in serum free Sf-900 (GIBCO) medium, and when FBS is replaced by BAF-V in the overlay medium. The percentage of L3 was not increased substantially in infected excised thoraces of mosquitoes when nutrient supplements, such as folic acid, p-aminobenzoic acid, glucose, lipid concentrate, hemin, or reduced glutathione, were added to the overlay medium containing BAF-V. These results suggested that 1-day-old, intracellularly lodged larvae developed to the L3 in infected excised thoraces of mosquitoes at almost the same rate as in intact mosquito, when excised thoraces were maintained alive under optimal conditions in a culture medium.     

In Vitro Culture of Phytomonas Sp. Isolated from Euphorbia characias. Metabolic Studies by 1H NMR

ABSTRACT. We describe the in vitro culture of Phytomonas species isolated from Euphorbia characias . The best choice between tested media was SDM-79, in which promastigotes, after 6 days of culture, reached cell densities as high as 4 × 10⁷ cells/ml. Cells growing in LIT or MTL medium showed longer division times and lower cell densities. We succeeded in obtaining Phytomonas sp. amastigote and spheromastigote forms in modified GRACE's medium, yielding transformation rates of up to 70%. Electron microscopy studies were performed in order to characterize the ultrastructural features of these forms obtained in vitro. On the other hand, metabolic studies based on qualitative (nuclear magnetic resonance spectroscopy) and quantitative metabolic methods (enzymatic assays) showed that promastigote forms secreted mainly ethanol, acetate, glycine, glycerol, piruvate and succinate in SDM-79 medium, whereas the major metabolites found after transformation in modified Grace's medium were ethanol, acetate, glycine, piruvate and smaller amounts of glycerol.     

A primary culture of haemocytes isolated from Gryllus bimaculatus (Orthoptera, Gryllidae) and their interactions with two intracellular parasites--Paranosema grylli (Microsporidia) and Adelina grylli (Coccidia)

Cricket haemocytes were derived from either haemolymph or haemopoietic organs (lymph glands) of insects and introduced to a primary culture. Varied isolation protocols, tissue culture vessels, media compositions and cell densities were tested to determine the optimal conditions for in vitro maintenance of haemocytes, and for subsequent light and electron microscopic analysis of monolayers. Freshly prepared Mitsuhashi and Maramorosh (MM;Sigma, Steinheim, Germany) insect medium (420 mOsm), buffered with sodium bicarbonate (pH 7.2) and supplemented with 10 % FCS, was found to be most appropriate for haemocyte maintenance. All tested tissue culture vessels (FLEXiperm units, multiwell plates and Thermanox slides, with the exception of Melineux agar plates), were suitable for cell attachment and haemocyte monolayers formation. Viability of cultured cells was confirmed by LIVE/DEAD Viability/Cytotoxity Kit for Eukaryotic Cells. Free circulating haemocytes were cultivated up to 27 days and then degraded. Infection with the microsporidian Paranosema grylli or the coccidian Adelina grylli caused noticeable swelling of host lymph glands (haemopoietic tissue) and increase in the number of cells comprising the glands. The cells derived from haemopoietic tissue were maintained for maximum 5 days; thereafter multiplication of bacteria normally inhabiting cricket lymph glands destroyed monolayers and killed the cells. Microsporidian and coccidian invasive stages (spores and sporozoites, respectively) were isolated from infected tissues, resuspended in MM medium and added to haemocyte monolayers in ratios 1 zoite per haemocytes or 10 spores per 1 haemocyte. Actively moving zoites contacted and penetrated the cultured cells. Unlike coccidian zoites, microsporidian spores were phagocytized by haemocytes. Application of fluorescent LIVE/DEAD kit allowed to visualize internalized parasites inside host cells as clearly shaped dark areas. The present study has demonstrated that 1) cricket haemocytes from both circulating haemolymph and lymph glands can be short-term cultivated on tissue culture vessel surfaces which made possible their further light and electron microscopic analysis; 2) short-term haemocyte cultures may be employed to study host-parasite interactions, in particular, to follow the initial steps of parasite internalization inside host cell; 3) Fluorescent assay with Viability/Cytotoxity Kit for Eukaryotic Cells (Molecular Probes, Oregon) allows to observe penetration of these parasites into cultured cells.     

