Improved Chemically Defined Basal Medium (CMRL-1969) for Primary Monkey Kidney and Human Diploid Cells

An improved tissue culture basal medium, CMRL-1969, supplemented with serum, has been evaluated by measuring the growth responses of primary cultures of trypsin-dispersed monkey kidney cells (PMKC) and of an established culture of a human diploid cell strain (HDCS). Medium H597, an early modification of medium 199 which has been used successfully in the preparation of poliomyelitis vaccine for 15 years, was used for comparison. In addition, parallel testing was done with Basal Medium Eagle (BME) widely used for the growth of HDCS. The improvements in basal medium CMRL-1969 are attributed to changes in amino acid concentrations, in vitamin composition, and, in particular, to enhanced buffering capacity. The latter has been achieved by the use of free-base amino acids and by increasing the dibasic sodium phosphate. The new medium has already been used to advantage for the production of polioviruses in PMKC where equivalent titers were obtained from cultures initiated with 70% of the number of cells required with earlier media. The population-doubling time was reduced in this system. Also, with small inocula of HDCS, the time required to obtain maximum cell yield was shorter with CMRL-1969 than with BME. Both media were supplemented with 10% calf serum. Maximum cell yields after repeated subcultivation in the new basal medium were greatly increased and the stability of the strain, as shown by chromosomal analysis, was not affected. Basal medium CMRL-1969 can be prepared easily in liquid or powdered form.     

A comparison of different nutrient media and supplementation with dexamethasone for mouse colon organ culture

Several complex nutrient media were compared for their effectiveness in maintaining viable and functional mouse colon mucosa in organ culture. The order of superiority for preserving survival of normal tissues for 14 days was: Williams' Medium E > Morgan's 199 > CMRL-1066 > Waymouth's MB 752/1 > Eagle's MEM > Trowell's T8. The ₃H-thymidine labeling index was highest in colon explants maintained in Morgan's 199 > Williams' Medium E > Waymouth's MB 752/1 > CMRL-1066 > Eagle's MEM. However, the very high labeling produced by Morgan's 199 medium was abnormal in comparison to in vivo levels. Supplementation with 1.0 µM dexamethasone almost always improved crypt survival and maintained normal DNA synthetic activity.Supported by National Cancer Institute contract No. 1-CP-75952 and grant No. CA-29602.     

Growth of Mycoplasma hyorhinis cultivar alpha on semisynthetic medium.

Serial passage of Mycoplasma hyorhinis cultivar alpha (formerly noncultivable strains) has been accomplished in modified CMRL-1066 medium with fetal bovine serum. In modified CMRL-1066 liquid medium, cultivar alpha strains grow at a similar rate and to equivalent titers when compared with BTS-7, the type strain of the species. Further experiments with BTS-7 demonstrate that the extent of growth obtained in the semidefined medium was comparable to growth in conventional mycoplasma medium. M. hyorhinis strains, including cultivar alpha strains, grow in serial passage when fetal bovine serum is replaced with bovine serum albumin, palmitic acid, and cholesterol. The results of these studies show that M. hyorhinis cultivar alpha strains are not nutritionally more fastidious than other mycoplasmas but that they are noncultivable on standard mycoplasma media because they are sensitive to high levels of inhibition activity by medium components.     

Different performances of soil phosphate tests for reflecting the effects of buffering capacity on uptake of native phosphate with time

The ability of two sodium bicarbonate (Colwell and Olsen) and two ammonium fluoride (Bray I and Bray II) soil tests to reflect the effect of phosphate buffering capacity of the soil on plant growth through time was studied on ten Argentine soils. The soils were divided into three groups (low, medium and high buffering capacity) according to a buffering index calculated from the slope of the Freundlich equation. The relation between phosphate extracted by soil tests and both relative yield and phosphate uptake of rye grass plants was affected by the phosphate buffering capacity of the soil. The effect of buffering on that relation was more marked for the sodium bicarbonate tests (specially Colwell) than for the Bray tests. This effect was consistent with time. Hence, adjustment for buffering would be more important for the sodium bicarbonate tests than for the Bray tests. Soils with high buffering capacity were able to sustain a greater rate of phosphate uptake. The effect of buffering on the relation between soil tests and both relative yield and phosphate uptake was greatest when the plants were young and decreased with time. This effect would therefore be very important for the early nutrition of annual pasture or crop species.     

