pH AND POPULATION DENSITY IN THE REGULATION OF ANIMAL CELL MULTIPLICATION

Sparse and dense cultures of chick embryo cells were affected differently by pH. The rates of cell multiplication and of thymidine-³H incorporation into DNA of dense cultures were increased as the pH was increased from 6.6 to 7.6. At pH higher than 7.6 the rate of multiplication decreased slightly in the dense cultures, but the rate of thymidine-³H incorporation continued to increase. The discrepancy was due in part to cell death and detachment at very high pH, and in part to a more rapid uptake of thymidine-³H at very high pH. Sparse cultures were much less sensitive to pH reduction and, when a suitably conditioned medium was used to minimize cell damage, very sparse cultures grew almost as well at pH 6.7 as at higher pH. The rates of cell multiplication and thymidine-³H incorporation at low pH decreased in the initially sparse cultures before they reached confluent cell densities. There was no microscope evidence of direct contact between plasma membranes of cells at these densities although the parallel orientation indicated that the cells were influencing locally each other's behavior. Even at much higher cell densities, electron microscopy revealed large intercellular gaps partly filled with a fragmentary electron-opaque material suspected to be glycoprotein. Wounding experiments showed that pH affected cell migration in a manner similar to its effects on cell multiplication. Low pH inhibited cell migration, but those cells which migrated into the denuded region multiplied as rapidly at low pH as at high pH. The effects of pH on growth were correlated with effects on the uptake of 2-deoxyglucose-³H. Dense populations of cells inhibited by low pH were stimulated to incorporate thymidine-³H by the addition of small amounts of diethylaminoethyl-dextran. Rous sarcoma cells at high cell density were less sensitive to pH than were normal cells at the same density, but were more sensitive than sparse normal cultures. The results suggest that cell growth is inhibited through the combined effects of both lowered pH and high cell density on cell surface permeability.     

Differences in reactivity of confluent and nonconfluent cultures of human endothelial cells toward thrombin-stimulated platelets or heparinized salt solution

Summary This study examined whether nonconfluent endothelial cell cultures reacted differently than confluent ones toward thrombin-stimulated platelets or a heparinized salt solution. The adherence to the endothelial cell cultures of⁵¹Cr-labeled human platelets stimulated at different thrombin concentrations was studied. There was significantly higher adherence of stimulated platelets to nonconfluent cultures compared with confluent ones. This was confirmed by scanning electron microscopy, which also revealed a tendency for the platelets to adhere at the cell periphery. Electron microscopy also showed that thrombin-stimulated platelets induced endothelial cell contraction. Part of the peripheral endothelial cell surface toward the bottom of the culture dish was inverted, facing the lumen of the dish. This phenomenon was particularly seen in nonconfluent cultures. When⁵¹Cr-labeled endothelial cultures were incubated with a mildly injurious fluid as heparinized sodium acetate and 20% serum, at 20° C for 30 min, the nonconfluent cultures showed significantly more cell detachment and release of⁵¹Cr than the confluent ones. We conclude that under the conditions of the present experiments there are differences in the reactivity of confluent and nonconfluent endothelial cell cultures. These differences probably reflect biological dissimilarities. In experiments where properties of cultured endothelium are studied, care should be taken that the degree of confluency is standardized.     

Production of reovirus type-1 and type-3 from Vero cells grown on solid and macroporous microcarriers

Two strains of reovirus were propagated in Vero cells grown in stationary or microcarriers cultures. Vero cells grown as monolayers on T-flasks or in spinner cultures of Cytodex-1 or Cultispher-G microcarriers could be infected with reovirus serotype 1, strain Lang (T1L), and serotype 3, strain Dearing (T3D). A regime of intermittent low speed stirring at reduced culture volume was critical to ensure viral infection of cells in microcarrier cultures. The virus titre increased by 3 to 4 orders of magnitude over a culture period of 150 h. Titres of the T3D reovirus strain were higher (43%) compared to those of the T1L strain in all cultures. Titres were significantly higher in T-flask and Cytodex-1 microcarrier cultures compared to Cultispher-G cultures with respect to either reovirus type. The viral productivity in the microcarrier cultures was dependent upon the multiplicity of infection (MOI) and the cell/bead ratio at the point of infection. A combination of high MOI (5 pfu/cell) and high cell/bead loading (>400 for Cytodex-1 and >1,000 for Cultispher-G) resulted in a low virus productivity per cell. However, at low MOI (0.5 pfu/cell) the virus productivity per cell was significantly higher at high cell/bead loading in cultures of either microcarrier type. The maximum virus titre (8.5 x 10(9) pfu/mL) was obtained in Cytodex-1 cultures with a low MOI (0.5 pfu/cell) and a cell/bead loading of 1,000. The virus productivity per cell in these cultures was 4,000 pfu/cell. The lower viral yield in the Cultispher-G microcarrier cultures is attributed to a decreased accessibility of the entrapped cells to viral infection. The high viral productivity from the Vero cells in Cytodex-1 cultures suggests that this is a suitable system for the development of a vaccine production system for the Reoviridae viruses.     

