Serum substitute in epithelial cell culture media: nonfat dry milk filtrate.

A number of milk types and milk fractions were investigated as possible substitutes for serum in cell culture media. A filtrate of reconstituted nonfat dry milk showed promise. Culture fluids containing 5% of the nonfat dry milk filtrate were used to propagate primary and continuous cell cultures, and the cell growth from these cultures was compared with that of cells grown in a serum-containing medium. The nonfat dry milk filtrate-supplemented medium supported the growth of all epithelial cells tested, but two fibroblast-type cultures failed to replicate. Cells grown in the medium containing the milk filtrate grew slowly for 2 to 3 days and then propagated to confluency in 6 to 8 days. Viable cell counts of 9 days were comparable to those of serum-grown cells that had been propagated for 7 days. Cells grown in the milk filtrate could be split 1 to 4 when subcultures were prepared. Cell growth could be stimulated by refeeding on days 2 to 3 or by the addition of 30 microM 2-mercaptoethanol to the growth medium. Virus susceptibility and titer comparisons with poliovirus 1, coxsackievirus B2, echovirus 7, and herpes simplex virus indicated that approximately the same data were obtained when either the nonfat dry milk filtrate-treated or the serum-treated cells were studied. The nonfat dry milk filtrate is inexpensive, is easily prepared, and is a substitute for serum in epithelial cell culture media.     

Concentrated Cultures of Leuconostoc citrovorum

Two single-strain cultures of Leuconostoc citrovorum were grown in a broth medium with automatic pH control. Culture concentrates were prepared by centrifugally harvesting the cells and resuspending them in 1/50th the original volume in 10% nonfat milk solids. The concentrates were stored in liquid nitrogen until analyzed. The maximum population attainable was approximately equal when cultures were grown at pH 6.0, 6.5, or 7.0 with sodium hydroxide or ammonium hydroxide as the neutralizer. Citrate was required in the growth medium for the cultures to be able to produce diacetyl subsequently in milk. At pH 6.0, the cells reached maximum population and ability to produce diacetyl. Organoleptic analysis by an experienced flavor panel showed a preference for cottage cheese creamed with a creaming mixture prepared with a culture concentrate rather than a normal culture. The culture concentrates maintained their viability and ability to produce diacetyl for at least 30 days when stored in liquid nitrogen.     

Effect of Benzyladenine, 2,4-Dichlorophenoxyacetic Acid, and d-Glucose on myo-Inositol Metabolism in Acer pseudoplatanus L. Cells Grown in Suspension Culture

Suspension cultures of Acer pseudoplatanus L. cells grown for 15 days in medium (T. Murashige and F. Skoog. 1962. Physiol. Plant. 15: 473-497) contained 3% sucrose, 1 mg/l 6-benzylaminopurine (BA), and 0.1 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D), referred to here as normal media, removed newly added myo-inositol-2-(3)H up to 100 mg/l in 24 hours and utilized up to 20% of this cyclitol for pectin biosynthesis. When the BA content of the growth medium was raised 10-fold, uptake of myo-inositol was drastically reduced and very little was available for pectin biosynthesis. Neither cell growth as measured by packed cell volume or by dry weight, nor monomer composition of pectic polysaccharides was affected by the increased level of cytokinin. Increasing, 2,4-d 10-fold instead of BA had little or no effect on myo-inositol uptake, although it did reduce the amount of myo-inositol utilized for pectin biosynthesis. Cells grown 15 days in normal media failed to remove added myo-inositol if 3% d-glucose was included. The net result was similar to that found in cells grown in the high BA condition. If a trace amount of d-galactose-1-(14)C was supplied to cells after 15 days of growth in normal, high BA, or high 2,4-d media, there was no significant variation in uptake and utilization of label among the three growth conditions.     