Growth of an ovarian cell line of Galleria mellonella and its response to immune-inducing factors

Antibacterial proteins are produced in the reproductive tracts of some insect species. The advent of a pupal ovarian cell line of the lepidopteran Galleria mellonella offered an opportunity for exploring the use of ovarian tissue culture to induce antimicrobial proteins in lieu of the larvae. The ovarian cell growth rates and cell yields were maximized by adjusting Grace's medium to pH 6.5, adding 15% (v/v) qualified heat-inactivated fetal calf serum, and lowering the sucrose concentration to 9.3 g/L. Five cell forms and biochemical profiles of the collective cell types were analyzed throughout the culture growth cycle. The final modified culture medium did not affect morphogenesis, whereas it increased the culture growth rate by 50% and the final cell yield threefold. The molting and immunoprotein-inducing hormone, 20-hydroxyecdysone, increased culture growth rate and altered the levels of cell types A and D. Neither 20-hydroxyecdysone nor the larval immunizing agents, apolipophorin-III or Bacillus subtilis, in combination or alone, induced antibacterial activity. The bacterium did induce immunity in both larval and adult stages.     

Construction of an apparatus for perfusion cell cultures which enables in vitro experiments under organotypic conditions.

The value of cultured cells in cell biological, pharmaceutical or biotechnological research depends on the degree of terminal cell differentiation. In conventional Petri dishes or tissue culture plates it is often difficult to achieve culture conditions which resemble the in situ situation of intact tissue, as regards optimal cell adhesion, exchange of nutrients and metabolic products. These limitations prompted us to develop simple laboratory tools which optimize the environment of cultured cells. A perfusion apparatus with various culture containers and compatible cell holder sets was constructed which allows the simulation of organotypic conditions. (i) The cells can be kept on individual and interchangeable support materials for an optimal cell attachment. (ii) Culture medium can be perfused during the whole culture period. (iii) One type of the new culture container can be perfused with different media at the apical and basal side of the cells, thus mimicking the organotypic environment that applies for epithelial monolayers. Cell culture experiments with renal collecting duct epithelia exhibited an excellent morphological appearance showing typical features of principal and intercalated cells.     

Practical methods for handling human periodontal ligament stem cells in serum-free and serum-containing culture conditions under hypoxia: implications for regenerative medicine

Stem cell-based therapies depend on the reliable expansion of patient-derived mesenchymal stem cells (MSCs) in vitro. The supplementation of cell culture media with serum is associated with several risks; accordingly, serum-free media are commercially available for cell culture. Furthermore, hypoxia is known to accelerate the expansion of MSCs. The present study aimed to characterize the properties of periodontal ligament-derived MSCs (PDLSCs) cultivated in serum-free and serum-containing media, under hypoxic and normoxic conditions. Cell growth, gene and protein expression, cytodifferentiation potential, genomic stability, cytotoxic response, and in vivo hard tissue generation of PDLSCs were examined. Our findings indicated that cultivation in serum-free medium does not affect the MSC phenotype or chromosomal stability of PDLSCs. PDLSCs expanded in serum-free medium exhibited more active growth than in fetal bovine serum-containing medium. We found that hypoxia does not alter the cell growth of PDLSCs under serum-free conditions, but inhibits their osteogenic and adipogenic cytodifferentiation while enabling maintenance of their multidifferentiation potential regardless of the presence of serum. PDLSCs expanded in serum-free medium were found to retain common MSC characteristics, including the capacity for hard tissue formation in vivo. However, PDLSCs cultured in serum-free culture conditions were more susceptible to damage following exposure to extrinsic cytotoxic stimuli than those cultured in medium supplemented with serum, suggesting that serum-free culture conditions do not exert protective effects against cytotoxicity on PDLSC cultures. The present work provides a comparative evaluation of cell culture in serum-free and serum-containing media, under hypoxic and normoxic conditions, for applications in regenerative medicine.     

