Long-term culture of human endothelial cells

Human umbilical vein endothelial cells can be grown in vitro for 28 passages (CPDL 58) in Medium 199 supplemented with newborn bovine serum and a partially purified growth factor derived from bovine brain. Newborn bovine serum is superior to fetal bovine serum for the proliferation of human umbilical vein endothelial cells seeded at low density in the presence of the growth factor. The endothelial cells, which can be passaged every 7 to 10 d at a 1-to-5 split ratio, retain their morphological and biochemical characteristics. The proliferation of cells seeded at low density (10(3)/cm2) is proportional to the concentration of the growth factor present in the medium. The growth factor, which has an isoelectric point between 5.0 and 5.5, can support cell proliferation at reduced serum concentrations; half-maximal growth is achieved in medium containing the growth factor and 3% serum. The brain endothelial cell growth factor does not stimulate DNA synthesis significantly in cultures of human skin fibroblasts.     

Karyometric observations of WISH cell cultures irradiated with 3 GHz microwaves.

WISH cell cultures 24 hours after passage were irradiated with 3 GHz microwaves (10 cm) at far field conditions in free space (anechoic chamber) for 30 minutes, at field power density 5 or 20 mW/cm2. Within 1,24 and 48 hours of the exposure to microwave fields the volumes of nuclei and nucleoli were measured with the use of a micrometer, and logvolumes and nucleo-nucleolar ratios were calculated. Under the applied irradiation conditions the culture medium temperature did not exceed 37 degrees C. In cultures irradiated at field power density 20 mW/cm2 increased number of cells with small nuclei and enlarged nucleoli was noted within 1 hour of the exposure. Within 24 and 48 hours after irradiation the nucleolar volume showed a slight decrease, whereas the nuclear volume increased. In cultures irradiated at field power density 5 mW/cm2 increased numbers of cells with enlarged nuclei and nucleoli were found. Analysis of the distribution curves of nuclear and nucleolar volumes suggests that non-thermal power densities of microwaves stimulate the metabolism of cell cultures. However, at higher power densities (20 mW/cm2) the stimulation phase is preceded by a period of reduced viability of cell cultures.     

Cryopreservation and in vitro expansion of chondroprogenitor cells isolated from the superficial zone of articular cartilage

Understanding the proliferation mechanisms of chondroprogenitor cells and their influence on cell differentiation is crucial in order to develop large-scale expansion processes for tissue engineering applications. Proliferation control mechanisms were mainly attributed to substrate limitation and cell-cell contact inhibition. The limiting substrates were found to be components of the FCS, with an optimal proliferation rate achieved in the presence of 40% FCS. In addition, the medium supply rate was found to be essential in reducing substrate limitation. In terms of FCS, 10 microL FCS cm(-2) h(-1) was the threshold feed rate required to prevent substrate limitation. Above this rate, maximum cell densities of 5.3 x 10(5) cells/cm2 were achieved, representing a 53-fold expansion. To reduce the need for high supply rates, the effect of specific growth factors was also investigated. Cell densities of 3.3 x 10(5) cells/cm2 were achieved in batch cultures using 40% FCS and 1 ng/mL TGF-beta1. Chondroprogenitor cells were expanded in this medium up to three passages without compromising their ability to differentiate and produce cartilage-like matrix in pellet cultures. In addition to substrate limitation, cell-cell contact, even at very sparse subconfluent densities, appeared capable of exerting some degree of growth inhibition. The cells exhibited deceleratory growth kinetics, characterized by a decrease of specific growth rates over time.     

