Daily Medium Changes Research Articles

Hormonal regulation of DNA synthesis in primary cultures of adult rat hepatocytes: Action of glucagon

Primary cultures of adult rat hepatocytes, grown in modified minimal essential medium (Eagle's) containing 10% calf serum, could be induced into DNA replication by combinations of epidermal growth factor (EGF), insulin and glucagon. The three hormones acted synergistically, and cells began entering DNA synthesis 48 h after hormone addition. The ability of the hormones to stimulate DNA synthesis was enhanced by plating cells at high cell concentrations or by conditioned medium, and was diminished by daily medium change. The contribution of glucagon to DNA synthesis was replaced by cAMP plus 1-methyl, 3-isobutyl xanthine or by adrenergic agents. Evidence is presented which suggests that all three hormones are required on the first day of culture, and that EGF and insulin are also required after the first day. This appears to be a useful system for studies on the hormonal initiation of growth in quiescent cells.

Replacement of mouse LM fibroblast choline by a sulfonium analog: Effects on membrane properties as determined by virus probes

A sulfonium analog of choline (‘sulfocholine’, a natural phospholipid constituent of diatoms) was metabolically incorporated into mouse LM fibroblasts cultured in serum-free medium. Subconfluent cultures of LM cells were able to utilize sulfocholine as sole choline source and to increase in cell number for 3 days of incubation; thereafter a decrease in cell number was observed. In contrast, cultures of LM cells seeded to confluency showed no decrease in cell number up to at least 10 days when maintained, with daily medium changes, in medium containing either choline or the sulfonium analog. Such confluent cultures, maintained for 7 days in sulfocholine-containing medium, showed virtually complete replacement of cellular phosphatidylcholine and greater than 50% replacement of cellular sphingomyelin by their respective sulfonium analogs. The functional exchangeability of natural phosphatidylcholine and sphingomyelin with their sulfonium analogs to participate in normal cell membrane-mediated activities was demonstrated by comparatively assaying the abilities of sulfocholine- and choline-maintained cells to incorporate and replicate certain animal viruses known to possess membrane-dependent steps in various phases of their replication cycles. No difference was detected between the abilities of sulfocholine- and choline-maintained cells to take up vesicular stomatitis virus or mengo virus, or to replicate vesicular stomatitis virus, mengo virus or mouse hepatitis virus.

Collagenase and collagenase inhibitor levels following changes in bone resorption in vitro.

Half-calvariae from 1-week-old mice were cultured for 3 days with daily medium changes. Collagenase and the synthesized collagenase inhibitor were measured in the media from untreated explants and from explants treated with agents that alter bone resorption. Collagenase from untreated explants was in a latent form that could be activated by either trypsin or 4-aminophenylmercuric acetate. The molecular weight of the latent collagenase was 60,000 by gel filtration, that of the activated enzyme 50,000, and that of the collagenase inhibitor 30,000. Activated enzyme from both untreated and treated explants gave the typical ¾ and ¼ fragments when reacted with collagen.

The effect of serum albumin and the effect of cell concentration on the in vitro growth of mouse and rat lymphocytes

The growth of mouse and rat T and B lymphocytes, activated by concanavalin A or lipopolysaccharide, is increased over growth in protein-free medium 5–20-fold by human or bovine serum albumin. The growth is dependent on the concentration of albumin. At a concentration of 2–4 mg/ml the growth rate is the same as in the presence of 10% fetal bovine serum. Of the other serum proteins (Cohn fractions) only human fraction VI supports growth somewhat while human fractions II–IV and bovine fraction VI do not support growth. The growth of mouse and rat lymphocytes is greatly suppressed if lymphocytes are cultured at high cell concentrations, and the growth-promoting ability of serum albumin cannot be detected under such conditions. The growth rate can be improved by daily adjustment of the pH, by daily refeeding, and by daily change of medium. The growth inhibitory activity can be removed largely by dialysis. It is concluded that the suppression of growth at high cell concentrations is caused by a combination of effects, i.e., a shift of pH, lack of nutrients, and accumulation of cellular metabolites.

