Analysis of correlation between some parameters depending on cell proliferation and life span of "stationary phase aging" cultures and maximum life span of mammals

Earlier we developed a "stationary phase aging" model and introduced a definition of life span of "stationary phase aging" cell cultures. In this model the cells grow after seeding in flasks without subcultivation and medium change. They reach cell saturation density, stop dividing, gradually degrade ("stationary phase aging") and perish. By the term "culture life span" we designate the time from cell seeding until culture death. We designate the culture as dead when the number of living cells is less than 10 per cent of their number at saturation density of cell culture. The life span of transformed Chinese hamster cells was found to be proportional to the duration of their growth from cell seeding up to saturation density, as well as to the number of cell culture doublings and to be inversely proportional to the velocity of cell culture doubling for the same growth period. Maximum life span of mammals is known to be proportional to pregnancy duration and to the age at puberty. We found that maximal life span of mammals was proportional to the number of cell population doublings and inversely proportional to the velocity of cell population doubling during embryonal period or for the time from zygote to growth termination. The dependences for cell cultures and for mammals are analogous to each other.     

Cholinergic differentiation in neurogenic basal forebrain cultures.

To study early events in the central nervous system (CNS) cholinergic development, cells from rat basal forebrain tissue were placed in culture at an age when neurogenesis in vivo is still active [embryonic day (E) 15]. The rapid mortality of these cells in defined medium, with 50% mortality after 5-10 h, was blocked completely by soluble proteins from the olfactory bulb (a basal forebrain target), extending earlier observations (Lambert, Megerian, Garden, and Klein, 1988). Treated cultures were capable of incorporating thymidine into DNA, and most cells incorporating 3H-thymidine (greater than 90%) also stained positive for neurofilament, confirming neuronal proliferation in the supplemented cultures. A small percentage of 3H-thymidine labelled cells were glial fibrillary acidic protein (GFAP) positive, but growth factors that support astroglial proliferation [epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and insulin-like growth factor (IGF-1)] were not sufficient for neuronal support. After 5 culture days with supplemented medium, almost 50% of the cells showed choline acetyltransferase (ChAT) immunofluorescence. The cholinergic neurons typically formed clusters separate from noncholinergic cells. These mature cultures did not develop if young cultures were treated with aphidicolin to block DNA synthesis. The data show that cultures of very young rat basal forebrain cells can be neurogenic, giving rise to abundant cholinergic neurons, and that early cell proliferation is essential for long-term culture survival.     

Adrenocorticotropic hormone controls Concanavalin A activation of rat lymphocytes by modulating IL-2 production

The initiation of DNA synthesis and secretion of Interleukin 2 (IL-2) was measured in isolated rat splenic lymphocytes following activation with Concanavalin A (ConA). The extent of 3H-thymidine incorporation into activated cells was tested when cultured with various concentrations of Adrenocorticotropic hormone (ACTH). A paradoxical dose-response curve resulted when ACTH caused a biphasic response of augmenting and inhibiting 3H-thymidine uptake in lymphocytes depending on the hormone concentration. Low levels of ACTH (0.001-1-nM) augmented 3H-thymidine uptake and high levels (10-1000 nM) reversed the effect. The optimal ACTH concentration was 10 pM ACTH in the presence of 5 ug/ml ConA and there was no ACTH effect on quiescent cells (no ConA). Conditioned media from splenic lymphocytes treated with various concentrations of ConA or ACTH was tested for increased uptake of 3H-thymidine by the IL-2 growth dependent Cytotoxic T Lymphocyte Leukemia (CTLL-2) cells. ConA conditioned medium could sustain the CTLL-2 cells indicating the presence of IL-2. Conditioned medium from splenic lymphocytes treated with both ConA and 100 pM ACTH further increased CTLL-2 cell proliferation indicating an additional increase of IL-2 secretion. The identity of IL-2 was confirmed by using an anti-rat IL-2 antibody to neutralize the growth potential of the conditioned medium. ACTH alone had no effect on the CTLL-2 cell proliferation indicating the effect is due solely to induced IL-2 found in the conditioned medium. IL-2 levels in the conditioned media were quantitated by ELISA assay; splenic lymphocytes produced 4.2 ng/ml to ConA only, 19.2 ng/ml in ConA plus 10 nM ACTH, and no detectable IL-2 at ConA plus 10 uM ACTH. These results demonstrated that ACTH modulates IL-2 secretion from activated lymphocytes, which is both biphasic and concentration dependent.     