CULTIVATION OF MAMMALIAN CELLS IN DEFINED MEDIA WITH PROTEIN AND NONPROTEIN SUPPLEMENTS

An improved chemically defined basal medium (CMRL-1415) has been used to advantage in studying the effects on trypsinized, newly explanted mouse embryo cells of certain glycoproteins from plasma and serum, certain nonprotein macromolecules, and various combinations of these, in stationary cultures. When protein and nonprotein fractions were separated from fetal calf serum, the entire growth activity was found to be associated with the protein. When 100 mg % of dialyzed, freeze-dried, supernatant solution of Cohn's fraction V (method 6) from human plasma was used as a supplement for CMRL-1415, there was considerable improvement in the cultures; and seromucoid prepared from calf serum had a similar effect. Supernatant V was further fractionated by gel filtration to give a threefold concentration of growth activity in a single, highly purified α₁-acid glycoprotein (orosomucoid). Starch gel electrophoresis of horse serum that was used to supplement the basal medium revealed a decrease of both α₁-acid glycoprotein and α₂-macroglobulin during the cultivation of mouse embryo cells. When horse serum was fractionated on DEAE-cellulose columns, the only fraction that showed growth activity was a slow α₂-globulin. When the α₂-macroglobulin of Schultze was prepared from horse serum by salt precipitation, it was equally effective. When the α₂-macroglobulin from horse serum was tested (at 100 mg %) in combination with α₁-acid glycoprotein from Supernatant V, seromucoid from calf serum, or unfractionated Supernatant V, the growth response was greatly in excess of that produced by any of these supplements tested separately. The α₂-macroglobulin from horse serum could be replaced by certain nonprotein macromolecules (e.g., dextran or Ficoll). Thus, dextran (mol. wt. 100,000 to 200,000) had no visible effect on the cells when used alone at 0.1 or 1%. But when these levels of dextran were used in combination with low molecular weight glycoproteins (e.g., unfractionated Supernatant V at 100 mg %), the cultures remained active and healthy for unusually long periods.     

Chemically defined and serum supplemented media effects on mouse embryo development

Mouse embryos at various stages of development were used to test three types of media: TC199, CMRL-1066 and TALP. The effect of 20% human cord serum (HCS) and fetal calf serum (FCS) were also compared in TC199 and CMRL-1066 media. Embryos were recovered at the two, four and eight cell stages and assessed for at least one cleavage progression in 24 hours. There was no difference in embryo growth rates between the media for four and eight cell embryos, however TALP significantly increased the proportion of two cell embryos which underwent at least one cleavage division. HCS significantly promoted a greater number of cleavage divisions compared with FCS. This study indicates that the defined medium (TALP) can be employed for equal or increased growth rates of early mouse embryos cultured in vitro compared to two serum supplemented media (TC199 and CMRL-1066).     

Stabilization of the synthetic media for plant tissue and cell cultures

In standardMurashige-Skoog medium, particularly at pH higher than 5.0 and after heat sterilization, there is a tendency for turbidity or a sediment to appear, and for the acidity to increase by 0.2 to 0.5 degrees pH. The sediment is an amorphous precipitate of ferric phosphate and partly also of ferrous phosphate. In a stock iron solution prepared by chelation of ferrous sulphate with an equimolar quantity of the complexone Na₂EDTA. up to 10% free Fe^II ions could be detected. By titration of a concentrated complexon solution it was found that in the presence of an excess of Na₂EDTA (at the approximate molar ratio Fe^II: Na₂EDTA 1: 2) chelation of this free iron takes place to such an extent that its concentration falls to as little as 0.1%. Media with iron stabilized in this way are quite clear and maintain the adjusted pH for up to several weeks. The heat sterilization, too, does not lead to any precipitation or to a shift in pH within the broad range of adjusted values pH 4.8 – 6.0. We also attempted to increase the relatively low buffering capacity of Murashige-Skoog medium. The addition of sodium citrate (1.25 mmol 1^-1) and particularly of citrate-phosphate buffer (at a final concentration of 1.97 mmol citric acid and 6.07 mmol dibasic sodium phosphate per litre of medium) to the Murashige-Skoog medium considerably increased its buffering capacity, so that at the end of the subculture interval of tobacco cell suspensions the adjusted acidity changed only slightly (pH 5.40 ± 0.15). A thorough evaluation of the growth parameters of tobacco batch cultures (cell counts, vital staining, kinetics of DNA and protein synthesis) failed to reveal any negative effect either of additional chelation or of the buffering components.     

