Use of a stationary bed reactor and serum-free medium for the production of recombinant proteins in insect cells.

Insect cells (Spodoptera frugiperda) have been cultured in a stationary bed reactor, packed with a fibrous polyester carrier. When the bioreactor was perfused with serum-supplemented medium, a cell density of 6 x 10(6) cells ml-1 packed carrier was reached. Scanning electron microscopy investigations have shown that the insect cells grew along the three-dimensionally oriented fibers of the Fibra-cel carrier. After infection of the logarithmically growing cells with a recombinant baculovirus (Autographa californica) containing the gene coding for beta-galactosidase, the medium in the bioreactor was changed to serum-free medium. At day 13 postinfection (p.i.), a beta-galactosidase level of 320 microgram ml-1 and, at day 17 p.i., a virus titer of 2.1 x 10(8) TCID50 units ml-1 (day 17 p.i.) were reached. In another bioreactor, operated in a similar way but with serum-containing medium, a beta-galactosidase concentration of 360 microgram ml-1 and a virus titer of 2.3 x 10(8) TCID50 units ml-1 were obtained. These results indicate the potential use of this production system for the production of recombinant protein and baculovirus in insect cells.     

Improvements in the Growth of BHK-21 Cells in Submerged Culture

A medium is described which will support the submerged culture of BHK-21 cells to 6.5 x 10(6) to 7 x 10(6) cells/ml in volumes up to 30 liters. Shaken-flask cultures were used to determine nutrient deficiencies in depleted medium. The final medium became limited by glutamine at 6.5 x 10(6) to 7 x 10(6) cells/ml, but increasing the glutamine concentration failed to improve cell yields. The value of the polyol Pluronic F68 as a protective substance is illustrated.     

Growth and differentiation of stage 24 limb mesenchyme cells in a serum-free chemically defined medium

A serum-free defined medium which supports the differentiation of chick limb mesenchymal cells has been developed. In this medium, stage 24 embryonic limb mesenchymal cells which are plated at high density (5 x 10(6) cells/35-mm culture dish) differentiate into chondrocytes. Morphologically, these cultures appear only slightly different from those in which the cells are maintained in serum-containing medium. DNA levels and proline incorporation in cultures grown in defined medium are indistinguishable from control cultures. The rate of radiolabeled sulfate incorporation, a monitor of the rate of proteoglycan synthesis, in Day 8 high-density cultures maintained in defined medium is approximately 70-80% of control values. Additionally, growth and differentiation of intermediate-density (2 x 10(6) cells/35-mm culture dish) and low-density (1 x 10(6) cells/35-mm dish) cultures are also supported by this defined medium. The availability of this medium allows exploration of bioactive factors which affect or modulate mesenchymal cell differentiation and subsequent development.     

Establishment and characterization of human colonic and gastric cancer cell lines

Two human cancer cell lines, DAIT-6 from a colonic cancer and IT-25 from a gastric cancer, derived from xenografts in nude mice have been established in tissue culture and maintained for over two years. In tissue culture, DAIT-6 cells grew in a monolayered sheet with a population doubling time of about 45.0 hr, and floated or piled up to form small buds above the monolayered surface in relatively confluent cultures. Chromosomal counts ranged from 40 to 108 with a modal number of 59. The cells secreted CEA (1.7 ng/1 x 10(6) cells/24 hr) and CA19-9 (540.5 u/1 x 10(6) cells/24 hr) in spent medium. The IT-25 cells grew in a monolayered sheet with a population doubling time of about 57.8 hr in tissue culture. The IT-25 cells also secreted CEA (0.5 ng/1 x 10(6) cells/24 hr) and CA19-9 (120.0 u/1 x 10(6) cells/24 hr) in spent medium. The xenografts for DAIT-6 and IT-25 in nude mice were histopathologically classified as a moderately differentiated tubular adenocarcinoma and a well differentiated tubular adenocarcinoma, respectively.     

Quantitative nucleic acid changes during phytohaemagglutinin-induced lymphocyte transformation in vitro. Dependence of the response on phytohaemagglutinin/serum ratio

1. Pig and human blood lymphocytes have been grown in culture without replenishment of medium, and stimulated to transform by phytohaemagglutinin. Quantitative nucleic acid changes during this process have been used as an index of transformation. 2. On the first day, cells attach to glass; then they detach and continue transforming. 3. The degree of transformation is dependent on the phytohaemagglutinin/serum ratio, and is independent of cell concentration within the range 0.5x10(6)-2.0x10(6) cells/ml. 4. At low phytohaemagglutinin/serum ratios there is no response; at high phytohaemagglutinin/serum ratios, inhibition appears after the cells have been cultured for a day.     