Proliferation of Buffalo rat liver cells in serum-free medium does not depend upon multiplication-stimulating activity (MSA).

A line of Buffalo rat liver cells (BRL 3A) that multiplies in the absence of serum produces a family of polypeptides termed MSA that can partially satisfy the serum requirement for growth of chick embryo fibroblasts. Temin, Pierson and Dulak (1972) proposed that BRL cells multiply in serum-free medium because they produce MSA. This does not appear to be the case. We have studied three BRL cell lines: 3A2 and 3A have diverged from the same original isolate from normal liver; 61t is a spontaneous transformant of a different isolate. All three cell lines showed a 10 fold increase in cell number during 5 days in serum-free medium. However, 3A-conditioned medium stimulated 3H-thymidine incorporation into DNA in chick embryo fibroblasts and human skin fibroblasts; 3A2- and 61t-conditioned media did not. After ion-exchange chromatography or gel filtration of the conditioned media and measurement of MSA by 3H-thymidine incorporation or radioreceptor assay, MSA again was found in the 3A medium but not in the 3A2 or 61t media. The absence of MSA in the 3A2 and 61t media was not due to inactivation of MSA by these two cell lines. Addition of partially purified MSA to 3A2 cells did not increase their multiplication rate in serum-free medium. We conclude that the ability of the BRL cells to multiply in serum-free medium is independent of the level of MSA in the medium.     

Observations consistent with autocrine stimulation of hybridoma cell growth and implications for large-scale antibody production

Existence of autocrine growth factors (aGFs) may influence the serum requirement for growth of hybridoma cells and thus significantly influence process economics. For the murine hybridoma cell line S3H5/2bA2, critical inoculum density (cID) and serum requirement for growth were inversely related for cultivation in both T flasks and spinner flasks. In spinner flasks, an inoculum density of 10⁶ cells/ml was necessary for the cells to grow in RPMI 1640 medium without serum supplement, and an inoculum density of 10³ cell/ml was necessary in RPMI 1640 medium with 10% serum. In T flasks, where the local cell density is higher than in spinner flasks, an inoculum density of 10⁶ cells/ml was necessary for the cells to grow in RPMI 1640 medium without serum supplement, and an inoculum density of 1 cell/ml was also necessary in RPMI 1640 medium with 10% serum. Further, immobilized cells at high local cell density could grow under conditions where cells in T flasks at corresponding overall cell density could not grow. The cells at high inoculum density were less sensitive to shear induced by mechanical agitation than the cells at low inoculum density. Taken together these observations support the existence of secreted aGF(s) by the hybridoma cell line used. Since the specific MAb production rate was independent of cultivation method and inoculum density, the existence of autocrine growth factors would suggest that the use of immobilized cells should improve the economics of MAb production.     

Dibutyryl cyclic AMP modulation of gap junctions in SW-13 human adrenal cortical tumor cells

Cultured human adrenal cortical adenocarcinoma cells (SW-13) form a confluent monolayer of epithelial-like cells when seeded into culture flasks. Following a 24-48 hr non-mitotic period, cells begin to divide and become confluent within a week after seeding at 5 X 10(4) cells/cm2. The SW-13 cells were exposed to dibutyryl cyclic AMP (DbcAMP), cyclic AMP (cAMP), sodium butyrate, and adrenocorticotropin (ACTH). The rate of SW-13 cell proliferation was measured with a DNA microfluorometric assay, as well as by procedures measuring the incorporation of 3H-thymidine. In addition, following administration of ACTH and DbcAMP, the fractional area of membrane covered by gap junctions was quantitated with freeze-fracture electron microscopic techniques. Dibutyryl cyclic AMP at a concentration of 1 X 10(-3) M decreased the growth rate of the cell population. There was a corresponding increase in the fractional area of gap junctions found on the cell membrane in 96-hr DbcAMP-treated cultures. ACTH (40 mU/ml) exposure failed to produce an increase in the fractional area of gap junctions or to alter the rate of cell proliferation. From these data it can be suggested that elevations in cAMP levels within the cell can be related to both the proliferation of gap junctions and the decrease in cell proliferation in the SW-13 tumor cell.     