The effect of pH on potentially lethal damage recovery in A549 cells

The radiation sensitivity and potentially lethal damage recovery (PLDR) capacity of A549 human lung carcinoma cells have been studied. For unfed monolayer cultures, radiation sensitivity was greater in plateau phase than in log phase of growth. PLDR was observed when plateau-phase cells were held in their own spent medium postirradiation, such that the dose-response curve with 24 h holding was similar to that for log-phase cells plated immediately after irradiation. The high PLDR capacity of A549 plateau-phase cells (recovery factor between 40 and 70 for 24 h holding after 10 Gy) was reduced 10-fold or more by alkalinizing the pH of the spent medium immediately after irradiation from a value of 6.5 +/- 0.1 to a value of 7.6. Medium alkalinization resulted in an increase in the rate of glycolysis, with subsequent reacidification to a pH of 7.3 within 2 h of the pH adjustment. No change in cell cycle distribution was observed in the plateau-phase cultures up to 32 h after change of medium pH, and no increase in cell density was found after 48 h. A slight increase in the rate of incorporation of radiolabeled thymidine into acid-precipitable material was observed at 4 and 24 h after alkalinization of the medium. While it is not possible at present to define a mechanism for this pH effect, our results demonstrate that, at least for this cell line, variables such as medium pH and glucose concentration can profoundly influence the observation of PLDR.     

Modulation of cell attachment to culture support by pH, fibronectin, hemin, and cobalt protoporphyrin

When chick embryo fibroblasts were seeded in the presence of minimum essential medium supplemented with 1% (v/v) horse serum, the rate of cell attachment, after 1 hr incubation, was less than 5% at pH 6.5 and about 50% and 80% at pH 7.5 and pH 8.3, respectively. If, however, cultures were pretreated with fibronectin, a substance that promotes cell adhesion, a high rate of cell attachment was also observed at pH 6.5. Two other compounds of totally different chemical nature, cobalt-protoporphyrin (CoPP) and hemin, also enhanced cell attachment at pH 6.5. CoPP was shown to increase the synthesis of proteins, but it did not affect the intracellular heme content of cells incubated at pH 6.5. The possibility that CoPP, and presumably also hemin, induce cell attachment by promoting the synthesis of a fibronectin like protein is discussed.     

The proliferative and morphologic responses of a colon carcinoma cell line (LIM 1215) require the production of two autocrine factors.

The role of autocrine growth factors in tumor cell growth has been difficult to prove. Our results indicate that more than one autocrine factor is required for the autonomous growth of the LIM 1215 colonic carcinoma cell line. Furthermore, the morphologic changes induced by epidermal growth factor (EGF) are also density dependent and appear to require a synergistic autocrine factor. The serum-free proliferation of the colonic carcinoma cell line LIM 1215 depends on cell density and the presence of EGF (A. Sizeland, S. Bol, and A.W. Burgess, Growth Factors 4:129-143, 1991). At cell densities below 10(4)/cm2, conditioned medium (from cells at a density of 10(5)/cm2) was required for the cells to elicit a mitogenic response to exogenous EGF. At higher cell densities (10(5)/cm2), the cells were independent of both exogenous EGF and conditioned medium. In addition, the EGF receptor was found to be phosphorylated on tyrosine in LIM 1215 cells proliferating at high density, suggesting that the autocrine production of transforming growth factor alpha (TGF alpha) and subsequent ligation to the EGF receptor was occurring. The proliferation of cells at high density was partly inhibited by TGF alpha antibodies but was almost completely inhibited by an antisense oligonucleotide to TGF alpha. The antisense inhibition could be overcome by the addition of EGF, indicating that the effect of the antisense TGF alpha oligonucleotide was on the production of autocrine TGF alpha. LIM 1215 cells were also observed to undergo morphologic changes (spreading and actin cable organization) in response to EGF. These changes were density dependent, but they occurred with a cell density dependence different from that of the proliferative response. These results suggest two possibilities: that the morphologic changes and proliferative responses have different sensitivities to the autocrine factors or that the actions of the autocrine factors are mediated through different signal transduction pathways.     