Induction of a Stress Protein in Developing Cell Cultures of the Rat Cerebellum

Abstract: We examined the ability of developing cere-bellar cell cultures to synthesize a 71,000 MW stress protein (SP71) in response to heat shock and Cd²⁺ treatment. The induction of SP71 synthesis appeared to be dependent on both the age of the culture and the stressor used. Heat shock induced SP71 synthesis in freshly prepared cells and in cell cultures at each age examined, whereas Cd²⁺ was effective only in cultures at 7 days of age and older. These findings are discussed with reference to the development of various cell types in these cultures.     

Factors promoting the establishment of primary cultures of liver cells fromXenopus larvae

Summary The requirements for establishment and survival of primary cultures of larval amphibian liver cells were investigated.Plating efficiency was found to be enhanced by a collagen substrate, by diluted conditioned medium from an adultXenopus kidney cell line and by high initial cell densities. Plating efficiency was highest at a tonicity of 165–220 mOsm/kg. In cultures with undiluted conditioned medium the increase in cell number was 50–60% greater than in controls, where it was about 2-fold between day 3 and 6 of culture. Conditioned medium from theXenopus kidney cell line is assumed to contain at least two components, which are effective at different concentrations and stimulate either plating efficiency and cell aggregation or cell proliferation.In cultures without collagen sheets, cell flattening is greatly reduced, indicating that cell shape is also dependent upon the substrate.     

An infectious agent causing “spontaneous” degeneration of swine cells in vitro

Summary “Spontaneous” cell degeneration occurred in clone C-19 of the IB-RS-2 swine kidney cell line at around the 20th passage after each cloning. An infectious cytopathogenic agent designated AgC-19 was isolated from such degenerating cultures; the agent was studied through its inoculation into cultures of the cell clone C-12 derived from the same line. AgC-19 was identified as a medium size ribonucleic acid lipovirus belonging to the Togavirus group, and it was shown to be closely related to hog cholera virus (HCV). The evidence indicated that, most likely, AgC-19 is a cytopathogenic mutant of an attenuated HCV that persistently infects the IB-RS-2 cell cultures. After extensive destruction of the cell monolayers, a few viable cells were able to restore the cultures. Such recuperated cultures were resistant to a reinfection with AgC-19. Therefore, the persistent non-cytopathogenic infection is not able to interfere with AgC-19. On the other hand, in relation to the resistant cell cultures, either AgC-19 had selected genetically resistant cells or it had induced resistance through homologous interference.     

The effects of cell density, attachment substratum and dexamethasone on spontaneous apoptosis of rat hepatocytes in primary culture

Summary The rates of spontaneous cell detachment, cell viability, and apoptosis in primary cultures of rat hepatocytes plated at high and low density were compared. Apoptosis was frequent in detached cells, and the rates of cell detachment and apoptosis were greater in high-density than in low-density cultures. Among attached cells, more cells had condensed or fragmented nuclei in high-density than in low-density cultures. Further, ladder-like DNA fragmentation was not seen in low-cell-density cultures but was clearly evident in high-density cultures. Bax was more highly expressed in cells cultured at high density, and on collagen vs. matrigel, whereas changes of Bcl-2 and Fas expression observed in culture appeared unrelated to the rate of apoptosis. The rate of hepatocyte apoptosis appeared to be identical in low-density cultures on collagen 1 and matrigel, but when cells were cultured at high density, matrigel suppressed apoptosis by more than 50% at 36 h. In hepatocytes cultured on collagen 1, dexamethasone (0.1 μM) suppressed apoptosis in both low- and high-density cultures; higher doses had no further effects. In high density cultures, aurintricarboxylic acid (10 μM) suppressed apoptosis and this improved cell attachment at 48 h. It is concluded that cell viability in primary cultures of rat hepatocytes grown on collagen I is dependent on optimal culture density and that the cell population is regulated, at least in part, by apoptosis. Corticosteroids suppress spontaneous apoptosis of cultured hepatocytes in a non-dose-dependent manner, whereas matrigel abolishes apoptosis induced by increasing cell density. Bax may be an important protein in the cell density and cell matrix-dependent regulation of apoptosis in cultured hepatocytes.     