Role of vitamin B 6 biosynthetic rate in the study of vitamin B 6 synthesis in Escherichia coli

Nutritional auxotrophs of Escherichia coli synthesize vitamin B(6) compounds at a rate of 1 x 10(-10) to 2 x 10(-10) moles per hr per mg (dry weight) of cells when they are suspended in minimal medium lacking their required nutrients. A few auxotrophs have been found to stop or reduce vitamin B(6) synthesis during such an experiment. These include thiamineless, citrate synthaseless, and pyridoxineless mutants as well as mutants which require four carbon compounds for growth. Glycolaldehyde was found to restore vitamin B(6) synthesis in the last named of these mutants without restoring normal growth. A class of pyridoxineless mutants which responded with normal growth to 0.4 mm glycolaldehyde or 0.15 x 10(-3) mm pyridoxol was also found. The results suggest that a thiamine pyrophosphate-requiring step as well as glycolaldehyde may be involved in pyridoxal phosphate biosynthesis.     

Effect of ammonia production on intracellular pH: Consequent effect on adenovirus vector production

Recombinant adenoviral vectors (AdV) have proven to be highly efficient for the delivery and expression of foreign genes in a broad spectrum of cell types and species both for vaccination and gene therapy in a number of specific applications. In this study, the effect of ammonia production on intracellular pH (pH(i)) and consequently inhibition of AdV production at high cell densities is assessed. Different specific ammonia production rates were obtained for 293 cells adapted to grow in glutamate supplemented medium (non-ammoniagenic medium) as compared with 293 cells growing in glutamine supplemented medium (ammoniagenic medium); pH(i) was observed to be lower during cell growth and AdV production at both high and low CCI in the ammoniagenic medium, where the specific ammonia production rate is higher. In addition, after infection at CCI of 3x10(6)cell/ml, the cell viability decreased significantly in the ammoniagenic medium, attributed to the activation of an acidic pathway of apoptosis. Furthermore, AdV DNA was observed to be degraded at the observed pH(i) in the ammoniagenic medium, decreasing significantly the amount of AdV DNA available for encapsulation. To elucidate the pH(i) effect upon AdV production, 293 cells were infected at a CCI of 1 x 10(6)cell/ml in the non-ammoniagenic medium with a manipulated pH(i) as observed at the time of infection at CCI of 3 x 10(6)cell/ml in the ammoniagenic (pH(i) 7.0) and non-ammoniagenic (pH(i) 7.3) media; AdV volumetric productivities were observed to be lower when the cells were exposed to the lower pH(i). Thus, the importance of controlling all the factors contributing to pH(i) on AdV production, such as ammonia production, has been established.     

A new sensitive flow-injection chemiluminescence method for the determination of H(2)-receptor antagonists.

Based on the chemiluminescence (CL) intensity generated from the potassium ferricyanide [K(3)Fe(CN)(6)]-rhodamine 6G system in sodium hydroxide (NaOH) medium, a new sensitive flow-injection chemiluminescence (FI-CL) method has been developed, validated and applied for the determination of three kinds of H(2)-receptor antagonists: cimetidine (CIMT), ranitidine (RANT) hydrochloride and famotidine (FAMT). Under the optimum conditions, the linear range for the determination was 1.0 x 10(-9)-7.0 x 10(-5) g/ml for CIMT, 1.0 x 10(-9)-5.0 x 10(-5) g/mL for RANT hydrochloride and 5.0 x 10(-9)-7.0 x 10(-5) g/mL for FAMT. During 11 repeated measurements of 1.0 x 10(-6) g/mL sample solutions, the relative standard deviations (RSDs) were all <5%. The detection limit was 8.56 x 10(-10) g/mL for CIMT, 8.69 x 10(-10) g/mL for RANT hydrochloride and 2.35 x 10(-9) g/mL for FAMT (S:N = 3). This method has been successfully implemented for the analysis of H(2)-receptor antagonists in pharmaceuticals.     