Culture-induced changes in osmolality of tobacco cell suspensions using four exogenous sugars

Suspension cultures of tobacco cells were grown in B5 media supplemented with sucrose, glucose, mannitol and sorbitol as exogenous sugars to examine culture-induced changes in the osmolality of the medium. Osmolality decreases were greatest in sucrose and glucose media during the 14 days in culture, and in glucose media were essentially linear, presumably reflecting the use of this sugar as a food source. Osmolality decreases occurred during the first week of culture in mannitol- and sorbitol-supplemented media, but later stabilized. Fresh weight of cultured cells in sucrose- and glucose-supplemented media increased by <200% during 14 days in culture, whereas cultured cells in mannitol- and sorbitol-supplemented media increased by only 39 and 48%, respectively. Cells transferred to the original liquid medium (B5 medium with 3% sucrose and 3% glucose) grew vigorously if they had been cultured in sucrose- and glucose-supplemented media; however, cells grown in mannitol- and sorbitol-supplemented media needed to be subcultured several times to recover their normal growth rate. By subculturing cells into increasingly higher conditions of sugars, cells tolerant to 560 mOsmol kg-1 H2O were obtained. The high osmolality-adapted cells increased by 140% in fresh weight in 8% glucose-supplemented medium. Glucose was best suited for producing the high osmolality required because sucrose concentrations at 10% sucrose and above resulted in cell death. To limit the decrease of osmolality in these suspension cultures requires changing the medium every 3 days to maintain osmolality above the 530 mOsmol kg-1 H2O needed to co-culture these as feeder cells with gametic and zygotic cells. This revised version was published online in June 2006 with corrections to the Cover Date.     

Injury and Death of Streptococcus lactis due to Freezing and Frozen Storage

Cells of Streptococcus lactis were harvested in the early stationary phase, washed, and resuspended in either skim milk (10% nonfat milk) or buffered distilled water (0.0003 m dipotassium phosphate, pH 7.2). Samples of each suspension were frozen and stored at -20 C for intervals up to 28 days. Colony counts of the frozen culture were made using lactic agar and a “restricted” lactic agar medium (Tryptone reduced to 0.5%) to determine injury and death. Death was determined by the difference in plate counts on lactic agar before and after freezing. Injured cells were determined by the difference in plate counts on the two plating media. Greatest injury of the cells occurred during early stages of frozen storage and decreased with time, and death continuously increased. Injury and death were more pronounced when cells were frozen in water than when frozen in 10% nonfat milk solids. Certain cultures survived better when frozen rapidly, whereas with others survival was greater when freezing was slow. Successive freezing, thawing, and propagation of the culture gradually eliminated cells which showed injury by freezing.     

Morphological and functional differentiation of cryopreserved lactating bovine mammary cells cultured on floating collagen gels

Cryopreserved bovine mammary epithelial cells prepared from lactating mammary tissue synthesize and secrete the milk proteins alpha_s1-casein, lactoferrin (Lf), and alpha-lactalhumin during in vitro culture on collagen gels in serum-free medium. Each milk protein is differently regulated by detachment and thickness of the collagen substratum, fetal calf scrum, and prolactin in the medium. Collagen detachment did not modulate lactoferrin secretion but strongly induced casein secretion, with detachment on day 6 (after formation of cell sheets) inducing casein secretion to 3 μg/ml medium, which was 2–3-fold higher than for cells on collagen detached on day 2 (prior to cell spreading to form sheets), and ten-fold higher than for cells grown on collagen not detached. Alpha-lactalbumin secretion was also induced, but only to low levels, in cells grown on detached but not on attached collagen. Cells grown on thin collagen gels secreted lower levels of lactoferrin and casein compared to cells on thick collagen. Lactoferrin but not casein secretion was increased in cells grown in the presence of fetal calf serum. Casein but not lactoferrin secretion was completely dependent on prolactin. Cells grown serum-free on collagen gels detached on day 6 of culture showed a polarized epithelial cell layer with high differentiation evidenced by the apical microvilli, tight junctions, and fat droplets surrounded by casein-containing secretory vesicles. An underlying layer of myoepithelial-like cells was also evident. These studies show for eryopreserved primary bovine mammary cells prepared from lactating mammary tissue the induction of highly differentiated and polarized cell morphology and ultrastructure with concomitant induction of the secretion of casein, lactoferrin. and alpha-lactalbumin in vitro, and that the non-coordinate regulation of milk protein secretion by substratum, prolactin, and serum likely involves alternate routing and control of secretion pathways for casein and lactoferrin.     