Effect of exogenous fatty acids on the retention of phospholipid acyl groups by mouse L fibroblasts

Exogenous oleic or linoleic acid, given at a high but nontoxic level (1 mg fatty acid/day for 20 · 10 6 cells in 50 ml medium), caused substantial redistribution of the otherwise permanently retained phospholipid acyls in mouse L fibroblasts. 18–40% of the preformed phospholipid acyls were shifted to triglycerides but most returned to phospholipids when the supply of exogenous fatty acid was removed. The phospholipid acyls could be reshuttled back to triglycerides again whenever an adequate amount of exogenous fatty acid was provided. Daily changes of medium containing oleic acid bound to bovine serum albumin caused a still greater total loss of phospholipid acyls into the medium. The removal of the prelabeled phospholipid acyls also occurred with phospholipid acyls which had been synthesized from [1- 14C]acetate 3 days earlier. The results demonstrate the fact that the apparent permanently retained phospholipid acyl groups found in L-cells could in fact be displaced through experimental manipulations.

Division of BALB/c mouse 3T3 and simian virus 40-transformed 3T3 cells in cellular aggregates

BALB/c mouse 3T3 cells and 3T3 cells transformed by simian virus 40 (SV40) were cultured as aggregates in agitated liquid medium. When maintained with daily medium changes, 3T3 cells incorporated [(3)H]thymidine into acid-insoluble material at a rate (1/4) that of 3T3 cells in logarithmic-phase flat cultures but 16 times that of the same cells in stationary-phase flat cultures. Similarly, SV40-transformed 3T3 cells in aggregates incorporated [(3)H]thymidine at (1/3) the rate of SV40-3T3 cells in logarithimic-phase flat culture and 14 times that of these cells in stationary-phase flat culture. Autoradiographs of aggregates of 3T3 and SV40-3T3 cells incubated for 2 hr in the presence of [(3)H]uridine and [(3)H]thymidine indicated that penetration of the nucleosides into aggregates during this period was limited to the outer four to six cell layers. Aggregates were also incubated in the presence of radiolabeled thymidine and uridine continuously for 4 days. Under these conditions, where nucleoside penetration was not limiting, 100% of the SV40-3T3 cells and 56% of the 3T3 cells incorporated [(3)H]thymidine into acid-insoluble material. The rates of cell division, cell loss, and net cell accumulation in aggregates of 3T3 and SV40-3T3 cells were measured by techniques not influenced by possible alterations in transport, pool size, or penetration. SV40-3T3 cells divided with a doubling time for the total population of 26.0 hr. The total cell number increased more slowly (doubling time of 48.3 hr) because of cell loss, which occurred with a half-time (time for 50% of the cells to be lost) of 53.3 hr. 3T3 cells in aggregates began to divide only after 3 days, then did so with a doubling time for the total population of 76.4 hr. Total cell number decreased (half-time of 26.3 hr) because this rate of cell division was exceeded by the rate of cell loss, which was constant with a half-time of 22.9 hr. The results suggest that 3T3 and SV40-3T3 cells display growth properties in aggregates consistent with their previously reported behavior in conventional flat culture: SV40-transformed 3T3 cells can proliferate under conditions of high cell density to a much greater extent than 3T3 cells can. Both cell lines, however, display an increased capacity to divide in aggregates relative to confluent flat culture, despite conditions of high cell density and absence of anchorage to an artificial solid substrate.

Metabolism of thyrotropin releasing factor in two clonal cell lines of nervous system origin

Immunoreactive thyrotropin releasing factor (TRF) was detected in homogenates of two clonal cell lines, BN1010-1 and BN1010-3, derived from a rat central nervous system tumor. TRF was present in logarithmically-growing cells; daily medium changes with slightly acid culture medium (pH 6.8) greatly increased the TRF content of these cells. In contrast, TRF could not be detected in stationary phase cells. TRF peptidases were <1% as active in homogenates of BN1010 cells as those in homogenates of guinea pig brain or hypothalamus. It is expected that these cells will provide an excellent model system for the study of various aspects of TRF metabolism.

Persistence of Bacteriophages in Experimentally Infected Cell Cultures

A bacteriophage, isolated from a live measles vaccine and designated ϕV-1 L , was intentionally introduced into cell culture systems. The bacterial virus persisted in the cell cultures after daily medium changes and trypsinization, and in cell lysates prepared by freezing and thawing. The results suggest that phages contaminating cell cultures may be carried over into the final product during the manufacture of viral vaccines.

