Channel Catfish Virus: a New Herpesvirus of Ictalurid Fish

Channel catfish virus was studied in ictalurid fish cell culture, the only system of fish, amphibian, avian, and mammalian cells found to be susceptible. Channel catfish virus infection resulted in intranuclear inclusions and extensive syncytium formation. Replication occurred from 10 to 33 C, but not higher. Best growth was from 25 to 33 C, and the amount of virus released nearly equalled the amount which remained cell-associated. The virus was labile to lipid solvents, and indirect determinations with labeled precursors and a metabolic inhibitor showed evidence of deoxyribonucleic acid. Electron microscopy showed progeny virus, about 100 nm in diameter, in various stages of development in cell nuclei by 4 hr. Present also were nuclear masses of exceptionally electron-dense lamellar material, with a unit dimension of 10 to 15 nm. Virus was enveloped at the nuclear membrane and in cytoplasmic vacuoles, resulting in virions having a diameter of 175 to 200 nm. Negative staining demonstrated icosehedral symmetry and 162 capsomeres. Our data indicate that channel catfish virus is a herpesvirus.     

Effect of glucocorticoid hormones on growth of human fibroblast cells and interferon production in a microcarrier culture system

Glucocorticoid hormones promoted the growth of fibroblast cells derived from human neonatal foreskins and prolonged their life span in a microcarrier culture system that used Eagle's minimum essential medium (MEM) supplemented with fetal calf serum (FCS). But, these hormones suppressed cell growth in conventional monolayer cultures. Precolostrum newborn calf serum (PNCS) was the only species that supported the serial propagation of fibroblast cells on microcarriers, possibly because of its high content of hydrocortisone (HC). Fibroblast cells grown on microcarriers in the presence of glucocorticoid hormones maintained their ability to produce interferon (IFN)-beta in a superinduction method with poly I: poly C and antimetabolites. These cells had more than 93% diploidy and no chromosomal aberration or translocation. Use of PNCS for the cultivation of human fibroblast cells has high potential for providing a microcarrier culture system for the mass production of human IFN-beta.     

A novel process for the production of a veterinary rabies vaccine in BHK-21 cells grown on microcarriers in a 20-l bioreactor

We studied BHK-21 cells growth in a 2-l bioreactor and investigated the effects of microcarrier concentration, type of growth medium, culture mode and serum concentration. The highest cell density reached was equal to 4x10(6) cells/ml and was achieved in minimum essential medium supplemented with Hanks' salts, non-essential amino acids and 5% fetal calf serum, using a perfusion culture mode and a microcarrier concentration of 4 g Cytodex 3/l. We studied rabies virus production (PV/BHK-21 strain) by BHK-21 cells grown at the optimal conditions determined previously. We analyzed the effects of multiplicity of infection (MOI) and type of medium used for virus multiplication in spinner-flasks and showed that the highest virus titer reached (when the cells were infected at a MOI of 0.3) in M199 medium supplemented with 0.2% of bovine serum albumin was equal to 8.2x10(7) Fluorescent Focus Units (FFU)/ml. When we grew the cells in a 2-l perfused bioreactor, we obtained a maximal virus titer of 3x10(8) FFU/ml. In addition, we scaled-up to a 20-l bioreactor and obtained similar results for cell density and virus titer. The experimental vaccine we developed meets WHO requirements for vaccine potency. Each run yielded about 40,000 doses of potent vaccine.     

Production of nerve growth-stimulating factor(s) from chick embryo heart cells : Use of Cytodex 3 microcarriers and serum-free media

Medium conditioned by embryonic chick heart cells is known to support extensive neurite outgrowth from autonomic and sensory neurons. In the present report we describe the use of microcarrier cell culture with serum-free media to scale up the production of the nerve growth-stimulating factors. A growth medium composed of DME /F10 supplemented with insulin, transferrin, human serum albumin and fibronectin in combination with a low molecular weight (MW) fraction of fetal calf serum (FCS) or a mixture of FGF, dexamethasone, calmodulin and thrombin supported the heart cell proliferation at a rate similar to that of medium with 10% FCS. Furthermore, the level of successively accumulated nerve growth activity measured in a bioassay with sympathetic ganglia proved to be nearly equivalent to what was obtained when cells were grown in medium containing serum. The results confirm the potential of microcarrier cell culture in serum-free media for the production and subsequent recovery of a specific cell product.     