The effect of EGF and the comitogen, norepinephrine, on the proliferative responses of fresh and cryopreserved rat and mouse hepatocytes

The effect of cryopreservation on the proliferative response of fresh and cryopreserved (CP) rat and mouse hepatocytes was studied. Of the parameters measured, incorporation of 3H-thymidine and bromodeoxyuridine (BdrU) incorporation were the most sensitive and LDH content was the least sensitive. The optimal seeding density for epidermal growth factor (EGF)-stimulated proliferative response in fresh rat and mouse hepatocytes was 1.8 x 10(4) cells/cm2 and 2.1 x 10(4) cells/cm2, respectively. 3H-thymidine incorporation by fresh rat and mouse hepatocytes was maximal in cultures treated with 10 and 5 ng/ml EGF, respectively. The cell attachment of fresh rat hepatocytes after 48 h was higher (68%) than CP (42%), therefore, the CP hepatocyte seeding density was increased to 7.1 x 10(4) cells/cm2 so that the cell number after 48 h was the same as fresh hepatocytes. Using the adjusted seeding density, the 3H-thymidine and BdrU incorporation into fresh and CP rat hepatocytes was equivalent. The attachment efficiencies of fresh and CP mouse hepatocytes were the same, therefore, no adjustment was needed. The proliferative response (3H-thymidine incorporation and DNA content) to EGF was the same in fresh and CP mouse hepatocytes. The comitogen, norepinephrine (NE), increased the proliferative response to EGF to the same extent in both fresh and CP rat hepatocytes. In summary, cryopreserved rat and mouse hepatocytes retain their ability to proliferate in culture. Adjustment and monitoring of the seeding density is of high importance, especially with rat hepatocytes, which lose some attachment capacity after cryopreservation. The secondary mitogenic effect of NE is also retained by cryopreserved rat hepatocytes, suggesting that these cells retain alpha1-receptor function.     

Growth efficiencies of freshwater bacterioplankton

The growth efficiency of freshwater bacteria was examined in continuous cultures. One series of experiments was carried out using generation times from 50 to 200 hours and aged, normal, and enriched media, all of natural origin. Another series of experiments examined the bacterial growth efficiency during the growth season in eutrophic Frederiksborg Slotssø, in relation to changes in the planktonic communities and to factors controlling the bacterial incorporation of ³H-thymidine. Attachment of bacteria to the inner surfaces of the experimental flasks was examined using various types of bottles, adding glass tubes to the bottles, and measuring ³H-thymidine incorporation and direct cell counts of attached and free-living bacteria. Attachment of bacteria varied, and in one example up to 36% of the thymidine incorporation was by attached bacteria after 4 days. It was calculated that 36% of attached bacteria caused an underestimation of the growth efficiency of 11%. The mean growth efficiency tended to decrease with generation time using enriched medium (47 to 19%) and aged medium (35 to 12%), and tended to decrease with medium quality (enriched > normal > aged media) from 37% to 27%. The only significant difference in growth efficiency occurred in relation to generation time, in samples with enriched medium (unpaired t-test, P < 0.05). The overall mean value for all generation times and media was 30% (SEM = 3%, n = 24). From April to October, the growth efficiency was determined 5 times in samples from Frederiksborg Slotssø. The overall mean value was 31% (SEM = 3%, n = 30), and there was no significant change in the growth efficiency during the period measured. In June, three bioassay experiments revealed that carbon limitation controlled bacterial incorporation of ³H-thymidine, whereas additions of phosphate and nitrate did not change the incorporation rates. The narrow range of growth efficiencies obtained in this study (mean 31%, SEM = 2%, n = 54) suggests that changes in substratequality in the media applied and in the eutrophic samples examined causes only subtle changes in the growth efficiency.     