昆虫细胞（Sf21）悬浮培养过程中生长限制性基质间歇补加技术的 …

基于ｒ２１昆虫细胞在浮过程中所表现出的生长代谢特征,提出以培养液中残糖浓度作为控制参数,并利用限制性基质（葡萄糖和蛋白水解物）的间歇补加技术调控细胞生长的方案。实际控制表明：与批培养相比,Ｓ１ｆ２１细胞在两种具代表性的昆虫水解物）的间歇补加调控细胞生长的方案。实际控制表明：与批培养相比,Ｓｆ２１细胞在两种具代表性的昆虫细胞培养基（ＩＰＬ－４１和ＴＣ－１００）中的生长期和稳定期部都到有效的延长。ＴＣ     

Studies on the Growth in Culture of Plant Cells: X. FURTHER STUDIES ON THE CONDITIONING OF CULTURE MEDIA BY SUSPENSIONS OF ACER PSEUDOPLATANUS L. CELLS

The standard synthetic culture medium (Stuart and Street, 1969)has been modified by adjustment of its initial pH to 6.4 andby the addition of gibberellic acid (0.25 mg/l) and of a mixtureof 15 L-amino acids formulated from an analysis of the conditionedmedium. The minimum effective density for the growth of sycamorecell suspensions in the standard medium is 9–15 x 10³cells ml^–1, for the modified synthetic medium it is 2.0x 10³ cells ml^–1, and for conditioned medium 1.0–1.25x 10³ cells ml^–1. Using either conditioned medium (Stuart and Street, 1969) orthe modified synthetic medium it is demonstrated that the growthof cultures initiated at low density is enhanced by a volatilefactor released from actively growing cell suspensions. In presenceof conditioned medium and this volatile factor cultures canbe established from stationary-phase cells at a density of 6x 10² cells ml^–1. The volatile factor can be absorbedin 40 per cent w/v KOH but attempts to replace the factor byair containing carbon dioxide at concentrations up to 5 percent have so far been unsuccessful.     

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.     

Evaluation of Methods for Reestablishment of L-Cell Suspension Cultures Directly from Liquid Nitrogen Stored Stocks

Methods were developed and evaluated for the preservation of tissue cells grown in suspension culture and the reestablishment of suspension cultures directly from inoculum stored at -175 C. The factors investigated were processing pH, temperature of processing, freezing medium, and method of inoculation of the starter suspension cultures from the frozen stock (-175 C). Three parameters, cell viability, cell size, and growth potential in suspension culture after freezing, were used to evaluate the various factors. The results indicate that cells processed at 4 C, frozen at 1 C per min to -50 C in a medium containing 5% dimethyl sulfoxide plus 10% bovine serum at concentrations of 2 x 10(7) to 4 x 10(7) cells/ml, and stored at -175 C will reestablish suspension cultures directly from frozen seed. A 1-ml amount of frozen stock inoculated into 99 ml of medium routinely produced 2 x 10(6) to 3 x 10(6) viable cells/ml (2 x 10(8) to 3 x 10(8) total cells) in suspension culture in 4 to 5 days. Inoculum preserved by this procedure grew equally well in either serum-free or serum-containing growth medium.     

In-vitro penetration of pig oocytes matured in culture by frozen-thawed ejaculated spermatozoa.

Pig oocytes matured in culture were inseminated with frozen-thawed ejaculated spermatozoa without preincubation in modified tissue culture medium (TCM) 199. High penetration rates (85-89%) and increased incidence of polyspermy were obtained at 25-100 x 10(6) spermatozoa/ml. Wide variation in penetration rates (16-89%) was observed in oocytes inseminated in medium containing 5mM caffeine and at 25-50 x 10(6) spermatozoa/ml obtained from 6 boars, regardless of sperm motility. At 25-50 x 10(6) spermatozoa/ml, penetration rates of oocytes were dependent upon the concentration of caffeine in the medium: there was no penetration without caffeine, but penetration was highest (89%) with 5mM caffeine. None of the oocytes was penetrated in the medium supplemented with heparin at 5-40 micrograms/ml. When heparin was included in the medium with 5mM caffeine, it inhibited the efficacy of caffeine to promote sperm penetration of oocytes.     