Autoclavable low cost serum-free cell culture media. The growth of L Cells and BHK cells on peptones

The growth of L-929 cells on a series of peptones, and protein hydrolysates was examined, and it was found that when MEM was supplemented with any of a series of peptones, cell growth was about as good as when serum was used as a supplement. Protein hydrolysates did not support cell growth very well and at higher concentrations actually reduced cell growth. L-929 and L-60TM cells were grown both as monolayers or stationary suspensions and in agitated systems in MEM supplemented with 0.5—1% bactopeptone. The addition of macromolecular compounds, insulin or oleic acid had no effect on cell growth. BHK cells were also grown on media supplemented with bactopeptone but richer media (MEM-alpha, F-12, or RMPI1640) gave higher cell yields. The cells did not form the monolayers observed with fetal calf serum, but a partial suspension system. Addition of a detergent Darvan #2 gave a totally suspension culture in both stationary and agitated systems. The production of Sindbis virus in BHK cells grown in serum-free media was examined and the yield of virus was found to be about the same as that produced in serum-supplemented systems. It is estimated that the cost of cell production media could be reduced by about 90% by the replacement of serum supplement by peptones.     

Predator Avoidance of Transgenic Channel Catfish Containing Salmonid Growth Hormone Genes

Transgenic channel catfish (Ictalurus punctatus) containing salmonid growth hormone genes can grow 33% faster than normal channel catfish under aquaculture conditions. However, before transgenic catfish are released and utilized by the private sector, their genetic impact on the natural environment must be examined. Predator avoidance is one of the major fitness traits determining potential environmental risk. To determine the predator avoidance ability and growth performance of transgenic catfish in a natural habitat, various densities of transgenic and nontransgenic channel catfish were communally stocked in 0.04-ha earthen ponds without supplemental feeding. Largemouth bass (Micropterus salmoides) and green sunfish (Lepomis cyanellus) were stocked as predators. Nontransgenic fry had better predator avoidance than transgenic channel catfish when data were pooled (p < .01). When data were not pooled, nontransgenic catfish had better predator avoidance in six trials and transgenic individuals had better predator avoidance in four trials. There was no difference in predator avoidance in three trials. Overall predator avoidance was also better for nontransgenic individuals (p < .01) when the fish were evaluated as 3.5-g fingerlings, more clearly than as fry, as transgenic individuals were more vulnerable in 3 of 4 trials at this life stage. There was no significant difference in growth performance between transgenic and nontransgenic channel catfish in ponds without supplemental feeding. These findings indicate that transgenic channel catfish could be used for commercial aquaculture without affecting the natural environment. Although transgenic channel catfish may be released to nature by accident, any ecological effect would be unlikely because the increased susceptibility of transgenic channel catfish to predators would most likely decrease or eliminate the transgenic genotype. Received March 8, 1999; accepted June 3, 1999.     

A low-serum medium with BSA and ferrous sulfate for the production of tissue plasminogen activator by human embryo lung cells

A low-serum medium containing bovine serum albumin (BSA) was investigated with respect to the growth of and tissue plasminogen activator (TPA) production by human embryo lung (HEL) cells on microcarrier beads and in collagen gel. BSA and ferrous sulfate were chosen as substitutes for fetal calf serum (FCS) through a simple screening test involving many substances. The growth promoting effects of BSA and ferrous sulfate were independent of each other and from the FCS concentration. Though BSA inhibited initial cell attachment to the carrier surface, it did promote the growth of cells attached to microcarrier beads. Cells grown on microcarrier beads in the low-serum medium containing BSA, ferrous sulfate and 3% FCS produced an amount of TPA similar to that produced by ones grown in the 10% FCS medium. Although cells on the dish surface did not grow at all on serum-free media containing BSA and ferrous sulfate, cells in the collagen gel were able to grow slightly on the serum-free medium. Cells grown on the low-serum medium in collagen gel produced more TPA over a long period than those in the microcarrier beads using the low-serum medium. The optimum concentration of proteose peptone in the TPA production medium for the collagen gel culture was similar to that for the dish surface culture.     