Investigating the establishment of primary cell culture from different abalone (<Emphasis Type="Italic">Haliotis midae</Emphasis>) tissues

The abalone, Haliotis midae, is the most valuable commodity in South African aquaculture. The increasing demand for marine shellfish has stimulated research on the biology and physiology of target species in order to improve knowledge on growth, nutritional requirements and pathogen identification. The slow growth rate and long generation time of abalone restrict efficient design of in vivo experiments. Therefore, in vitro systems present an attractive alternative for short term experimentation. The use of marine invertebrate cell cultures as a standardised and controlled system to study growth, endocrinology and disease contributes to the understanding of the biology of economically important molluscs. This paper investigates the suitability of two different H. midae tissues, larval and haemocyte, for establishing primary cell cultures. Cell cultures are assessed in terms of culture initiation, cell yield, longevity and susceptibility to contamination. Haliotis midae haemocytes are shown to be a more feasible tissue for primary cell culture as it could be maintained without contamination more readily than larval cell cultures. The usefulness of short term primary haemocyte cultures is demonstrated here with a growth factor trial. Haemocyte cultures can furthermore be used to relate phenotypic changes at the cellular level to changes in gene expression at the molecular level.     

Gametic differentiation in Chlamydomonas reinhardi: cell cycle dependency and rates in attainment of mating competency

Withdrawal of a utilizable nitrogen source during mid G₁ of the cell cycle induces gametic differentiation in synchronously grown vegetative cultures of Chlamydomonas reinhardi. Cell division accompanies gametic differentiation in such cultures, and the ability of mid G₁ vegetative cells to form gametes is matched by their ability to undergo a round of cell division after nitrogen withdrawal. Synchronously grown cultures require up to 19 hr in nitrogen-free medium to complete a round of division and to form mating-competent cells. Asynchronously grown liquid cultures require less time after nitrogen withdrawal (generally 5–8 hr) to achieve mating competency. In these cultures cell division did not necessarily accompany gametic differentiation since gametic differentiation took place in induced cultures at high cell concentrations which prevented cell division. Maximum mating competency was achieved in less than 2 hr after induction of vegetative cells grown on agar plates. Little cell division was observed during that short induction interval. The relationship between the attainment of mating competency (gametogenesis) and other physiological events resulting from nitrogen withdrawal is discussed.     

Continuous production of acetone and butanol by Clostridium acetobutylicum growing in turbidostat culture

Turbidostat cultures of Clostridium acetobutylicum were analysed with respect to their fermentation products after steady states were obtained at various cell densities. It was found that at low densities the fermentation of glucose was essentially acidogenic in nature, whereas acetone and butanol were the major end-products when the cultures were maintained at a high cell density.     

PHYSIOLOGICAL CHARACTERISTICS OF SPIRULINA PLATENSIS (CYANOBACTERIA) CULTURED AT ULTRAHIGH CELL DENSITIES1

Photosynthetic activity and growth physiology of Spirulina platensis (Nordstedt) Geitler cultures maintained at ultrahigh cell densities (i.e. above 100 mg chlorophyll-L^?1) in a newly designed photobioreactor were investigated. Nitrogen (NaNO₃) in standard Zarouk medium was characterized as a major nutrient-limiting factor in such cultures. The effect of ultrahigh cell density on photoinhibition of photosynthesis, as reflected by chlorophyll fluorescence and photosynthetic oxygen evolution, was studied: elevating the population density may arrest photoinhibition induced by high photon flux density, as well as low temperature. The relationship between incident irradiance and oxygen production rate was linear in situ for cultures at the optimal cell density, indicating that light limitation rather than light saturation or photoinhibition is the dominant condition outdoors in cultures of ultrahigh cell densities. In contrast with other reports, the extent of biomass loss at night due mainly to dark respiration was found to be relatively small when cell density was optimal, exerting only a minor effect on overall net productivity. Measurements of oxygen consumption at night revealed low rates of respiration, which may be explained by the low value of the volumetric mass transfer coefficient (K_La) of oxygen. Hence, reduced oxygen tension may play a role in preventing full expression of the respiratory potential in ultrahigh cell density cultures in which photoadaptive strategy may explain cell composition. Ultrahigh cell densities optimized with respect to the intensity of the light source, the length of the light path, and the extent of stirring represent the key for obtaining high output rates of cell mass and some natural products.     