Preparation of Poliovirus Labeled with Phosphorus-33

Phosphorus-33 ((33)P), a weak (0.25 Mev) beta-emitting isotope of phosphorus with a half-life of 25 days, has been used to label poliovirus in cell culture. HeLa cell monolayers were depleted of phosphate and then labeled by incubating at 37 C in a medium (LM) containing about 10 muCi of (33)P as orthophosphate per ml. Labeled cells were infected at a high multiplicity with poliovirus type 1 and incubated for 8 hr in LM medium. Virus from infected cells was then concentrated and purified. Virus purity was confirmed by comparison of virus infectivity and radioactivity after CsCl density gradient centrifugation and by observing purified virus preparations with electron microscopy. With the method described, yields of about 10(10) to 5 x 10(10) plaque-forming units (PFU) of highly purified poliovirus with specific activities of about 3 x 10(-4) to 10(-3) disintegrations per min per PFU have been obtained from 1.5 x 10(8) to 3.0 x 10(8) HeLa cells.     

Levels of true thymidine kinase and nucleoside phosphotransferase in two strains of Tetrahymena pyriformis under different growth conditions.

Activities of typical thymidine kinase and nucleoside phosphotransferase are both present in logarithmically growing tetrahymena pyriformis, GL-1 and ST strains, contrary to previous reports. 2. Activities of thymidine kinase and nucleoside phosphotransferase are also found in both GL-1 and ST strains grown in the defined medium, PPL medium and Neff's medium. 3. The specific activities of both enzymes are very much influenced by the growth state. Both the specific activities of thymidine kinase and nucleoside phosphotransferase decrease steadily from the start of the experiments when the cell numbers were about 2-3 x 10(4) cells/ml in the PPL medium, while in the Neff's medium, the specific activities of thymidine kinase increase up to when the cell numbers reached 3-5 x 10(5) cells/ml and then decreased, but the specific activities of nucleoside phosphotransferase continuously decreased when the cell concentrations were 2-6 x 10(4) cells/ml. 4. In the PPL medium, the final cell numbers reached are about 6.5 x 10(5) cells/ml, while in the Neff's medium, the cell numbers increase further (to about 2 x 10(6) cells/ml). 5. No striking difference in activities of thymidine kinase and nucleoside phosphotransferase was observed when the cells were transferred from the defined medium to the Neff's medium, contrary to that reported by others for the activity of thymidylate synthetase.     

Bovine colostrum or milk as a serum substitute for the cultivation of a mouse hybridoma

A mouse-mouse hybridoma was grown in serum-free medium supplemented with bovine milk or colostrum. Bovine colostrum supported growth of the hybridoma whereas bovine milk alone did not support cellular proliferation. For growth in medium supplemented with colostrum, the maximum cell concentration achieved was 1.4 x 10(6) cells/mL in 2.2% colostrum, which is 44% of that obtained in 9% serum. When cells were grown in media containing milk and low amounts of serum (<1%) the maximum cell concentration in 2.2% milk with 0.4% serum was 2 x 10(6) cells/ml, whereas it was only 0.2 x 10(6) cells/ml and 1.3 x 10(6) cells/ml in 2.2% milk alone and 0.4% serum alone, respectively. Similar behavior was observed for growth in media containing colostrum and low amounts of serum. The monoclonal antibody production in media containing combinations of serum and milk or colostrum was comparable to that obtained in media with higher serum concentrations. Experiments performed with conditioned media suggest that the rapid decrease in viability, after the maximum cell concentration has been reached, is partially due to the presence of some inhibitory components generated during the cell culture rather than due to depletion of some serum components.     

High-Density culture of mouse-human hybridoma in serum-free defined medium

Mouse-human hybridoma 4H11 cells producing anti-Pseudomonas sp. monoclonal antibody (IgA) grew in a serum-free medium supplemented with insulin, transferrin, ethanolamine, and selenite (ITES). The hybridoma could be applied to high-density culture in a serum-free medium supplemented with ITES, 0.5% BSA, egg yolk VLDL, and artificial blood FC-43 in a culture vessel equipped with hollow-fiber modules for medium exchange. Total cell density reached 1.1 x 10(7) cells/mL (viable cell density was 7.6 x 10(6) cells/mL), and the IgA productivity was around 20 mug/10(6) cells/day in the serum-free medium, which corresponded to the levels in serum-supplemented medium.     