Persistence of Bacteriophages in Experimentally Infected Cell Cultures

A bacteriophage, isolated from a live measles vaccine and designated varphiV-1(L), was intentionally introduced into cell culture systems. The bacterial virus persisted in the cell cultures after daily medium changes and trypsinization, and in cell lysates prepared by freezing and thawing. The results suggest that phages contaminating cell cultures may be carried over into the final product during the manufacture of viral vaccines.

Studies on contract inhibition of growth in the mouse fibroblast, 3T3. I. Changes in cell size and composition during 'unrestricted' growth.

ABSTRACT Growth of the contact-inhibited 3T3 and its SV40-transformed derivative, 3T3T is unbalanced. Despite obvious exponential increase in cell number, cell volume, RNA and protein show a marked decrease with increasing population density in both cell types, greater in 3T3 than 3T3T. Daily medium change does not correct this growth pattern but merely seems to equalize initial growth rates of the 2 cell types and prolong the period during which the cell number continues to increase. The evidence suggests that the lag period that ensues following plating of the cells is a period of intense metabolic activity during which a large proportion of cellular protein and RNA is synthesized in preparation for exponential growth.

The quantitative utilization of amino acids and glucose and contact inhibition of growth in cultures of the human diploid cell, WI-38.

ABSTRACT There are many reports in the literature showing that contact inhibition of growth is affected by the culture medium. A quantitative study of amino acid and glucose uptake by the human diploid cell line, WI-38 was carried out to determine more precisely what effect nutritional factors have on contact inhibition of growth. Eagle’s minimal essential medium (MEM) was found to support higher cell yields than Eagle’s basal medium (BME) and for growth to continue beyond 96 h a medium change was essential. However, analysis of the used growth media showed that neither amino acids nor glucose were fully depleted after 96 h. The rate of glucose utilization was in the range 65–100 μg/mg dry wt./h and this agreed very closely with the results of other authors. The pattern of amino acid uptake also closely resembled that for other cell lines except that the utilization of cystine was higher. The nutritional requirements were further studied as the results from the medium analyses failed to explain the growth-promoting activity of MEM. Daily medium changes greatly increased cell yields even though the medium nutrients were not exhausted. This effect was dependent upon fresh medium being used and the only medium component found to be of importance was the amino acid complement. These results are discussed in relation to the low saturation density of diploid cells in culture and a possible explanation is proposed in terms of differences in the cell membrane between normal and altered cells.

Enhanced interferon production from chick embryo cells aged in in vitro

The plaque-forming efficiency of Sindbis virus decreased as much as 1000-fold, and plaque size was diminished markedly, when tested on chick embryo cell monolayers aged in vitro. The plaquing efficiency and plaque size of Newcastle disease virus was unaffected. The reaction(s) associated with aging in vitro which lead to lowered plaquing efficiency are slowed considerably when cell monolayers are: (1) established in Simpson-Hirst medium rather than standard growth medium; (2) held at 31°; (3) given a regimen of daily medium changes; or (4) trypsinized and used as young secondary cultures. A loss in the average yield of virus per cell accompanies the loss in plaquing efficiency of Sindbis virus on aged monolayers. Adding actinomycin D to the aged cells at the time of infection eliminated completely the inhibition of Sindbis virus replication. Cells aged for 7 days in vitro were found to produce up to 32 times more interferon than cells 1–2 days old and were more sensitive to the action of interferon. The decrease in efficiency of Sindbis virus plaquing and yield in aged cells is accounted for by their development of an enhanced capacity to synthesize interferon upon appropriate stimulation. The process of contact inhibition and its concomitant regulation of macromolecular synthesis seems implicated in the aging phenomenon in that it may produce a generalized state of “enhanced derepressibility” in the cell.

Maturation of schistosome eggs in vitro.

EGG-LAYING in vitro by Schistosoma japonicum and S. mansoni in serum media was not described by early investigators1–4, and presumably did not occur. Later it was reported that a few eggs were laid when flukes were kept for up to two months in a glass apparatus with provision for daily change of medium (horse serum)5. A method of maintaining S. mansoni flukes so that a regular output of 30–40 eggs a day was obtained has recently been published6–8. Some cell division was seen, but the eggs did not mature in serum media, saline or Tyrode's solution.