Muscle cell cultures from chicken breast muscle have increased specific activities of creatine kinase when incubated at 41 degrees C compared with 37 degrees C

Chicken muscle cell cultures were incubated at 41 degrees C, the physiological chicken body temperature, and compared with cultures incubated at 37 degrees C, the typical cell culture incubation temperature. The cultures incubated at 41 degrees C show not only an increase in creatine kinase (CK)-specific activity but also a marked increase in the percentage of adult muscle CK isozyme (MM-CK) in 7-day muscle cultures. Muscle cell cultures incubated in the presence of cytosine arabinoside (ara-C), a cell proliferation inhibitor, do not have the mononucleated cell overgrowth seen at 41 degrees C and thus exhibit a further increase in creatine kinase-specific activity compared with cultures incubated at 41 degrees C in the absence of ara-C. These results suggest that muscle cell cultures incubated at 41 degrees C are more highly differentiated than those incubated at 37 degrees C.     

Strategies to increase the use of entomopathogens

Synchronized cultures of the TN-368 insect cell line were infected with a nuclear polyhedrosis virus from the alfalfa looper, Autographa californica, during different phases of the cell cycle. Cultures exposed to virus during the middle and late S phase have higher percentages of infected cells than cultures inoculated with virus in the G₂ phase. The amount of virus produced from each infected cell (polyhedra and plaque forming units) is not significantly different between cultures infected at all phases of the cell cycle.     

High-frequency stable transformation of cotton (Gossypium hirsutum L.) by particle bombardment of embryogenic cell suspension cultures

Stable transformation of cotton (Gossypium hirsutum L.) at a high frequency has been obtained by particle bombardment of embryogenic cell suspension cultures. Transient and stable expression of the β-glucuronidase (GUS) gene was monitored in cell suspension cultures. Transient expression, measured 48 h after bombardment, was abundant, and stable expression was observed in over 4% of the transiently expressing cells. The high efficiency of stable expression is due to the multiple bombardment of rapidly dividing cell suspension cultures and the selection for transformed cells by gradually increasing the concentrations of the antibiotic Geneticin (G418). Southern analysis indicated a minimum transgene copy number of one to four in randomly selected plants. Fertile plants were obtained from transformed cell cultures less than 3 months old. However, transgenic and control plants from cell cultures older than 6 months produced plants with abnormal morphology and a high degree of sterility. Received: 20 January 1999 / Revision received: 1 October 1999 / Accepted: 11 October 1999     

Yeasts preservation: alternatives for lyophilisation

The aim of the study was to compare the effect of two low-cost, low technology traditional methods for drying starter cultures with standard lyophilisation. Lyophilised yeast cultures and yeast cultures preserved in dry rice cakes and dry plant fibre strands were examined for viable cell counts during 6?months storage at 4 and 25?°C. None of the yeast cultures showed a significant loss in viable cell count during 6?months of storage at 4?°C upon lyophilisation and preservation in dry rice cakes. During storage at 25?°C in the dark, yeast cultures preserved in dry rice cakes, and lyophilised cultures of Saccharomyces cerevisiae and Issatchenkia orientalis showed no significant loss of viable cells up to 4?months of storage. Yeast cultures preserved in dry plant fibre strands had the greatest loss of viable count during the 6?months of storage at 25?°C. Preservation of yeasts cultures in dry rice cakes provided better survival during storage at 4?°C than lyophilisation. The current study demonstrated that traditional methods can be useful and effective for starter culture preservation in small-scale, low-tech applications.     

Effect of liver cell coat acid mucopolysaccharide on the appearance of density-dependent inhibition in hepatoma cell growth.

Yoshida ascites hepatoma 66 (AH 66) cells grown in monolayer cultures show a lack of density-dependent inhibition of growth. When acid mucopolysaccharide (AMPS) isolated from rat liver cell coats is added to growing cultures at concentrations of 50–100 μg/ml, AH 66 cell cultures became markedly inhibited and exhibited density-dependent inhibition of growth at a cell density of 19 × 10⁴ cells/cm². Inhibition reached 84% below control levels. Inhibition is a density-related phenomenon since cells at densities below 19×10⁴ cells/cm² do not exhibit inhibition of growth. AH 66 cells inhibited in the plateau state are capable of resuming growth when AMPS is removed from the cultures. When AMPS is added at a concentration of 0.5 μg/ml, growth of tumor cells is promoted. Promotion reaches 78% above control levels. It is suggested that AMPS may play an important part in the regulation of cellular proliferation.     