Growth and differentiation of alveolar bone cells in tissue-engineered constructs and monolayer cultures

The ability to enhance bone regeneration by implanting autologous osteoblasts in combination with an appropriate scaffold would be of great clinical interest. The aim of our study was to compare the growth and differentiation of alveolar bone cells in tissue-engineered constructs and in monolayer cultures, as the basis for developing procedures for routine preparation of bone-like tissue constructs. Alveolar bone tissue was obtained from four human donors and explant cultures of the cells were established. Expanded cells were seeded on macroporous hydroxyapatite granules, and cultured in medium supplemented with osteogenic differentiation factors for up to 3 weeks. Control monolayer cultures were established in parallel, and cultured in media with or without osteogenic supplements. Cell proliferation, alkaline phosphatase (AP) activity and gene expression of AP, osteopontin and osteocalcin were determined under different culture conditions at weekly intervals. Cells in tissue constructs exhibited growth patterns similar to those in control monolayer cultures: enhanced proliferation was noted during the first 2 weeks of cultivation, followed by a decrease in cell numbers. AP activity at 3 weeks was higher in all cultures in osteogenic medium than in control medium. Gene expression levels were stable in monolayer cultures in both types of media whereas, in tissue constructs, they exhibited patterns of osteogenic differentiation. Light and scanning electron microscopy examination of the cell-seeded constructs showed uniform cell distribution, as well as cell attachment and growth into the interior region of the hydroxyapatite granules. Our results show that bone-like constructs with viable cells exhibiting differentiated phenotype can be prepared by cultivation of alveolar-bone cells on the tested hydroxyapatite granules.     

Three-dimensional culture of human tumor cells under standardized medium conditions

The 3-dimensional culture of human tumor spheroids under standardized medium conditions may reveal information on specific biological parameters that could be masked in serum-supplemented media. Spheroids derived from human tumor cells are growth retarded in media free of serum. Ex-Cyte IV is a substance derived from human blood that can be used to improve growth in tissue culture. In this study the growth of spheroids from four different human tumor cell lines was studied when grown in medium free of serum, medium supplemented with varying concentrations Ex-Cyte IV, and medium supplemented with foetal calf serum (FCS). The parameters used for comparisons were growth rate, growth enhancement, clonogenicity and cell cycle distribution.The four cell lines showed different growth rates in serum-free medium, which were increased to different extents when Ex-Cyte IV or FCS were added. The growth enhancing effect induced by Ex-Cyte IV was differently concentration dependent for each cell line. The clonogenicity of cells grown as spheroids in serum-free medium was lower than in spheroids grown in supplemented media. There was no difference in clonogenicity between the differently supplemented media. All four cell lines responded to growth in serum-free medium with a drop in the S-phase and G2M phase.The present study provides a novel approach to the study of human tumor cells in 3-dimensional culture under defined conditions. The human serum derived substance Ex-Cyte IV may provide a method to obtain information on specific biological parameters that could be masked in serum-supplemented media.     

Maintenance of midgut epithelial cells from dendroctonus valens (coleoptera: Scolytidae) in vitro

This article describes the culture of epithelial cells from anterior and posterior midgut regions of adult Dendroctonus valens. Culture conditions were established, and cell adherence was improved by means of a new technique that allowed the cells to grow between two glass coverslips. Cytoplasmic projections occur as anterior midgut cells grow to confluence; these projections were not observed in cells of the posterior midgut. The optimal culture medium for the maintenance of these epithelial cells was Roswell Park Memorial Institute 1640 medium at 25 degrees C. Cells in Grace's medium died in 24 h. Cultures did not require CO(2) atmosphere, but culture development was favored by the microaerophilic environment and the dark conditions in which the cells were grown, between the coverslips.