Differential preimplantation development of rhesus monkey embryos in serum-supplemented media

Several media, some augmented with amino acids, have been formulated recently, based on simplex optimization, to support the preimplantation development of mouse embryos. For the highly limited studies on preimplantation development of nonhuman primate embryos, a complex medium (CMRL-1066) has been employed. Our objective was to compare the developmental ability of rhesus monkey embryos in a simple medium containing amino acids, KSOM/AA, with the complex media used previously. Zygotes (99) were recovered following in vitro fertilization (IVF) from six monkeys, allocated to either CMRL or KSOM/AA both containing 10% fetal calf serum (FCS), and monitored daily until reaching the expanded or hatched blastocyst stage. The distribution of cells between the inner cell mass (ICM) and trophectoderm was determined at the end of culture by differential nuclear staining. Although a greater number of embryos cultured in KSOM/AA vs. CMRL developed to the morula stage (80%) and beyond (66% to expanded blastocyst), the differences were not significant. Such embryos in KSOM/AA did, however, develop at a significantly faster rate, on average, reaching the expanded blastocyst stage 26 hr earlier than did embryos cultured in CMRL. KSOM/AA embryos hatched in less time and had a higher percentage (43 vs. 34) of cells allocated to the ICM. These results indicate that a simple medium, KSOM/AA, in the presence of serum, supports the development of rhesus monkey embryos at high efficiency and at a faster rate than that observed for embryos cultured in the complex medium, CMRL-1066. © 1996 Wiley-Liss, Inc.     

Obtaining Suspensions of Animal Cells in Metaphase from Cultures Propagated on Glass

Cells of some strains continuously cultivated in vitro have a pronounced tendency to lose their attachment to glass during mitosis. If division of these cells is arrested in meta-phase by treatment with colchicine, many of the cells are set free and float into the medium. Advantage has been taken of this property to collect large numbers of suspended cells in c-metaphase. By applying standard procedures of staining with orcein and manual squashing to these cells, preparations are obtained that are useful for critical chromosome analysis. The procedure consists in treatment of cultures with colchicine (final concentration 0.1 μg/ml) for 18-24 hr; agitation in hypotonic medium (quarter-strength Tyrode's or CMRL-1066) for 5 min; addition of 1:3 acetic-alcohol for 5-10 min; removal and centrifugation of the resulting fluid mixture from the culture; and resuspension of the sedimented cells in a small volume of the fluid. Advantages of the method are: large numbers of metaphase plates can be examined on a single slide, the chromosomes are contracted and well separated, and cytoplasmic boundaries are preserved.     

Increased degradation rate of nitrososureas in media containing carbonate

The stability of two nitrosoureas, tauromustine and lomustine, has been investigated in different media and buffers. All media tested, except Leibovitz's L-15 medium, significantly increased the degradation rate of the investigated nitrosoureas at pH 7.4. Sodium bicarbonate seems to be the cause of the observed increase of the degradation rate, since it provides the main buffering capacity of all the media except for Leibovitz's L-15 medium, which is based on phosphate buffer. Other ingredients in the media, such as amino acids, vitamins, and inorganic salts, or the ionic strength of a buffer, did not have any major effect on the degradation rate of the nitrosoureas. These results suggest that media containing carbonated buffer should be avoided when the anti-tumor effect of nitrosoureas is to be investigated in different cell cultures.     