Cholinergic differentiation in neurogenic basal forebrain cultures

To study early events in the central nervous system (CNS) cholinergic development, cells from rat basal fore brain tissue were placed in culture at an age when neurogenesis in vivo is still active [embryonic day (E) 15]. The rapid mortality of these cells in defined medium, with 50% mortality after 5–10 h, was blocked completely by soluble proteins from the olfactory bulb (a basal forebrain target), extending earlier observations (Lambert, Megerian, Garden, and Klein, 1988). Treated cultures were capable of incorporating thymidine into DNA, and most cells incorporating ³H-thymidine (>90%) also stained positive for neurofilament, confirming neuronal proliferation in the supplemented cultures. A small percentage of ³H-thymidine labelled cells were glial fibrillary acidic protein (GFAP) positive, but growth factors that support astroglial proliferation [epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and insulin-like growth factor (IGF-1)] were not sufficient for neuronal support. After 5 culture days with supplemented medium, almost 50% of the cells showed choline acetyltransferase (ChAT) immunofluorescence. The cholinergic neurons typically formed clusters separate from noncholinergic cells. These mature cultures did not develop if young cultures were treated with aphidicolin to block DNA synthesis. The data show that cultures of very young rat basal forebrain cells can be neurogenic, giving rise to abundant cholinergic neurons, and that early cell proliferation is essential for long-term culture survival.     

Senescence and cell density of human diploid fibroblasts influence metabolism of insulin-like growth factor binding proteins

In order to analyze changes in metabolism of insulin-like growth factor binding proteins (IGFBPs) related to cell senescence and cell density, we compared human diploid fibroblasts (HDF) in the proliferatively vigorous first half (young cells) and senescent HDF in the last 10% (old cells) of the replicative lifespan after seeding cells over an eighfold range and proliferation to high density. Increasing the seeding cell density of both young and old HDF led to elevated rates of IGFBP-3 secretion, an increasing ratio of the 42/38 kDa species of IGFBP-3, and degradation of all species of IGFBPs derived from both the fetal bovine serum component of the culture medium and from HDF. At a given seeding density old HDF produced more IGFBP-3 and degraded more IGFBPs than young HDF. IGFBP-4 was degraded by a protease that appeared to be different from the protease(s) involved in degradation of the other IGFBPs. Young HDF at all seeding densities contained a cell-associated 29 kDa IGFBP, whereas this protein could not be detected in old cells. Thus, although certain changes in IGFBP metabolism are similar in young HDF seeded at high densities and in old HDF, young and old phenotypes can be distinguished by characteristic qualitative and quantitative changes in IGFBPs derived from fetal bovine serum and from HDF. © 1994 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America

     

The Role of Cytokinin in the Regulation of Growth, DNA Synthesis and Cell Proliferation in Cultured Soybean Tissues (Glycine max var. Biloxi)

Changes in protein content and cell proliferative activity were followed after a cytokinin-requiring strain of cultured Glycine max tissue was transferred to freshly prepared media which either contained or lacked cytokinin. Cell numbers doubled within the first two days after transfer, both in the presence and absence of cytokinin. However, after the second day no further increase in cell number was observed in the absence of cytokinin, while cell numbers continued to increase logarithmically in the presence of cytokinin. The size of the cell population attained after the first six days of growth was a function of the cytokinin concentration of the culture medium. However, the amount of ³H-thymidine incorporated into nuclear DNA bore no relation to the rate of cell proliferation. Tissues cultured on medium lacking cytokinin incorporated the greatest amount of ³H-thymidine per microgram of DNA, while the actively dividing tissues incorporated somewhat less. Using autoradiography and isopycnic CsCl gradient centrifugation, it was shown that the radioactivity derived from ³H-thymidine was associated with nuclear DNA in the cytokinin-deprived cells. Biochemical measurements demonstrated that cells cultured for six days without cytokinin had approximately twice the DNA content of the actively proliferating cells cultured on cytokinin-containing medium. Furthermore, in autoradiographs labeled cells were found to average nearly three times as many silver grains per nucleus in tissues cultured without cytokinin as the cytokinin-grown tissues. This suggests that the ³H-thymidine incorporation in the non-proliferating soybean cells results from nuclear DNA synthesis and that some of the cells became polypoid in the absence of cytokinin. These findings would be consistent with the idea that cytokinin acts as a specific trigger for cytokinesis.     