Formation of bridges and large cellular clumps in CHO-cell microcarrier cultures: Effects of agitation dimethyl sulfoxide and calf serum

We have investigated conditions that inhibit the tendency of CHO K1 cells to form cellular bridges between microcarriers and dense clumps of cellular overgrowth in microcarrier cultures. Microcarrier aggregation by cellular bridge formation was found to occur only during periods of rapid cell growth. The level of microcarrier aggregation decreased with increasing agitation intensity. Dense masses of cellular overgrowth formed inside bridges connecting the microcarriers and in clumps that protruded off the microcarrier surface. To replace cells that were continuously sheared from the microcarriers, cell growth occurred preferentially in areas of overgrowth after confluent microcarriers were maintained in a serum-free medium. This ultimately led to poor surface coverage as bare spots developed on the microcarrier away from the areas of dense cellular overgrowth. The development of bare spots was inhibited when confluent microcarriers were maintained in medium supplemented with 1% serum. The development of cellular overgrowth was inhibited by dimethyl sulfoxide. Thus, maintaining confluent microcarriers in medium supplemented with 1% dimethyl sulfoxide and 1% calf serum resulted in microcarriers that appeared similar to monolayer cultures. There was also a decrease in bridging in cultures supplemented with either 1% calf serum or 1% dimethyl sulfoxide/1% calf serum compared to serum-free cultures.     

Purification and Quantitation of Channel Catfish (Ictalurus punctatus) Immunoglobulin M

A method is described for purification of channel catfish IgM from immune catfish serum. This should also be a feasible method for purification of IgM from other teleost fish species. The IgM concentration of adult channel catfish was determined to be 11 mg IgM per ml of serum.     

Suppression of endocytosis in polyclonally activated Con A-stimulated T lymphocytes by 25-hydroxycholesterol

A new high quality young-calf serum, Hy-clone calf serum (HcCS), was tested for use in hybridoma culture. This calf serum alone had little growth promoting activity and was much inferior to fetal calf serum (FCS). Red cell lysate (RCL) used in combination with the young-calf serum showed very good growth promoting activity. Growth was increased about threefold over that in the presence of FCS. However, HcCS and RCL could not substitute for feeder cells when hybridomas were cultured as single cells under conditions of limiting dilution. It is thought likely that the potent growth promoting factor in red cell lysate is hemoglobin.     

Serum effects on the response of mammalian cells to the exotoxins of Pseudomonas aeruginosa and Corynebacterium diphtheriae.

The response of mammalian cells to Pseudomonas and diphtheria exotoxins was studied. A method was developed whereby the sensitivity of cells to these two toxins could be quantitated. The method is versatile and can be used to study the effects of toxins on many cellular metabolic or transport processes. The type of serum used in the culture medium significantly influenced the response of cells to the toxins. Calf, horse, and human sera protected cells while fetal calf serum did not. Precipitation with (NH4)2SO4 demonstrated the probable presence of toxin-specific antibody in the protective calf serum while none was detected in the nonprotective fetal calf serum. The level of antibody in calf serum, as titrated by hemagglutination, was sufficient to account for all the observed protection. It is suggested that fetal calf serum be used for all future cell culture studies of bacterial toxins.     

5-Methylthioribose. Its effects and function in mammalian cells

The growth responses of 5-deoxy-5-methylthioribose on a 5'-deoxy-5'-methylthioadenosine phosphorylase containing cell line (BW5147) and the methylthioadenosine phosphorylase-deficient cell line (L1210D) were examined. Methylthioribose was shown to dramatically affect these cells, increasing their growth rate, saturation density, and viability. It was also found that methylthioribose could satisfy the methylthio dependence of the enzyme-deficient cell line, L1210D. A model is proposed to explain the selective growth of methylthioadenosine phosphorylase-deficient cells in medium lacking a methylthio donor but containing fetal calf serum. It is hypothesized that cellularly exported methylthioadenosine is degraded to methylthioribose in the presence of medium containing serum of high methylthioadenosine phosphorylase activity (i.e. fetal calf serum). The resultant methylthioribose can then be used to satisfy the methylthio requirement of these cells. To test this theory, various purified preparations of bovine liver methylthioadenosine phosphorylase were used to artificially increase the specific activity of methylthioadenosine phosphorylase in horse serum. In each case, it was demonstrated that only medium containing serum of enzyme activity nearly equal to that of the glutathione-stimulated fetal calf serum activity, supported the growth of methylthio-dependent cells in the absence of methylthio compounds. The data suggest that the degradation of methylthioadenosine and subsequent formation of methylthioribose represents an essential process in the growth of mammalian cells.     