Cytosolic pH Variations in Perfused Rat Liver at 4°C: Role of Intracellular Buffering Power

The effect of low temperature on cytosolic pH regulation and buffering capacity was evaluated in the isolated rat liver. The pH changes were followed by phosphorus-31 nuclear magnetic resonance. Cooling from 37 to 4°C, with Krebs–Heinseleit perfusion at an external pH of 7.35, induced an alkaline shift in cytosolic pH (pH_cyt) of 0.13 or 0.75 pH units in the presence or absence of bicarbonate, respectively (dpH_cyt/dT values were 0.004 and 0.022 unit/°C). With 4°C perfusion, in the presence or absence of bicarbonate, acute changes of external pH (from 7.40 to 5.90) did not affect pH_cyt. In contrast, intracellular loading with isobutyric acid or NH₄Cl induced rapid pH_cytvariations. The intrinsic buffering power value (10 to 50 slykes) measured in the absence of bicarbonate depended on pH_cyt. The larger value was observed for pH_cyt7.30, a value near the pK value of the imidazole group of intracellular proteins at 4°C. The presence of bicarbonate modified the amplitude of the pH_cytchange by increasing the total buffering power. It was demonstrated that during hypothermia, ionic carriers are inactivated and the charged forms of molecules are unable to cross the cell membrane; thus, the pH_cythomeostasis depends essentially on intracellular buffering power.     

Explant culture of rat esophagus in a chemically defined medium

Summary Esophagus from adult male CDF rats was cultured for a period of 28 d in CMRL-1066 medium supplemented with pyruvic acid, HEPES buffer, β-retinyl acetate, and antibiotics. Morphological, radioautographic, and biochemical studies indicated that the survival of the tissue in serum-free medium was equivalent to that in medium containing 5% heat-inactivated fetal bovine serum. There was a relatively constant uptake of [³H]thymidine into DNA and [³H]leucine into protein of the esophageal explants during the incubation. Only the basal cells of the epithelium incorporated [³H]thymidine into their nuclei. The normal morphology of the tissue was preserved when the explants were maintained at both 37 and 30° C, and in either 50 or 20% O₂. Ninety-five percent O₂ was highly toxic to the cells of the explants. This culture system should be suitable for a variety of investigations in esophageal cell differentiation and carcinogenesis.     

pH and buffer capacities of apoplastic and cytoplasmic cell compartments in leaves

After opening the stomata in CO₂-free air, darkened leaves of several plant species were titrated with CO₂ at concentrations between 1 and 16%, in air in order to reversibly decrease cellular pH values and to calculate buffer capacities from pH changes and bicarbonate accumulation using both gas-exchange and fluorescence methods for analysis. After equilibration with CO₂ for times ranging between 4.4 and 300 s, fast CO₂ release from bicarbonate indicated catalysis by highly active carbonic anhydrase. Its time constant was below 2.5 s. Additional CO₂ was released with time constants of about 5, 15 and approximately 300 s. With CO₂ as the acidifying agent, calculated buffer capacities depend on assumptions regarding initial pH in the absence of an acid load. At an initial stroma pH of 7.7, the stromal buffer capacity was about 20 mM pH-unit⁻¹ in darkened spinach leaves. At an initial pH of 7.5 it would be only 12 mM pH-unit⁻¹, i.e. not higher than expected solely on the basis of known stromal concentrations of phosphate and phosphate esters, disregarding the contribution of other solutes. At a concentration of 16%, CO₂ reduced the stromal pH by about 1 pH unit. Buffering of the cytosol was measured by the CO₂-dependent quenching of the fluorescence of pyranine which was fed to spinach leaves via the petiole. Brief exposures to high CO₂ minimized interference by effective cytosolic pH regulation. Cytosolic buffering appeared to be similar to or only somewhat higher than chloroplast buffering if the initial cytosolic pH was assumed to be 7.25, which is in accord with published cytosolic pH values. The difference from chloroplast pH values indicates the existence of a pH gradient across the chloroplast envelope even in darkened leaves. Apoplastic buffering was weak as measured by the CO₂-dependent quenching of dextran-conjugated fluorescein isothiocyanate which was infiltrated together with sodium vanadate into potato leaves. In the absence of vanadate, the kinetics of apoplastic fluorescence quenching were more complex than in its presence, indicating fast apoplastic pH regulation which strongly interfered with the determination of apoplastic buffering capacities. At an apoplastic pH of 6.1 in potato leaves, apoplastic buffering as determined by CO₂ titration with and without added buffer was somewhat below 4 mM pH-unit⁻¹. Thus the apoplastic and cytosolic pH responses to additions of CO₂ indicated that the observed cytoplasmic pH regulation under acid stress involves pumping of protons from the cytosol into the vacuole of leaf cells, but not into the apoplast. Received: 27 November 1998 / Accepted: 22 March 1999     