Seeding density modulates migration and morphology of rabbit chondrocytes cultured in collagen gels

The cultures of rabbit chondrocytes embedded in collagen gels were conducted to investigate the cell behaviors and consequent architectures of cell aggregation in an early culture phase. The chondrocyte cells seeded at 1.0 x 10(5) cells/cm(3) underwent a transition to spindle-shaped morphology, and formed the loose aggregates with a starburst shape by means of possible migration and gathering. These aggregates accompanied the poor production of collagen type II, while the cells seeded at 1.6 x 10(6) cells/cm(3) exhibited active proliferation to form the dense aggregates rich in collagen type II. Stereoscopic observation was performed at 5 days to define the migrating cells in terms of a morphology-relating parameter of sphericity determined for individual cells in the gels. The frequency of migrating cells decreased with increasing seeding density, while the frequency of dividing cells showed the counter trend. The culture seeded at 1.0 x 10(5) cells/cm(3) gave the migrating cell frequency of 0.25, the value of which was 25 times higher than that at 1.6 x 10(6) cells/cm(3). In addition, the analysis of mRNA expression revealed that the chondrocyte cells seeded at 1.0 x 10(5) cells/cm(3) showed appreciable down-regulation in collagen type II relating to differentiation and up-regulation in matrix metalloproteinases relating to migration, as compared to the cells seeded at 1.6 x 10(6) cells/cm(3). These data supports the morphological analyses concerning the cell migration and aggregate formation in the cultures with varied seeding densities. It is concluded that the seeding density is an important factor to affect the cell behaviors and architecture of aggregates and thereby to modulate the quality of cultured cartilage.     

Behavior of confluent endothelial cells after irradiation. Modulation of wound repair by heparin and acidic fibroblast growth factor

Image analysis was used to study the repair process of a circular mechanical lesion of confluent human endothelial cells in culture after irradiation (10 Gy) prior to wounding. Coverage of denuded areas 48 and 96 h after injury of endothelial cells was identical in control and irradiated cultures, although the labeling index was lowered by 80 to 95% in irradiated cultures. The cell density of non damaged irradiated areas was decreased by 50%. When cultures were submitted to increasing doses of radiation (5.0-30 Gy), the labeling index of the cells diminished rapidly between 0 and 5.0 Gy and reached a plateau at 10 Gy. The decrease in cell density (50% of control at 96 h) was identical at each dose of radiation. Thus cell migration alone could be sufficient for the repair of the lesion, while cell proliferation would mainly maintain the original cell density. The addition of heparin to the culture medium slowed down cell migration and proliferation, but the speed of repair was identical in irradiated and non-irradiated cultures. Acidic fibroblast growth factor plus heparin accelerated equally the repair process whether the cultures were irradiated or not. In irradiated cultures the presence of acidic fibroblast growth factor and heparin maintained cell density in confluent areas at a level similar to that in non-irradiated damaged control cultures without addition of mitogens. Thus heparin and acidic fibroblast growth factor play a role in cell proliferation, in the maintenance of the cell monolayer integrity and in restoring a continuous layer by rapid cell migration and elongation after irradiation.     

Behavior of confluent endothelial cells after irradiation. Modulation of wound repair by heparin and acidic fibroblast growth factor

Image analysis was used to study the repair process of a circular mechanical lesion of confluent human endothelial cells in culture after irradiation (10 Gy) prior to wounding. Coverage of denuded areas 48 and 96 h after injury of endothelial cells was identical in control and irradiated cultures, although the labeling index was lowered by 80 to 95% in irradiated cultures. The cell density of non damaged irradiated areas was decreased by 50%. When cultures were submitted to increasing doses of radiation (5.0–30 Gy), the labeling index of the cells diminished rapidly between 0 and 5.0 Gy and reached a plateau at 10 Gy. The decrease in cell density (50% of control at 96 h) was identical at each dose of radiation. Thus cell migration alone could be sufficient for the repair of the lesion, while cell proliferation would mainly maintain the original cell density. The addition of heparin to the culture medium slowed down cell migration and proliferation, but the speed of repair was identical in irradiated and non-irradiated cultures. Acidic fibroblast growth factor plus heparin accelerated equally the repair process whether the cultures were irradiated or not. In irradiated cultures the presence of acidic fibroblast growth factor and heparin maintained cell density in confluent areas at a level similar to that in non-irradiated damaged control cultures without addition of mitogens. Thus heparin and acidic fibroblast growth factor play a role in cell proliferation, in the maintenance of the cell monolayer integrity and in restoring a continuous layer by rapid cell migration and elongation after irradiation.     