Demonstration of growth in porcine thyroid cell culture

Eagle's minimum essential medium supplemented with 20 per cent newborn calf serum (N.C.S.) allows porcine thyroid cell survival but not cell growth in vitro. In NCTC 109 medium supplemented with 20 per cent N.C.S. these cells actively grow and may be serially propagated. Cell population doubling time expressed as DNA doubling value is 3.5 days at 37 degrees C in 95 per cent air-5 per cent CO2. Thyrotropin does not affect porcine thyroid cell multiplication in vitro but stimulates the plating efficiency in primary cultures to about 130 per cent of controls. Cell selection was obtained by replacing media with Earle's balanced salt solution. This operation provoked death of nearly all cells by day 18 but subsequent addition of growth medium resulted in proliferation of epithelial cell clones. From generation 2 to generation 8, cells produce thyroglobulin but they do not actively trap iodide nor form follicles when thyrotropin is added to the media. Cell selection, demonstration of growth, as well as freeze-storage techniques described in this paper permit selection and storage of porcine thyroid cells and the potential constitution of cell collections.     

Comparative analysis of casein synthesis during mammary cell differentiation in collagen and mammary gland development in vivo

Substrata upon which epithelial cells are cultured modulate their morphology,growth, and ability to differentiate. Mouse mammary epithelial cells cannot be induced to synthesize caseins, a marker of cell differentiation, when grown on a plastic surface. An analysis was made of the effect of time within a collagen matrix on the ability of normal mammary epithelial cells to be induced to synthesize caseins and that response was compared to mammary gland development in vivo. Primary cultures of mammary cells from unprimed virgin BALB/c mice were embedded in rat-tail collagen gel mixtures and maintained in growth medium. Induction medium containing lactogenic hormones was added at various times. The cells were monitored every 3-7 days over a period of 8 weeks for cell growth, casein synthesis, and ability to grow in vivo in cleared mammary fat pads. Casein accumulation was assayed quantitatively by an ELISA competition assay and qualitatively by the immunoblot procedure using specific antisera prepared against purified mouse caseins. No marked differences in cell numbers and transplantability potential were observed among cells cultured for various times in collagen. Mammary cells grown in collagen for up to 8 weeks retained the capacity to grow in vivo as normal ductal outgrowths. The duration of culture within collagen prior to hormonal stimulation did influence the kinetics of casein synthesis. Cells cultured for 1 week in growth medium did not accumulate detectable levels of casein until after 3 weeks of induction, whereas cells cultured for 2 or 4 weeks responded by accumulating caseins after 2 weeks and 3 days of induction, respectively. While the levels of total caseins that accumulated under optimal conditions of induction in culture approached levels found during lactation in vivo, the relative proportion of specific casein polypeptides synthesized in culture was altered from alpha casein (43K) in favor of the beta casein (30K) species. These results suggest that a period of culture within collagen is required to permit mammary epithelial cells to become responsive for hormone-induced differentiation. It is possible that during growth within the collagen the cells synthesize and deposit extracellular matrix components important in modulating gene expression.     

Cell culture factors influencing in vitro expression of mouse mammary tumor virus.

Several cell culture factors were found to influence in vitro expression of mouse mammary tumor virus (MMTV) in the mouse adenocarcinoma cell line Mm5mt/c1. Cells were propagated in a variety of commercially available cell culture media to which dexamethasone (DXM) was added as a stimulator of MMTV production. Culture seeding density, culture medium type, and glucose concentration each influenced MMTV production when expressed on a per cell basis. Maximum cell growth occurred in cultures grown in RPMI-1640 medium containing insulin. Those media which provided good cell growth were not necessarily optimal for virus expression. Addition of insulin did not stimulate MMTV synthesis although dexamethasone alone was stimulatory in all media used; however, maximum MMTV expression was obtained when dexamethasone and insulin were used in concert. Equivalent levels of MMTV-specific cell membrane antigen, MMTV-specific protein, and virus particles were produced at incubation temperatures of 32 degrees, 34 degrees or 37 degrees C; however, higher levels of virus-related RNA-dependent DNA polymerase (RDDP) activity were recovered from cultures incubated at 32 degrees and 34 degrees C than at 37 degrees C. Decreased levels of RDDP were attributed to enzyme thermolability at 37 degrees C incubation.     