Effect of oviductal epithelial cells on fertilization of pig oocytes in vitro

The incidence of polyspermy is reduced by co-culture of pig oocytes with oviductal cells. It is not known whether the effect is due to soluble factors secreted into the medium. Oviductal epithelial cell monolayers and cell-conditioned media were prepared and their effects on fertilization of pig oocytes were examined. In vitro matured pig oocytes were inseminated with ejaculated boar spermatozoa at a concentration of 1 x 10(5) or 1 x 10(6) cells/ml and co-cultured in one of 5 culture systems: an oviductal epithelial cell monolayer, a fibroblast monolayer, an oviductal epithelial cell-conditioned medium, or a fibroblast-conditioned medium, and medium alone (modified-TCM199). In all 5 systems, the majority (range 85 to 100%) of the oocytes were penetrated by sperm. When oocytes were inseminated with spermatozoa at a concentration of 1 x 10(5) cells/ml, the percentages of monospermic oocytes were significantly higher in the oocytes co-cultured with oviductal epithelial cells and fibroblasts than that of the oocytes cultured without these cells. In contrast, when oocytes were inseminated with spermatozoa at a concentration of 1 x 10(6) cells/ml, the percentages of monospermic oocytes were significantly higher in the oocytes co-cultured with epithelial cells than those cultured with the fibroblasts and in the control medium. The suppressive effect on polyspermy was observed in the oviductal epithelial cells-conditioned medium when oocytes were inseminated with spermatozoa at both concentrations of 1 x 10(5) and 1 x 10(6) cells/ml. The effect was absent in the fibroblasts-conditioned medium. Moreover, the effect of the epithelial cells was maintained during the culture period, whereas the proportion of monospermic oocytes co-cultured with fibroblasts showed a gradual decrease, reaching 0% after 16 h. These results suggest that a soluble factor(s) derived from the oviductal epithelial cells decreased the number of spermatozoa penetrating the oocytes without suppressing the high rate of fertilization.     

Quantitative analysis of low-resistance junctions between cultured cells and correlation with gap-junctional areas

Electrophysiological studies of low-resistance junctions between Novikoff hepatoma cells grown in suspension cultures were carried out and correlated with gap-junctional areas per inferface determined by freeze-fracture. The mean coupling coefficient between isolated cell pairs was 0.773 +/- 0.025 (SEM) in 67G medium and 0.653 +/- 0.028 in M67 medium; the respective means for the central pairs of four-cell chains were 0.714 +/- 0.034 and 0.595 +/- 0.026. Mean estimates of nonjunctional resistances for cell pairs were 3.0 +/- 0.32 x 10(7) ohm (67G) and 2.01 +/- 0.01 x 10(7) ohm (M67), and the respective estimates for specific nonjunctional resistances were 158.6 +/- 8.1 ohm-cm2 (67G) and 133.0 +/- 812 ohm-cm2 (M67). Mean estimates of junctional conductances were 0.409 +/- 0.058 x 10(-6) mho (67G) and 0.211 +/- 0.018 x 10(-6) mho (M67) for pairs and 0.291 +/- 0.063 x 10(-6) mho (67G) and 0.212 +/- 0.04 mho (M67) for four-cell chains. The mean area of gap junction per interface for separate cell populations was 0.187 +/- 0.049 micron 2 and 0.269 +/- 0.054 micron 2 for cells fixed in loose pellets and in suspension, respectively. When compared with the mean junctional conductance, these values gave specific junctional conductance estimates of 1.13 x 10(2) mho/cm2 and 0.78 x 10(2) mho/cm2, respectively. These values are higher than most previous estimates, but are consistent with the hypothesis that gap-junctional particles contain central hydrophilic channels, about 2 nm in diameter, which have cytoplasmic resistivity.     

Cryopreservation of epididymal spermatozoa collected by needle biopsy from cynomolgus monkeys (Macaca fascicularis)

We have examined the motility, morphology, and cryopreservation of epididymal spermatozoa collected by needle biopsy from cynomolgus monkeys (Macaca fascicularis). At collection, epididymal sperm (23 x 10(6) +/- 4 x 10(6) sperm/sample; 611 x 10(6) +/- 116 x 10(6) sperm/ ml; n = 18) were alive (79 +/- 2%), motile (67 +/- 2%), and exhibited intact membranes (65 +/- 2%). Sperm maintained at room temperature in handling medium exhibited decreased motility over time, but head-to-head agglutination was limited. Tris egg-yolk extender containing 6% glycerol and dimethylsulfoxide (DMSO) did not significantly affect functional morphology, whereas extender containing propanediol significantly reduced motility, survival, and membrane integrity. Cryostorage reduced all measures of functional morphology independent of cryoprotectant. Post-thaw motility was superior for glycerol and DMSO compared to propanediol. Variation in glycerol concentration (4, 6, and 8%) produced equivocal effects on sperm functional morphology post-thaw. Needle biopsy may be a useful technique for laboratory and field-based collection of spermatozoa from nonhuman primates.