Production of live attenuated varicella-zoster virus in human embryonic lung cells using microcarrier

Summary Attenuated varicella-zoster virus was propagated in human fetal embryonic lung cells grown on microcarriers to produce live attenuated varicella vaccine. We have investigated the characteristics of cell growth and virus production in microcarrier culture system at various culture conditions. The cell-associated and cell-free virus yields in microcarrier were comparable to those in the stationary tissue cultures.     

Primary suspension culture of calf anterior pituitary cells on a microcarrier surface

In order to achieve a steady-state primary culture system for mammalian cells, with the potential to eventually correlate and control cell function and growth, a serious evaluation of various suspension systems was made. Calf anterior pituitary cells were employed as a differentiated cell type and successfully cultivated in a microcarrier suspension culture system. DEAE-Sephadex was demonstrated to be a satisfactory type of microcarrier. The cells readily attached to the bead and, after a short lag period, they actively proliferated on the bead surface to yield growth of a predominantly epithelial cell type. Under specific conditions the microcarrier supported primary cell growth up to levels of 2 × 10⁶ cells per ml. High bead concentrations inhibited cell growth. The inhibition could be overcome by using proportionately higher cell inoculum so that a concentrated culture with 5 × 10⁶ cells per ml was achieved. The inhibitory effect of high bead concentration was found to be due to the absorption of serum protein and certain growth enhancing factors. The fact that the growth enhancing factors were released from cells during the period of trypsinization and were both thermostable and nondialyzable, seems to suggest one approach to a dialysis culture system. In addition, relatively trauma-free primary cell cultures can be achieved by using explant culture without prior trypsinization. In microcarrier suspensions direct growth of primary rat mammary tumor cells was also demonstrated.     

Transforming growth factor-beta1 is responsible for maturation-dependent spontaneous apoptosis of cultured gastric pit cells

In this study, we established a system of high concentration serum-dependent spontaneous apoptosis of guinea pig gastric pit cells in primary culture, which seems to mimic the spontaneous apoptosis of matured gastric pit cells at gastric surface in vivo. In addition to induction of the spontaneous apoptosis, cell growth was inhibited in the presence of 10% serum compared with 0.5% serum. Transforming growth factor-beta1 (TGF-beta1), which is known to cause both apoptosis and growth inhibition in mammalian cells, was present in serum of both fetal calf and guinea pig. The addition of recombinant TGF-beta1 to the culture medium containing 0.5% fetal calf serum caused both induction of apoptosis and inhibition of cell growth. On the other hand, immunodepletion of TGF-beta1 from fetal calf serum caused inability to induce both the spontaneous apoptosis and inhibition of cell growth. These data suggest that TGF-beta1 is involved in the spontaneous apoptosis of guinea pig gastric pit cells in primary culture.     

Comparisons of microcarrier cell culture processes in one hundred mini-liter spinner flask and fifteen-liter bioreactor cultures

Microcarrier cell culture process can be used to culture anchorange-dependent cells in large bioreactor vessels. The process performance in large bioreactors is usually less prominent than that in spinner flask vessels and bench scale reactors. In this study we investigated the microcarrier cell culture processes in 100?ml spinner flask and 15-liter bioreactor cultures, including the kinetics for cell attachment, cell growth and the production of Japanese encephaltilis vaccine strain (Beijing-1) virus. Under a fixed concentration of microcarrier and cell density used in inoculations, the attachment kinetics of Vero cells on Cytodex 1 microcarrier in a 15-liter bioreactor vessel was 2 folds slower than with 100?ml spinner flask culture. Virus replication in 15-liter bioreactor culture also revealed an approximately one day lag-time compared to 100?ml spinner flask culture. Findings presented herein provide valuable information for designing and operating microcarrier cell culture processes in large bioreactor vessels.     