Primary cultures of normal rat kidney proximal tubule epithelial cells for studies of renal cell injury

Summary Normal rat kidney proximal tubule epithelial cell cultures were obtained by collagenase digestion of cortex and studied for 10 days. To assess the purity of the seeding suspension, we histochemically demonstrated γ-glutamyltranspeptidase in >95% of the starting material. To identify cell types in cultures, we investigated several markers. Cells stained positively for lectinArachis hypogaea (rat proximal tubule) and negatively forLotus tetragonolobus (rat distal tubule). Intermediate filament expression of cytokeratin confirmed the epithelial differentiation of the cultured cells. Using indirect immunofluorescence, we found that cultures were negative for vimentin and Factor VIII. Cells exhibited activities of two brush border enzymes, γ-glutamyltranspeptidase and leucine aminopeptidase, and Na⁺-dependent glucose transport activity. Multicellular domes were evident in the Week 2 of culture. Proliferation was studied by comparing growth factor-supplemented serum-free medium to cells grown in serum; growth enhancers included insulin, hydrocortisone, transferrin, glucose, bovine albumin, and epidermal growth factor. Cells proliferate best in medium with 5 or 10% serum and in serum-free medium supplemented with insulin, hydrocortisone, transferrin, glucose, and bovine albumin. Proliferation was assessed by determining cell number (population doublings). By light microscopy, the cells were squamous with numerous mitochondria, a central nucleus, and a rather well-defined homogeneous ectoplasm. By electron microscopy, the cells were polarized with microvilli and cell junctions at the upper surface and a thin basal lamina toward the culture dish. These data show that the proximal tubule epithelial cells retain a number of functional characteristics and that they represent an excellent model for studies of normal and abnormal biology of the renal proximal tubule epithelium. This project was supported by grant 2-R01-DK15440-16A1 from the National Institutes of Health, Bethesda, MD, and by grant N0001 4-88-K-0427 from the Department of the Navy, Washington, DC.     

Functional differentiation and primary metabolism of mouse mammary epithelial cells in extended-batch and hollow-fiber culture

Growth, expression of functional differentiation (as characterized by synthesis and secretion of milk proteins), and primary metabolism were studied for a mouse mammary epithelial cell line, COMMA-1D, in extended-batch and hollow-fiber reactor cultures. Batch cultures were performed on Costar polycarbonate membrane inserts, allowing basal and apical exposure to medium. Protein production was induced in both batch and hollow-fiber cultures in hormonesupplemented medium. In batch cultures, high levels of protein production and secretion were maintained for 18 days. Once differentiation was induced, the rate of deinduction was low, even in medium containing epidermal growth factor (EGF) and serum; cells continued to express and secrete proteins for at least 12 days after prolactin and hydrocortisone were removed. Cells in both batch and hollow-fiber cultures were highly glycolytic and exhibited low rates of glutaminolysis. In batch culture on membrane inserts, cells showed polarized metabolism between the apical and basal side, maintaining significant gradients of glucose and lactate. Medium hormonal composition and subsequent differentiation affected both glucose uptake and lactate yield for COMMA-1D in batch culture. (c) 1992 John Wiley & Sons, Inc.     