The chronic effects of db-cAMP on cell cycle parameters and cell size in asynchronous culture of Chinese hamster ovary cells

N⁶,O^2′-dibutyryl adenosine 3′,5′-cyclic-phosphate (db-cAMP) has been shown to convert Chinese hamster cells of ovarian origin (CHO-K₁) from compact, randomly oriented cells growing in multilayers to elongated fibroblast-like cells which grow in monolayers. This compound also has been reported to have a variety of effects on the cell cycle. Most such studies have employed synchronized cells to determine cell cycle effects, and consequently have been limited to the short-term effects of the compound. We have looked for chronic effects on the cell cycle in cultures exposed continuously to db-cAMP from the initiation of the cultures until they had reached or approached the plateau phase. This was done by combined autoradiography and Feulgen microspectrophotometry plus measurements of the protein content of mitotic cells to detect any influence on cell size. The overall results were that continuous exposure to db-cAMP had at most only minor effects on the cell cycle and cell size when the culture medium was renewed daily. Somewhat greater effects were found on plateau-phase cells in cultures in which the medium was not renewed. In this case fewer cells appeared to remain in the cell cycle in the cultures with db-cAMP. Comparison with our earlier results with Chinese hamster V79 cells led to the conclusions that cell cycle parameters and cell size at mitosis were less altered during culture growth in CHO cells, but that CHO cells seemed to be less able to maintain cells in the cell cycle in crowded cultures.     

Metabolic active-high density VERO cell cultures on microcarriers following apoptosis prevention by galactose/glutamine feeding

The control of cell death occurring in high density cultures performed in bioreactors is an important factor in production processes. In this work, medium nutrient removal or feeding was used to determine at which extension apoptosis could be, respectively, involved or prevented in VERO cell cultures on microcarriers. Glutamine and galactose present in the VERO cell culture medium was consumed after, respectively, 6 and 12 days of culture. Kinetics studies showed that fresh medium replacement and, to some extent, galactose or glutamine depleted-fresh medium replacement provided a nutritional environment, allowing the VERO cell cultures to attain high densities. Galactose was shown to be a more critical nutrient when cultures reached a high density. In agreement with that, VERO cell cultures supplemented with galactose and/or glutamine were shown to confirm previous findings and, again at high densities, galactose was shown to be a critical nutrient for VERO cell growth. These observations also indicated that in VERO cell cultures, for feeding purposes, the glutamine could be replaced by galactose. The inverse was not true and led, at high densities, to a decrease of cell viability. In the absence of glutamine and galactose, apoptosis was observed in VERO cell cultures by cytofluorometry, Acridine orange staining or light and electron microscopy, reaching high levels when compared to cultures performed with complete medium. VERO cells apoptosis process could be prevented by the galactose and/or glutamine feeding and, at high densities, galactose was more efficient in protecting the cultures. These cultures, prevented from apoptosis, were shown to synthesize high levels of measles virus following infection. Our data show that apoptosis prevention by glutamine/galactose feeding, led to high productive and metabolic active VERO cell cultures, as indicated by the high cell density obtained and the virus multiplication leading to higher virus titers.     

Process design and development of a mammalian cell perfusion culture in shake-tube and benchtop bioreactors

The development of mammalian cell perfusion cultures is still laborious and complex to perform due to the limited availability of scale-down models and limited knowledge of time- and cost-effective procedures. The maximum achievable viable cell density (VCD_max), minimum cell-specific perfusion rate (CSPR_min), cellular growth characteristics, and resulting bleed rate at steady-state operation are key variables for the effective development of perfusion cultures. In this study, we developed a stepwise procedure to use shake tubes (ST) in combination with benchtop (BR) bioreactors for the design of a mammalian cell perfusion culture at high productivity (23 pg·cell⁻¹·day⁻¹) and low product loss in the bleed (around 10%) for a given expression system. In a first experiment, we investigated peak VCDs in STs by the daily discontinuous medium exchange of 1 reactor volume (RV) without additional bleeding. Based on this knowledge, we performed steady-state cultures in the ST system using a working volume of 10 ml. The evaluation of the steady-state cultures allowed performing a perfusion bioreactor run at 20 × 10⁶ cells/ml at a perfusion rate of 1 RV/day. Constant cellular environment and metabolism resulted in stable product quality patterns. This study presents a promising strategy for the effective design and development of perfusion cultures for a given expression system and underlines the potential of the ST system as a valuable scale-down tool for perfusion cultures.