Effects of cell density and cell growth alterations on cyclic nucleotide levels in cultured human diploid fibroblasts.

cGMP and cAMP concentrations were studied in cultures of two strains of normal human diploid lung fibroblasts, WI38 and KL-2, under various conditions which alter growth rate. Higher levels of cAMP were found in fibroblasts grown in medium with low (0.1 – 1.0%) serum concentration and thus exhibiting a decreased rate of growth. A rise in cAMP also preceded the decreased growth rate when medium was not changed for 4 days or longer (starvation). The reinitiation of cell growth by addition of fresh medium containing the standard 10% serum to either starved or serum-restricted cells was preceded by a rapid drop in cAMP level. Cellular cAMP levels increased to a moderate extent as sparse cultures first increased in density, but did not continue to rise as the culture approached saturation density. cGMP levels were inversely related to cell density: much higher cellular cGMP levels were found at low density than at higher cell density, whether cells were rapidly proliferating under standard growth conditions or had their growth arrested by omission of medium change or restriction of serum. Thus, under these conditions the steady state levels of cGMP appear to be related to cell density rather than rate of cell proliferation. However, a transient but appreciable increase in cGMP did occur upon the addition of fresh medium containing 10% serum to starved or serum-restricted cells, a condition leading to reinitiation of cell proliferation. Smaller but significant increases in cGMP were also evident following routine addition of fresh medium with serum to growing cells fed every other day and following mild EDTA-trypsin treatment of confluent WI38 fibroblasts. Thus, at least dual control mechanisms appear to be involved in the regulation of cGMP levels. Comparison of mid- and late-passage WI38 cells revealed no significant differences either in the levels of cGMP at sparse densities or in the density-dependent change in levels. These results suggest that levels of both cAMP and cGMP are influenced by cell density and also by conditions which alter the rate of cell proliferation.     

Ultrasonic cell separator as a cell retaining device for high density cultures of plant cell

A system of ultrasonic filter device consisted of an ultrasonic generator, ultrasonic cell separation chamber (resonator) and a guide column, which was developed for suspension cultures of a plant cell. The key operation parameters affecting the efficiency of separation of cells from medium fluid were found to be the voltage of ultrasonic generator, the convective flow rate, and the distance between transducer and reflector. In the high density cultures ofAloe saponaria (>17 g DCW/L), the ultrasonic filter was so efficient that the cell holding time in the separation chamber was 10-fold higher than the case without ultrasonic wave at a convective flow rate of 0.24 cm/min. Furthermore, in perfusion type of high cell density cultures, cell aggregates were observed to be densely held in the ultrasonic chamber by ultrasonic force overcoming both gravitational and drag forces by pump. The accumulated cells were finally overflowed after the holding capacity of the chamber was reached. Back pressure was applied periodically to the resonator to flush cells back to bioreactor. The ultrasonic cell separator could operate over 75 min at a convective flow rate of 0.1 cm/min and at a cell concentration of 17 g DCW/L.     