Selection of microcarrier diameter for the cultivation of mammalian cells on microcarriers

The kinetics of mammalian cell growth in a microcarrier culture are affected by the distribution of cells on microcarriers. It has been shown previously that a critical cell number per microcarrier is required for the growth of FS-4 cells on microcarriers. It is advantageous to alter the cell distribution on microcarriers to allow for a larger fraction of microcarriers to acquire enough cells to initiate normal growth. This can be achieved by selecting the diameter of the microcarriers employed. It has also been shown previously that the critical cell number could be reduced by choosing a better culture medium to support low density growth. However, even if all cells inoculated into a culture are capable of growing to confluence, it is still necessary to select the microcarrier diameter ration ally to improve the growth kinetics. The method of selecting the microcarrier diameter is discussed. By employing a improved medium as well as using microcarriers of selected diameter, the multiplication ratio was in creased to 15- to 16-fold for FS-4 cells, as opposed to 3- to 4-fold typically obtained in a batch culture.     

Hybridoma cell growth and anti-neuroblastoma monoclonal antibody production in spinner flasks using a protein-free medium with microcarriers

The main disadvantages of foetal calf serum as the world-wide common serum supplement for cell growth are its content of various proteins of variable concentrations between batches as well as its high cost. The use of serum-free and protein-free media is gradually becoming one of the goals of cell culture especially for standardizing culture conditions or for simple purification of cell products like monoclonal antibodies. The mouse hybridoma cells 14/2/1 were cultivated either in protein-free UltraDOMA medium or in serum-containing RPMI medium with and without microcarriers to generate high quantities of monoclonal antibodies against neuroblastoma tumour cells. Cell growth rate, IgG production, viability, glucose and lactate concentrations, attachment rate and doubling time have been used as investigation criteria. Modifications of culture procedures (static or stirred), inoculum density, and microcarrier concentration caused an improvement of monoclonal antibody production. The kinetics of antibody synthesis was best in spinner culture with 2 ml of microcarriers in protein-free medium. These results of short-term microcarrier culture in stirred spinner flasks indicate that IgG yields in protein-free medium 2.5-fold higher to those in serum-supplemented medium can be achieved.     

原代地鼠肾细胞及狂犬病毒不同培养方式的研究

目的通过比较原代地鼠肾细胞在转瓶、微载体、细胞工厂的3种培养方式的培养效果,为原代地鼠肾细胞选择一种易扩大规模、培养高质量细胞的培养方式,进而提高狂犬病毒的产量。方法消化取得的细胞悬液分别在转瓶、微载体、细胞工厂中培养,通过显微镜观察细胞形态、计数等结果比对培养的差别。结果细胞工厂可以很好地培养原代地鼠肾细胞和狂犬病毒;而细胞在微载体上贴附性差,生长不好。结论实验结果表明细胞工厂可以取代转瓶,用于大规模培养原代地鼠肾细胞扩大狂犬病毒的产量。     

Factors affecting cytotrophoblast cell viability and differentiation: Evidence of a link between syncytialisation and apoptosis

A relationship between cytotrophoblast differentiation (syncytialisation) and apoptosis is hypothesised to exist, but has not been clearly determined. To address this, we explored the effects of cAMP, an inducer of syncytialisation, on human choriocarcinoma cell differentiation and viability under three different culture conditions related to diverse survival status: no serum, 10% fetal calf serum or 10% charcoal-stripped fetal calf serum. 8-Br-cAMP increased BeWo cell viability in culture media without serum, but viability was decreased in a dose- and time-dependent manner when serum was present. The appearance of apoptotic nuclei fragments were only observed when BeWo cells were cultured in media containing serum combined with 8-Br-cAMP treatment. In addition, the ratio of FasL to Fas expression following treatment with 8-Br-cAMP increased by 20-fold in 10% charcoal-stripped fetal calf serum media and 65-fold 10% fetal calf serum media, and activation of caspase-3 also required media with serum. The markers of syncytialisation (syncytin 1 expression and human chorionic gonadotropin secretion) were induced significantly by 8-Br-cAMP, and were higher in 10% fetal calf serum media than in 10% charcoal-stripped fetal calf serum media, than in the absence of serum. Syncytia formation was stimulated by 8-Br-cAMP and this required serum in the media. We now show that factors contained within serum are necessary for cAMP-stimulated cytotrophoblast differentiation, that syncytialisation involves apoptotic events, and that a lack of serum based factors could switch the cellular program away from differentiation.