Buffering as a means for increasing growth and butanol production byClostridium acetobutylicum

Summary The objective of this work was to optimize butanol formation in the acetone-butanol-ethanol (ABE) fermentation by examining the level of buffering as it affects the dissociation of butyric acid to the less toxic butyrate anion. Experiments were carried out in batch culture using chemically defined (P2) or complex media containing various buffering agents. These included salts of acetate, citrate, phosphate, nitrate, or bicarbonate, representing a range of pK _a values and buffering capacities. Growth in highly buffered medium was found to increase the stationary phase cell density, carbohydrate utilization, and the final butanol concentration. At higher levels of buffering, increased growth and elevated concentrations of butyric acid were required to initiate solventogenesis, suggesting the involvement of a critical threshold level of undissociated butyric acid.     

Explant culture of rat colon: A model system for studying metabolism of chemical carcinogens

Summary An explant culture system has been developed for the long-term maintenance of colonic tissue from the rat. Explants of 1 cm² in size were placed in tissue-culture dishes to which was added 2 ml of CMRL-1066 medium supplemented with glucose, hydrocortisone, β-retinyl acetate, and either 2.5% bovine albumin or 5% fetal bovine serum. The dishes were placed in a controlied-atmosphere chamber which was gassed with 95% O₂ and 5% CO₂. The chamber then was placed on a rocker platform which rocked at 10 cycles per min causing the medium to flow intermittently over the epithelial surface. The explants were incubated at 30°C. The viability of the tissue was measured both by incorporation of specific precursors into cellular macromolecules and by monitoring of tissue morphology with light and electron microscopy. Cultured rat colon was able to metabolize benzo[a]pyrene, 7,12-dimethylbenz[a]anthracene, aflatoxin B₁, dimethylnitrosamine, 1,2-dimethylhydrazine, and methylazoxymethanol acetate into chemical species that bind to cellular DNA and protein.     

Cytotoxicity of cysteine in culture media

Summary When added to Eagle’s Minimum Essential Medium supplemented with 10% bovine serum (MEM-10BS), 1mM cysteine was highly toxic to cultured cells. This toxicity was eliminated by (a) preincubation of the medium at 37°C for 24 hr before use, or (b) presence of 5mM pyruvate. Similar results were obtained with freshly prepared CMRL 1066 supplemented with 10% bovine serum (CMRL-10BS), which contains 1.5 mM cysteine as an original ingredient. Medium L 15 supplemented with 10% bovine serum (L-10BS), which contains both 1 mM cysteine and 5 mM pyruvate, supported cell growth. On incubation of MEM-10BS supplemented with 1 mM cysteine (MEM-10BS-1CySH) or CMRL-10BS without cells for one day, the cysteine concentrations decreased to about one-tenth or less of the original concentrations. The cysteine concentration in L-10BS did not decrease so much on similar incubation. Pyruvate reduced the rate of disappearance of the cysteine in MEM-10BS-1CySH or CMRL-10BS as assayed with p-chloromercuribenzoate, although less than that in L-10BS. This effect of pyruvate was concentration dependent. These paradoxical effects of pyruvate on cysteine, i.e. the reduction of its cytotoxicity and the stabilization as an SH compound, are probably due to the formation of a dissociable complex between these two compounds, which is not cytotoxic and resistant to oxidation. This work was supported by Grants-in-Aid for Cancer Research from the Ministry of Education, Science and Culture, Japan, and a grant from the Princess Takamatsu Cancer Research Fund.     

Effects of Bicarbonate on Growth of Neisseria gonorrhoeae: Replacement of Gaseous CO2 Atmosphere

The effect of NaHCO₃ on the growth of Neisseria gonorrhoeae cultures was studied in a liquid and a semisolid growth medium. With a broth culture, NaHCO₃ (0.009 M) greatly reduced the lag phase and also increased the total growth. The same concentration of bicarbonate supported rapid growth when added to the semisolid medium if the plates were individually incubated in sealed plastic bags. In a container with a large air space, a higher concentration of NaHCO₃ was necessary to support growth. The assimilation of ¹⁴C-labeled NaHo₃ by growing cultures was also investigated.     