In vitro culture of cryopreserved bovine mammary cells on collagen gels: synthesis and secretion of casein and lactoferrin

The preparation, cryopreservation, and culture on type I collagen gels of lactating bovine mammary cells with prolonged milk protein synthesis and secretion in vitro is described. Cryopreserved cells prepared as acinar fragments from either lactating or developing mammary glands attached to the collagen substratum within 24-48 hr after plating in serum and hormone supplemented medium. During continued culture in hormone-supplemented (insulin, cortisol, and prolactin) serum-free medium outgrowth of cells from the attached acinar fragments was observed beginning on day 2, with continued outgrowth to near confluence by day 6. Two morphologically distinct cell types were evident; initial outgrowth was by large polygonal cells that were subsequently overlain by spindle-shaped cells. Cells from both lactating and developing mammary glands sustained substantial milk protein secretion for at least 14 days in culture. Alpha S1-casein synthesis and secretion in cultures of lactating mammary cells was dependent on a critical minimum cell population density, below which alpha S1-casein was not secreted. In contrast, lactoferrin (LF) secretion into the medium increased linearly with the increase in cell population density. Cells cryopreserved up to 16 months secreted LF at levels comparable to fresh cultures of the same cells.     

PROTEIN AND AMINO ACID COMPOSITION OF THE OBLIGATE HALOPHILE APHANOTHECE HALOPHYTICA (CYANOPHYTA)1

Total protein was determined for cells of Aphanothece halophytica Fremy harvested during early log, mid-log and linear growth phases in media containing 1, 2, and 3 M NaCl. Cells grown in medium containing 1 M NaCl showed a progressive increase in protein content up to a maximum of 76% of dry weight (linear phase). Total protein also increased in cells grown in 2 M NaCl. medium (56.5–72.0%). Cells grown in 3 M NaCl medium showed a progressive decrease in total protein (59.9–43%). Although amounts of protein varied, the percentages of the respective amino acids of hydrolyzed bulk protein were consistent to within 1% for linear phase cells grown in 1, 2, and 3 M NaCl cultures. Percentages of acidic amino acids were 2.3–2.6 times greater than those of the basic amino acids. The amino acid composition of phycocyanin was similar to that of bulk protein. Free amino acids varied with both age of the culture and the concentration of NaCl. The high quantity and quality of the protein observed suggest that A. halophytica might be a useful food organism.     

Naphthoquinone contents of in vitro cultured plants and cell suspensions of Dionaea muscipula and Drosera species

In vitro cultured carnivorous plants were grown on a hormone-free medium. They produced the following naphthoquinones: Dionaea muscipula (plumbagin: 5.3%), Drosera rotundifolia (7-methyljuglone: 0.6%), D. binata (plumbagin: 1.4%), and D. capensis (7-methyljuglone: 0.5%). A red, slow-growing suspension culture of D. muscipula was maintained in a modified McCowns Woody Plant (McC) medium and produced plumbagin (2.59%) after 30 days growth. A suspension culture of D. rotundifolia grew slowly as multicoloured small aggregates only in a modified Murashige and Skoog (MS) medium. No quantifiable amounts of naphthoquinones were produced. Several cell lines of D. capensis were developed. Green aggregates grown in a modified MS medium contained 7-methyljuglone (0.33%) and differentiated into plants when placed onto hormone-free medium. Pink cultures grown in modified McC medium contained 7-methyljuglone (1.24%), while dark red cultures produced ca. 1% in both modified McC and MS media. Though the latter medium was significantly better with regard to biomass production, cells excreted a mucin when cultured in both media (0.21 g dry mucin/g dry cells in McC) and (0.16 g dry mucin/g dry cells in MS). Effects of the presence or absence of light during the growth period of 30 days showed that there was no effect on biomass and only slight effects on mucin production and naphthoquinone contents.     