Production of Choristoneura fumiferana Nucleopolyhedrovirus in C. fumiferana (CF-2C1 Cells) in a 3 Litre Bioreactor Using Serum-free Medium

The purpose of this study was to develop a cell culture process in a bioreactor for the production of a viral insecticide for the spruce budworm, Choristoneura fumiferana . Several cell lines were tested for their growth in serum-free medium suspension cultures. One cell line, CF-124T-2C1 (CF-2C1), was successfully adapted to grow in suspension cultures in SFM. Serum-free Ex-Cell 405 medium produced a much higher cell density (6.3 x 10 6 cells ml -1 ) than the Grace's medium supplemented with 10% fetal bovine serum (2.5 x 10 6 cells ml -1 ). Also, a higher yield of virus was obtained in the former medium. Ex-Cell 405, was used to study the growth of CF-2C1 cells and the production of C. fumiferana nucleopolyhedrovirus (CfMNPV) in a 3 l bioreactor. Under these conditions, a specific growth rate ( μ) of 0.027 h -1 was obtained during the exponential growth phase, and the specific carbon dioxide evolution rate, as determined by on-line measurement, was 0.9 x 10 -16 mol cell -1 s -1 and 1.78 x 10 -16 mol cell -1 s -1 during growth and infection phases, respectively. Virus production in bioreactor cultures infected at 1.3 x 10 6 cells ml -1 was consistently lower than that obtained in Erlenmeyer shake flasks. Only 26% of the cells were infected in the bioreactor compared to 44% in the shake flasks. However, a higher yield of occluded virus was obtained in the bioreactor cultures than in shake flasks. The production of occlusion bodies (OB) achieved in bioreactor cultures was 2 x 10 6 OB ml -1 .     

Hormonal regulation of rat granulosa cell deoxyribonucleic acid synthesis: effects of estrogens

Although it is widely accepted that estrogens exert a major trophic effect on follicular growth, their mechanism of action has not been established. We examined the effect of estrogen treatment in vivo or in vitro on DNA synthesis in rat granulosa cells cultured under defined conditions (DMEM:F12, collagen-coated plastic wells). Treatment with diethylstilbestrol (DES) in vivo (silastic implants containing 5 mg DES) for at least 2 days was required to observe a significant stimulation of 3H-thymidine incorporation by insulin (1 microgram/ml) in culture. Rat thecal/interstitial cells (TI) were isolated from DES-treated rats and cultured under the same conditions as granulosa cells. Conditioned media from TI cells stimulated DNA synthesis in granulosa cell cultures (as much as twofold). This effect was markedly amplified by estradiol treatment (1 microgram/ml) of the TI cell cultures. Addition of estradiol to granulosa cell cultures enhanced the effect of conditioned medium from nontreated TI cells. Conditioned medium from estradiol-treated TI cells stimulated DNA synthesis in granulosa cells from both DES-treated and nontreated rats. Estradiol had no effect when added directly to purified granulosa cell cultures but stimulated 3H-thymidine incorporation in crude preparations of ovarian cells. The stimulatory effects of TI cell-conditioned medium and insulin were reflected in the final cell densities achieved after 9 days in culture. We conclude that the mitogenic actions of estrogens in the ovary involve sensitization of granulosa cells to locally present mitogens like insulin and a TI cell-derived growth factor.     