Effects of cell density and cell proliferation on acid cholesterol esterase and cathepsin activity of cultured human skin fibroblasts

We tested the effects of fibroblast cell density and proliferation on the activities of acid cholesterol esterase and cathepsins, the lysosomal enzymes which degrade low-density lipoprotein. Rates of cell proliferation were increased by: (1) fibroblast conditioned medium, (2) increasing the time since subculture from 3 to 7 days, and (3) decreasing the plating density of cells. Cathepsin activity was consistently decreased as cellular proliferation was increased by these various methods. Changes in acid cholesterol esterase activity were more variable. For example, acid cholesterol esterase activity was consistently a positive function of cell density only at densities under 3 micrograms protein/cm2, while cathepsin activity increased up to densities of 16 micrograms protein/cm2. However, the activities of both enzymes were lower at cell densities of under 3 micrograms cell protein/cm2 compared to confluent cultures. Sparse fibroblast cultures may provide a unique model system to study low-density lipoprotein metabolism since, at low cell density, LDL receptor activity is high while lysosomal activity is low, making it possible that lysosomal degradation could become the rate-limiting step in the process of LDL degradation rather than receptor-mediated internalization of the lipoprotein. This might then allow an accumulation of lipoprotein-derived cholesteryl esters in the cell. Such a model could be relevant to the propensity of arterial cells to become foam cells during atherogenesis.     

Defined protein-free NS0 myeloma cell cultures: stimulation of proliferation by conditioned medium factors

A chemically defined, protein-free, and animal-component-free medium, designated RITM01, has been developed for NS0 myeloma cells. The basal medium used was a commercial serum-free and protein-free hybridoma medium, which was supplemented with phosphatidylcholine, cholesterol, beta-cyclodextrin, and ferric citrate. Increasing the amino acid concentration significantly improved cell growth. An NS0 cell line, constitutively producing a human IgG1 antibody, reached a peak cell density of 3 x 10(6) cells mL(-1) in this medium. The antibody yield was 195 mg L(-1) in batch culture, which is a 3-fold increase compared to that of a standard serum-supplemented medium, even though the cell yield was the same. The increase in antibody yield was a consequence of a longer growth phase and a slight increase in specific antibody production rate at low specific proliferation rates. Adaptation of the NS0 myeloma cell line to the protein-free conditions required about 3 weeks before viability and cell densities were stabilized. Most probably, changes in gene expression and phenotypic behavior necessary for cell survival and proliferation occurred. We hypothesize that mitogenic factors produced by the cells themselves are involved in autocrine control of proliferation. To investigate the presence of such factors, the effect of conditioned (spent) medium (CM) on cell growth and proliferation was studied. Ten-fold concentrated CM, harvested at a cell density of 2 x 10(6) cells mL(-1), had a clear positive effect on proliferation even if supplied at only 2.5% (v/v). CM was found to contain significant amounts of extracellular proteins other than the antibody. Fractionation of CM on a gel filtration column and subsequent supplementation of new NS0 cultures with the individual fractions showed that factors eluting at 20-25 kDa decreased the lag phase and increased the peak cell density as compared to control cultures. Identification of autocrine factors involved in regulation of proliferation may lead to completely new strategies for control of growth and product formation in animal cell processes.     

Influences of construct properties on the proliferation and matrix synthesis of dedifferentiated chondrocytes cultured in alginate gel

To investigate whether the chondrocytes-alginate construct properties, such as cell seeding density and alginate concentration might affect the redifferentiation, dedifferentiated rat articular chondrocytes were encapsulated at low density (LD: 3 x 10(6) cells/ml) or high density (HD: 10 x 10(6) cells/ml) in two different concentrations of alginate gel (1.2% or 2%, w/v) to induce redifferentiation. Cell viability and cell proliferation of LD culture was higher than those of HD culture. The increase in alginate gel concentration did not make an obvious difference in cell viability, but reduced cell proliferation rate accompanied with the decrease of cell population in S phase and G2/M phase. Scan electron microscopy observation revealed that chondrocytes maintained round in shape and several direct cell-cell contacts were noted in HD culture. In addition, more extracellular matrix was observed in the pericellular region of chondrocytes in 2% alginate culture than those in 1.2% alginate culture. The same tendency was found for the synthesis of collagen type II. No noticeable expression of collagen type I was detected in all constructs at the end of 28-day cultures. These results suggested that construct properties play an important role in the process of chondrocytes' redifferentiation and should be considered for creating of an appropriate engineered articular cartilage.     