The turkey myogenic satellite cell: optimization of in vitro proliferation and differentiation

Myogenic satellite cells were isolated from the pectoralis major muscle of young growing tom turkeys. These cells were capable of proliferating and forming large multinucleated myotubes in vitro. Of 36 media-sera combinations evaluated, McCoy's 5A medium containing 15% chicken serum (CS) promoted the greatest level of proliferation and subsequent myotube formation when cells were induced to differentiate (P less than 0.05). Myotube formation was maximized following exposure of cultures to Dulbecco's Modified Eagle's Medium (DMEM) containing 1% horse serum (HS; DMEM-1% HS) for 4 days. Satellite cells grown under these conditions generally resulted in cultures containing greater than 90% fused nuclei. Cells plated in the presence of DMEM-10% HS resulted in greater attachment and larger cultures (and consequently a greater fused nuclei number) when transferred to growth media than similarly grown cultures plated in McCoy's 5A medium-10% CS, regardless of substrata tested (P less than 0.05). The greatest proliferation and myotube formation was seen in cultures grown in gelatin-coated wells. Proliferation was maximized in McCoy's 5A medium containing 18% CS, although this was not significantly different than the proliferation with media containing 15% CS (P greater than 0.05). Our results (1) document that the postnatal myogenic satellite cell can be isolated from the turkey in sufficient quantities for biological studies and (2) identify culture conditions which optimize proliferation and differentiation of these cells in vitro.     

A serum-free, chemically-defined medium for function and growth of primary neonatal rat heart cell cultures

A serum-free, chemically defined medium for supporting rhythmic contraction, maximum survival, and moderate growth of cardiac cells was achieved by using a combination of hormones and growth supplements in a mixture of equal volumes of Ham's F12 and Dulbecco's modified Eagle's medium. The hormones and growth supplements included insulin, transferrin, selenium, fetuin, bovine serum albumin, hydrocortisone (HC), L-thyroxine (T4), and epidermal growth factor (EGF). Cardiac cells were grown on fibronectin-precoated plates using the above serum-free medium. Cells grown in this medium exhibited a higher beating rate and were maintained for a longer time compared to those cells grown in serum. The effects of T4, EGF, and HC on beating rate and survival time of both cultures of mixed cell population and enriched myoblast cell population were studied. In the enriched myoblast cell cultures grown in serum-supplemented medium, the beating rate ranged from 40 to 200 beats/min, and these cultures survived for 30 d. When these enriched cell cultures were grown in serum-free hormone-supplemented medium, the beating rate ranged from 190 to 240 beats/min, and these cultures survived for more than 90 d. These results show that some hormones affect growth, whereas others affect function.     

Bovine colostrum or milk as a serum substitute for the cultivation of a mouse hybridoma

A mouse-mouse hybridoma was grown in serum-free medium supplemented with bovine milk or colostrum. Bovine colostrum supported growth of the hybridoma whereas bovine milk alone did not support cellular proliferation. For growth in medium supplemented with colostrum, the maximum cell concentration achieved was 1.4 x 10(6) cells/mL in 2.2% colostrum, which is 44% of that obtained in 9% serum. When cells were grown in media containing milk and low amounts of serum (<1%) the maximum cell concentration in 2.2% milk with 0.4% serum was 2 x 10(6) cells/ml, whereas it was only 0.2 x 10(6) cells/ml and 1.3 x 10(6) cells/ml in 2.2% milk alone and 0.4% serum alone, respectively. Similar behavior was observed for growth in media containing colostrum and low amounts of serum. The monoclonal antibody production in media containing combinations of serum and milk or colostrum was comparable to that obtained in media with higher serum concentrations. Experiments performed with conditioned media suggest that the rapid decrease in viability, after the maximum cell concentration has been reached, is partially due to the presence of some inhibitory components generated during the cell culture rather than due to depletion of some serum components.     

The Influence of Calcium or Manganese on the Resistance to EDTA, Polymyxin B or Cold Shock, and the Composition of Pseudomonas aeruginosa Grown in Glucose- or Magnesium-depleted Batch Cultures