DNA synthesis in cultures of atrial and ventricular cardiomyocytes in the rat

By means of 3H-thymidine autoradiography DNA replicative activity has been studied in cultured atrial and ventricular myocytes, and non-muscle cells from hearts of 2-week-old rats (age when cell proliferation in the myocardium is already significantly depressed). PAS-reaction was used as a cytochemical marker of cardiomyocytes: atrial myocytes are richer in glycogen than ventricular cells. Labeling indices of atrial myocytes after a 24 hour exposure to 3H-thymidine were higher than ventricular ones: on day 6 of culturing--47 and 5%, and on day 11-34 and 8%, respectively. After 10 days of culturing the number of binucleated atrial myocytes, non-typical for atrial myocardium in vivo, increased by 25-40% as compared with 8-13% on days 2-3 in culture. In 10-day cultures, 3- and 4-nucleated atrial myocytes were observed. Both mononucleated and binucleated atrial and ventricular myocytes incorporated 3H-thymidine. To find out whether the deeper inhibition of replicative activity in ventricular myocytes influences fibroblasts and endothelial cells from ventricles, the proliferative activity of non-muscle cells was studied. Non-muscle cells, both in atrial and ventricular cultures, behaved as a totally proliferating population (labeling indices on the 6th day are about 75-90%) and their growth rate decreased during the formation of the contact-inhibited monolayer. These cells, contrary to myocytes, are predominantly mononucleated in all the periods studied. The deeper depression of replication in ventricular myocytes appears to be related with their higher level of differentiation as compared to myocytes of the atrial myocardium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Why does a cell monolayer replicate the metal tray pattern of the thermostat?

Cells freshly seeded into the closed culture flasks or dishes and placed on the metal tray with holes of the thermostat or incubator are seen to form the layer with uneven density: with high density corresponding to the flask bottom regions above the metal and low density corresponding to the flask bottom region above the holes in the tray. The effect was shown using several cell lines with different degrees of transformation and saturation density, including Swiss 3T3. The main cause of this effect is the difference between the temperature inside the thermostat and the lower temperature of the flasks with culture medium (rather than between the metal framework and the air), together with a high heat conduction of the metal. The reverse difference in the temperatures (higher temperature of the culture flasks) leads to the formation of the reverse pattern of the cell layer, with higher density corresponding to the holes. The temperature differences exert their influence presumably during the first 10-15 minutes after the cells seeding, when the process of cell sedimentation is involved possibly by creating the microcurrents in the medium.     

Evaluation of various serum and animal protein free media for the production of a veterinary rabies vaccine in BHK-21 cells

We have carried out the adaptation of BHK-21 cells to two serum free (Ex Cell 520 and HyQ PF CHO) and three animal protein free media: Ex Cell 302, HyQ PF CHO MPS and Rencyte BHK. After a direct switch or a gradual adaptation, we have achieved BHK-21 cells growth in the following media: HyQ PF CHO, HyQ PF CHO MPS, Rencyte BHK and Ex Cell 302. The most suitable media for BHK-21 cells growth, with respect to cell density and specific growth rate, were HyQ PF CHO and HyQ PF CHO MPS. Hence we have selected these media to study cell growth and the production of rabies virus. Kinetic studies of cell growth in spinner flasks using the selected media have shown that a maximal cell density of 2x10(6) cells x ml(-1) was reached in both media. For rabies virus production, the viral titer obtained was 1.7x10(6) FFU x ml(-1) in HyQ PF CHO as well as in HyQ PF CHO MPS medium. The optimization of rabies virus production by BHK-21 cells grown in a 2 l bioreactor using the selected media, pointed to the following parameters: culture mode, perfusion rate and multiplicity of infection (MOI), as being the critical factors for achieving a good virus yield. When tested in mice, the activity of the experimental vaccines prepared on HyQ PF CHO MPS medium has shown a protective activity that meets WHO requirements.     

Simultaneous localization of 3H-thymidine incorporation and acid phosphatase activity in mouse spleen: EM radioautography and cytochemistry.

Simultaneous localization of 3H-thymidine incorporation and acid phosphatase (AcP) activity was undertaken by combined radioautography and cytochemistry in the spleen of mice at different ages. The localization of radiolabelled thymidine was used to determine the site of DNA synthesis (cell proliferation), while AcP activity as a marker for cell lysis/death. For EM radioautography (EMRAG), the tissue sections were incubated in a medium containing 3H-thymidine and processed for radioautography, while the lanthanide-based method for the ultrastructural localization of AcP activity was employed. Quantitation of AcP activity was carried out by X-ray microanalysis. In all tissue sections examined, mostly of the labelled nuclei were observed in the hematopoietic cells. Few mitochondria of these cells were labelled. The labeling index was expressed as the percentage of labelled cells over the total number of counted cells. The labeling indices dropped considerably from day one after birth and progressively until the 10th month. The result of AcP activity correlated well with the result of a previous work (Olea, 1991). The localization of radiolabelled thymidine and AcP activity were not hindered by the simultaneous exposure of the same tissue section to 3H-thymidine and AcP cytochemical media. Interestingly enough, the spleen actively participates both in hematopoiesis and erythrophagocytosis. Prominently, it is most active during the early postnatal life. However, their influence declined considerably at the later stage of life (adult stage).     