Toxicity of Al to Desulfovibrio desulfuricans

The toxicity of Al to Desulfovibrio desulfuricans G20 was assessed over a period of 8 weeks in a modified lactate C medium buffered at four initial pHs (5.0, 6.5, 7.2, and 8.3) and treated with five levels of added Al (0, 0.01, 0.1, 1.0, and 10 mM). At pH 5, cell population densities decreased significantly and any effect of Al was negligible compared to that of the pH. At pHs 6.5 and 7.2, the cell population densities increased by 30-fold during the first few days and then remained stable for soluble-Al concentrations of <5 x 10(-5) M. In treatments having total-Al concentrations of > or =1 mM, soluble-Al concentrations exceeded 5 x 10(-5) M and limited cell population growth substantially and proportionally. At pH 8.3, soluble-Al concentrations were below the 5 x 10(-5) M toxicity threshold and cell population density increases of 20- to 40-fold were observed. An apparent cell population response to added Al at pH 8.3 was attributed to the presence of large, spirilloidal bacteria (accounting for as much as 80% of the cells at the 10 mM added Al level). Calculations of soluble-Al speciation for the pH 6.5 and 7.2 treatments that showed Al toxicity suggested the possible presence of the Al(13)O(4)(OH)(24)(H(2)O)(12)(7+) "tridecamer" cation and an inverse correlation of the tridecamer concentration and the cell population density. Analysis by (27)Al nuclear magnetic resonance spectroscopy, however, yielded no evidence of this species in freshly prepared samples or those taken 800 days after inoculation. Exclusion of the tridecamer species from the aqueous speciation calculations at pHs 6.5 and 7.2 yielded inverse correlations of the neutral Al(OH)(3) and anionic Al(OH)(4)(-) monomeric species with cell population density, suggesting that one or both of these ions bear primary responsibility for the toxicity observed.     

Effect of different fetal bovine serum concentrations on the replicative life span of cultured chick cells

The effect of Eagle's minimal essential medium, containing different fetal bovine serum (FBS) concentrations, on the proliferation and replicative life span of cultured chick cells has been studied. Our results showed that the rate of chick cell proliferation and the cell density at stationary phase increased as a function of serum concentration between 5 and 30% FBS. The replicative life span of cultured chick cells was dependent on the FBS concentration between 5 and 20% in a medium volume of 0.20 ml/cm2. The maximum replicative life span of chick cells was obtained by serially propagating cells in a medium volume of 0.20 ml/cm2 containing 20 or 30% FBS, or, alternatively, in 0.53 ml/cm2 containing 10, 20 or 30% FBS. Cells grown in medium containing 5% serum had a calendar life span of 35 days, whereas cells propagated in medium containing higher serum concentrations had a calendar life span of 50 days. These results reenforce the concept that, although the kinetics of cell population aging can be affected by the culture medium composition, the aging of cells in culture is controlled by alterations within the cell.     

Variation in the growth medium during the culture cycle of Tetrahymena: with special reference to ammonia (NH3), ammonium (NH4+), and pH1

The ciliate Tetrahymena pyriformis was grown in a peptone medium without added glucose. The interrelationship between increasing cell density and pH of the growth medium was studied from mid-log to the stationary phase, i.e. from 50,000 to 1,000,000 cells/ml, by continuous registration of the pH of the growth medium. The present findings correlate with the known physiological, biochemical, and structural changes occurring in Tetrahymena as it passes through the culture cycle. The ammonia production of the cells and the buffer capacity of the growth medium were determined throughout the growth cycle. The results revealed that the ammonia excreted by the cells can explain the increase in pH of the medium from 6.8 to about 8.3 normally seen during the culture cycle. Moreover, neither the increased pH nor the raised level of ammonia were found to be the responsible factor for cessation of cell proliferation in the stationary growth phase although these factors may affect cell proliferation in concentrations well beyond the range found in normal cultures.     