In vitro Growth and Shoot Multiplication of Achras zapota in a Controlled Carbon Dioxide Environment

The culture vessels with multiplying shoots of Achras zapota L. on Schenk and Hildebrandt (SH) medium containing 8.88 M 6-benzylaminopurine (BAP) with or without sucrose were kept under varied CO₂ concentrations ranging from 0.6 to 40.0 g m^–3 using different concentrations of sodium bicarbonate (NaHCO₃), sodium carbonate (Na₂CO₃), potassium bicarbonate (KHCO₃), and potassium carbonate (K₂CO₃) in small acrylic chambers. Complete absence of carbon source caused death of shoots within 20 d. Under elevated concentrations of CO₂ (10.0 and 40.0 g m^–3) the shoots grew photoautotrophically on sucrose-free medium. The growth of cultures was better at 40.0 g (CO₂) m^–3 than on 3.0 % sucrose under ambient air of growth room. However, the best response was obtained at 10.0 g (CO₂) m^–3 and 3.0 % sucrose where maximum number of shoots, shoot length, fresh and dry mass, total number of leaves and leaf area was observed.     

Photoautotrophic growth of soybean cells in suspension culture IV. Free amino acid pools and the effect of nitrogen on chlorophyll levels

A photoautotrophic soybean suspension culture was used to study free amino acid pools during a subculture cycle. Free amino acid analysis showed that the intracellular concentrations of asparagine, serine, glutamine, and alanine reached peaks of 200, 10, 9 and 7 mM, respectively, at specific times in the 14-day subculture cycle. Asparagine and serine levels peaked at day 14 but glutamine level rose quickly after subculture, peaking at day three and then declined gradually. Roughly similar patterns were found in the conditioned culture medium although the levels were 1000-fold lower than those found in cells. Photoautotrophic (SB-P) and photomixotrophic (SB-M) cultures were quantitatively similar with regard to free asparagine and serine but not glutamine or free ammonia. Heterotrophic (SB-H) cells had 81–85% less free asparagine on day seven than did SB-M or SB-P cells. Hence, similar to the phloem sap of a soybean plant, asparagine, glutamine, alanine and serine were the predominant amino acids in photoautotrophic soybean cell cultures. Varying the amount of total nitrogen in culture medium for two subcultures at 10, 25, 50, and 100% Of normal levels showed that growth was inhibited only at the 10 and 25% levels but that growth on medium containing 50% of the normal nitrogen was as good as that on 100% nitrogen. Moreover, cellular chlorophyll content correlated exceptionally well with initial nitrogen content of the medium. Thus, the photosynthesis of SB-P cells was not limited by chlorophyll content. SB-P cells grown for two subcultures on 10% nitrogen contained very low free amino acid levels and only 1% of the free ammonia levels found in cells growing on a full nitrogen complement.Abbreviations SB-P photoautotrophic soybean cells (no sucrose, high CO₂, high light) - SB-M photomixotrophic soybean cells (1% w/v sucrose, high light) - SB-H heterotrophic soybean cells (3% sucrose, dark)     

Effects of nitrogen source on crude oil biodegradation

Summary The effects of NH₄Cl and KNO₃ on biodegradation of light Arabian crude oil by an oil-degrading enrichment culture were studied in respirometers. In poorly buffered sea salts medium, the pH decreased dramatically in cultures that contained NH₄Cl, but not in those supplied with KNO₃. The ammonia-associated pH decline was severe enough to completely stop oil biodegradation as measured by oxygen uptake. Regular adjustment of the culture pH allowed oil biodegradation to proceed normally. A small amount of nitrate accumulated in all cultures that contained ammonia, but nitrification accounted for less than 5% of the acid that was observed. The nitrification inhibitor, nitrapyrin, had no effect on the production of nitrate or acid in ammonia-containing cultures. When the culture pH was controlled, either by regular adjustment of the culture pH or by supplying adequate buffering capacity in the growth medium, the rate and extent of oil biodegradation were similar in NH₄Cl- and KNO₃-containing cultures. the lag time was shorter in pH-controlled cultures supplied with ammonia than in nitrate-containing cultures.