Cultures were batch grown in simple salts media in which growth was limited either by depletion of glucose and magnesium (C/Mg-dep) or by glucose alone (C-dep). Cultures were also grown in these media supplemented by calcium and/or manganese.
All cultures grown in the C-dep media were sensitive to ethylenediaminetetraacetic acid (EDTA), polymyxin and also to cold shock but were relatively resistant to ethyleneglycol-bis(2-aminoethyl ether)-N, N-tetraacetic acid (EGTA). Inclusion of calcium or manganese in the growth medium enhanced lysis by EDTA. Cultures grown in the basic C/Mg-dep media were resistant to EDTA, EGTA, polymyxin and to cold shock. Sensitivity to these agents was retained by cultures grown in C/Mg-dep media supplemented with Ca²⁺ and/or Mn²⁺. Cells grown in C/Mg-dep media with added Mn²⁺ were more sensitive to EDTA and polymyxin than those from the unsupplemented C/Mg-dep media but still resistant compared with C-dep cultures. All cultures from supplemented C/Mg-dep media were more sensitive to EGTA than those from any of the C-dep media.
Whole cells and cell walls from these various media had differing amounts of cell wall, phosphorus, amino sugar, carbohydrates, readily extractable lipid (REL), total phospholipid (PL), and especially differences in cell wall divalent metal cation content.
The differences in PL, REL and amino sugars and carbohydrate did not correlate with the response of C-dep and C/Mg-dep bacteria to EDTA, EGTA or polymyxin. The results are discussed in relation to the hypothesis that the sensitivity of Pseudomonas aeruginosa to polymyxin and EDTA is more dependent on outer membrane cation content rather than on other components, e.g. PL and lipopolysaccharide.     

Biochemical and morphological characterization of growth and differentiation of normal human neonatal keratinocytes in a serum-free medium

Growth and differentiation of keratinocytes in a serum-free medium (keratinocyte growth medium or KGM) was studied and compared to that under conditions in which serum and feeder cell layers were used. Cells were grown in KGM containing 0.1 mM calcium (KGM/low calcium), KGM containing 1.2 mM calcium (KGM/normal calcium), or Dulbecco's modified Eagles medium containing 5% fetal calf serum and 1.8 mM calcium in presence of mitomycin treated 3T3 M cells (DMEM/5% FCS). Plating efficiency and rate of growth were similar in the three media till confluence. In postconfluent cultures, protein and DNA content of cells attached to the plate in KGM/low-calcium dishes decreased as an increased number of cells were shed into the medium. Cell shedding was much less evident in the presence of normal calcium. Cells grown in KGM/low calcium had a higher rate of cell proliferation (3H-thymidine incorporation into cellular DNA) than cells grown in normal calcium. Transglutaminase activity, involucrin content, and cornified envelope formation were greatest in cells grown in KGM/normal calcium, intermediate in cells grown in DMEM/5% FCS, and least in cells grown in KGM/low calcium. Keratin profiles from cells grown in KGM/low calcium showed a lower percentage of high molecular weight bands compared to the keratin profiles from cells grown in the presence of normal calcium. Keratinocytes in KGM/low calcium grew as a monolayer of cuboidal cells with few features of differentiation, whereas cells grown in KGM/normal calcium stratified into multilayered islands (3-5 layers) surmounted by 2-4 layers of enucleated cells with thickened cornified envelopes. Cells grown in KGM/normal calcium also contained tonofilaments and lamellar bodies unlike cells grown in KGM/low calcium. Cells grown in DMEM/5% FCS also formed stratified layers comparable to cells grown in KGM/normal calcium but lacked cornified cells, keratohyalin granules, tonofilament bundles, and lamellar bodies. These studies indicate the usefulness of serum-free conditions for the culture of human keratinocytes and confirm the importance of extracellular calcium in keratinocyte differentiation.     

Analysis of cell size and DNA content in exponentially growing and stationary-phase batch cultures of Escherichia coli.

Escherichia coli strains were grown in batch cultures in different media, and cell size and DNA content were analyzed by flow cytometry. Steady-state growth required large dilutions and incubation for many generations at low cell concentrations. In rich media, both cell size and DNA content started to decrease at low cell concentrations, long before the cultures left the exponential growth phase. Stationary-phase cultures contained cells with several chromosomes, even after many days, and stationary-phase populations exclusively composed of cells with a single chromosome were never observed, regardless of growth medium. The cells usually contained only one nucleoid, as visualized by phase and fluorescence microscopy. The results have implications for the use of batch cultures to study steady-state and balanced growth and to determine mutation and recombination frequencies in stationary phase.