Maintenance of the keratocyte phenotype during cell proliferation stimulated by insulin

Keratocytes normally express high levels of aldehyde dehydrogenase and keratocan. They proliferate and lose their keratocyte markers when they become fibroblastic during corneal wound healing. Keratocytes cultured in fetal bovine serum also become fibroblastic, proliferate, and lose these markers. In this report, we studied the effects of three serum growth factors, fibroblast growth factor-2, insulin, and platelet-derived growth factor-BB, on keratocyte proliferation and the maintenance of the keratocyte markers in 7-day cultures in cells plated at low (5,000 cells/cm2) and high (20,000 cells/cm2) density in serum-free medium. Keratocyte proliferation was measured by [3H]thymidine incorporation and by DNA content of the cultures. Cytosolic aldehyde dehydrogenase and keratocan accumulated in the medium were quantified by Western blot. The results showed that all the growth factors stimulated proliferation, but insulin stimulated proliferation more consistently. The keratocyte markers aldehyde dehydrogenase and keratocan were maintained after 7 days in culture in all growth factors, but keratocyte cell morphology was only maintained in medium containing insulin. Most of the proteoglycans were degraded in cultures of keratocytes plated at low density and cultured in the absence of growth factors. This degradation was prevented when keratocytes were cultured in the presence of the growth factors or when keratocytes were plated at high density. The results of this study show that insulin can expand keratocytes in vitro, maintain their phenotype, and prevent proteoglycan degradation.     

Studies of long-range density effects on the proliferation of 3T3 and RLCW cells in recirculated medium.

Swiss mouse 3T3 cells and rat liver-derived RLCW cells were grown in monolayers and perfused with culture medium. A flow-rate dependent increase in the growth rate was observed both by 3H-thymidine uptake and by a rise in cell numbers. The characteristics of the response were dependent on the recirculating volume and on whether serum was present in the culture medium. In RLC cultures perfused with serum-supplemented medium the growth promoting effect decreased with increasing density of the cells. In the absence of serum, recirculation of NCTC medium had no effect on RLCs but increased growth was observed in recirculated MEM. In 3T3 cultures, a linear response was observed over a limited density range in the presence of 10% serum-supplemented medium indicating that substances present in the serum substantially modify the behaviour of the monolayer to perfusion. In serum-free medium the effect of perfusion on 3T3 cultures was confined to a small density range and was consistent with the more rapid removal of a diffusible inhibitor from the pericellular environment by recirculating the medium. Treatment of the perfusing medium with immobilised proteinases (trypsin, chymotrypsin, protease) did not alter the response except in the presence of putrescine.     

Studies of Long-Range Density Effects on the Proliferation of 3T3 and RLCW Cells in Recirculated Medium

Swiss mouse 3T3 cells and rat liver-derived RLCW cells were grown in monolayers and perfused with culture medium. A flow-rate dependent increase in the growth rate was observed both by ³H-thymidine uptake and by a rise in cell numbers. The characteristics of the response were dependent on the recirculating volume and on whether serum was present in the culture medium. In RLC cultures perfused with serum-supplemented medium the growth promoting effect decreased with increasing density of the cells. In the absence of serum, recirculation of NCTC medium had no effect on RLCs but increased growth was observed in recirculated MEM. In 3T3 cultures, a linear response was observed over a limited density range in the presence of 10% serum-supplemented medium indicating that substances present in the serum substantially modify the behaviour of the monolayer to perfusion. In serum-free medium the effect of perfusion on 3T3 cultures was confined to a small density range and was consistent with the more rapid removal of a diffusible inhibitor from the pericellular environment by recirculating the medium. Treatment of the perfusing medium with immobilised proteinases (trypsin, chymotrypsin, protease) did not alter the response except in the presence of putrescine.