Stimulation of uptake of tritiated thymidine into mouse marrow cells in culture by a factor from L-cell conditioned medium

Uptake of ³H-thymidine into suspension cultures of mouse marrow cells was stimulated by the addition of serum-free conditioned medium harvested from cultures of mouse L-cells. Characterization of the “conditioning factor activity” (CFA) by gel filtration, ion exchange chromatography, velocity sedimentation, polyacrylamide gel electrophoresis and susceptibility to trypsin digestion indicated that the CFA detected by stimulation of ³H-thymidine uptake is the same as the CFA detected previously by its ability to stimulate colony formation by marrow cells in vitro. The ³H-thymidine uptake assay was used to investigate the kinetics of disappearance of CFA as a function of time in the presence of mouse marrow cells. The CFA recoverable from the cultures decreased rapidly during the first day, and approached background levels by the fifth day. There was no evidence of inhibitory substances in the depleted media. Even if the cultures continued to receive fresh conditioned medium at daily intervals, ³H-thymidine uptake decreased sharply after the fifth day, indicating that the marrow cells had lost their capacity to respond to CFA.     

Studies of alpha-protein in human cell cultures

alpha-Protein growth fraction (AGF) eliminates the 60- to 90-day adaptive phase required to establish actively growing cultures of HeLa (Gey), human heart (Girardi), KB (Eagle) and other established cell lines in serum-free chemically defined medium A3. AGF is effective at less than 0.4 microgram per ml. By using the procedures described in the text, it is possible to culture HeLa cells in very simple media such as Eagle's basal medium. The properties of AGF are such that it may be adsorbed on glass or plastic flasks. Glass flasks treated with AGF retain full activity after washing with acetone, and treatment with ethyl ether and chemically defined medium. Adsorbed AGF is destroyed by trypsin. AGF can detoxify protamines, polylysines or histones. It will reverse the aggregation response induced by adding complexes composed of carboxymethylcellulose (CMC) and basic proteins. The results support the contention that highly adsorptive AGF functions at the cell surface and is capable of modifying the response of the cell to its environment.     

Cardiac tissue engineering: cell seeding, cultivation parameters, and tissue construct characterization.

Cardiac tissue engineering has been motivated by the need to create functional tissue equivalents for scientific studies and cardiac tissue repair. We previously demonstrated that contractile cardiac cell-polymer constructs can be cultivated using isolated cells, 3-dimensional scaffolds, and bioreactors. In the present work, we examined the effects of (1) cell source (neonatal rat or embryonic chick), (2) initial cell seeding density, (3) cell seeding vessel, and (4) tissue culture vessel on the structure and composition of engineered cardiac muscle. Constructs seeded under well-mixed conditions with rat heart cells at a high initial density ((6-8) x 10(6) cells/polymer scaffold) maintained structural integrity and contained macroscopic contractile areas (approximately 20 mm(2)). Seeding in rotating vessels (laminar flow) rather than mixed flasks (turbulent flow) resulted in 23% higher seeding efficiency and 20% less cell damage as assessed by medium lactate dehydrogenase levels (p < 0.05). Advantages of culturing constructs under mixed rather than static conditions included the maintenance of metabolic parameters in physiological ranges, 2-4 times higher construct cellularity (p &le 0.0001), more aerobic cell metabolism, and a more physiological, elongated cell shape. Cultivations in rotating bioreactors, in which flow patterns are laminar and dynamic, yielded constructs with a more active, aerobic metabolism as compared to constructs cultured in mixed or static flasks. After 1-2 weeks of cultivation, tissue constructs expressed cardiac specific proteins and ultrastructural features and had approximately 2-6 times lower cellularity (p < 0.05) but similar metabolic activity per unit cell when compared to native cardiac tissue.