An improved chemically defined culture medium for strain L mouse cells based on growth responses to graded levels of nutrients including iron and zinc ions

Nutritional factors were evaluated for effects on growth of mouse fibroblast cells in suspension in a chemically defined medium. Quantitative requirements for each of the essential amino acids, choline, inorganic phosphate, iron, and zinc were established. An improved chemically defined medium was formulated on the basis of the findings yielding populations of L cells in excess of 5 × 10⁶ per ml without nutrient replenishment. When spent medium was replaced periodically, yields approaching 30 × 10⁶ cells per ml were attained. The efficiency of utilization of most amino acids in the new medium appears to be 2- to 3-fold better than results reported by others.     

Developement of serum-free media in CHO-DG44 cells using a central composite statistical design

A serum free medium was developed for the production of recombinant antibody against Botulinum A (BoNTA) using dihydrofolate reductase deficient Chinese Hamster Ovary Cells (CHO-DG44) in suspension culture. An initial control basal medium was prepared, which was similar in composition to HAM’s F12: IMDM (1:1) supplemented with insulin, transeferrin, selenium and a lipid mixture. The vitamin concentration of the basal medium was twice that of HAM’s F12: IMDM (1:1). CHO-DG44 cells expressing S25 antibody grew from 2 × 10⁵ cells to maximum cell density of 1.04 × 10⁶ cells/ml after 5 days in this control medium. A central composite design was used to identify optimal levels and interaction among five groups of medium components. These five groups were glutamine, Essential Amino Acids (EAA), Non Essential Amino Acids (NEAA), Insulin, Transferrin, Selenium (ITS), and lipids. Fifty experiments were carried out in four batches, with two controls in each batch. There was little effect of ITS and Lipid concentrations over the range studied, and glutamine concentration showed a strong interaction with EAA. The optimal concentrations of the variables studied were 2.5 mM Glutamine, 7.4 mM (2×) EAA, 1.4 mM (0.5×) NEAA, 1× ITS supplement, 0.7× Lipids supplement. The maximum viable cell density attained in the optimized medium was 1.4 × 10⁶ cells/ml, a 35% improvement over the control culture, while the final antibody titer attained was 22 ± 3.4 μg/mL, a 50% improvement.     

Growth characterizations of a human monocytic leukemia cell line in a serum-free medium

A clone, AH-01S, derived from a human monocytic leukemia cell line, THP-1, grew rapidly in a serum-free medium containing insulin, transferrin, ethanolamine, and sodium selenite. In batch culture using the serum-free medium, the AH-01S cells proliferated at a specific growth rate (μ) of 0.30 to 0.50 (1/day) from a cell concentration of 1 × 10⁴ cells/ml to 1.6 × 10⁶ cells/ml, an increase of 160 times. A higher cell concentration of 0.45 × 10⁷ cells/ml (cell volume ratio was 0.5%) was obtained in spinner flask culture using the serum-free medium. A mean specific growth rate 0.50 (1/day) was also observed in a culture in a fully instrumented cell culture fermentor. However, μ decreased drastically after the cell concentration reached 1.5 × 10⁶ cells/ml. Analyses of medium composition during cultivation revealed that under lower cell concentration, l-glutamine was the main carbon source while glucose was converted to lactate almost stoichiometrically, and that the production of lactate from glucose decreased at higher cell concentrations. To obtain cultures of 1 × 10⁹ cells, 1,200 to 1,300 mg of a carbon source (glucose) and 400 to 500 of amino acids were consumed during high cell concentration cultivation of the AH-01S cells in the serum-free medium.     

SILICON METABOLISM IN DIATOMS. VI. SILICIC ACID UPTAKE BY A COLORLESS MARINE DIATOM,NITZSCHIA ALBA LEWIN AND LEWIN12

Cells of the colorless, heterotrophic diatom Nitzschia alba, after removal from the culture medium, were able to absorb silicic acid from a salt solution lacking carbon, nitrogen, and phosphorus. Silicic acid uptake continued for approximately 12 hr. At low cell densities (ca. 2.5×10⁵ cells/ml), the cell number doubled under these conditions. At high cell densities (ca. 2 ×10⁶ cells/ml) no cell division resulted. When such cells were washed with a salt solution, their ability to absorb silicic acid was somewhat impaired. The degree of impairment became progressively more pronounced after subsequent washing treatments. A heat-stable factor washed from the cells and present in the first “wash water” was able to restore completely the ability of washed cells to absorb silicic acid. The factor was not identified. Aspartic acid (5 ± 10^-4 M) or glutamine (5 ±10^-4 M) when added to the saline solution similarly promoted complete recovery. A t such concentrations, these substances had only a slight effect on unwashed cells. A solution of 5 ±10^-4 M Na glutamate (or aspartic acid plus glutamine) had an men more pronounced effect, and in addition promoted cell division or growth" of unwashed cells. Several other amino acids and other compounds tested were apparently without effect.     

Large-scale rotating filter perfusion system for high-density growth of mammalian suspension cultures

Summary A system has been developed for growth and maintenance of mammalian cells in suspension culture at high density. In principle, the maintenance of constant levels of required nutrients coupled with the removal of toxic cell byproducts can support much higher suspension cell densities than may be obtained in conventional spinners. The system consisted of 4- or 40-liter reaction vessels equipped with a vertically supported rotating cylindrical filter. Agitation was provided by the magnetically driven, rotating filter. Fresh medium was supplied at a rate of 10 to 20 ml/h per 10⁹ cells and the expended medium free of cells was withdrawn through the rotating filter. Both pH and dissolved O₂ and CO₂ were monitored and regulated. Walker 256 carcinosarcoma cells have been grown in these reactors to densities 10-to 30-fold greater than that obtained in Bellco spinners. In addition to high cell densities, the yield of cells per liter of medium used was 2- to 3-fold that obtained in the conventional systems. Both 4-and 40-liter versions of this reactor have been operated without the use of antibiotics. The 40-liter reactor also has been modified for chemostat operation. In a single run, for example, the Walker cell density was maintained between 6 and 10×10⁶ cells/ml with a total yield of 8.7×10¹¹ cells from 360 liters of medium.     

Cell Culture Derived AgMNPV Bioinsecticide: Biological Constraints and Bioprocess Issues

We have studied parameters for optimizing the Spodoptera frugiperda (Sf9) cell culture and viral infection for the production of Anticarsia gemmatalis multiple nucleopolyhedrosis virus (AgMNPV) polyhedra inclusion bodies (PIBs) in shaker-Schott or spinner bottles and bioreactors. We have assayed the k_La of the systems, initial cell seeding, cell culture volume, dissolved oxygen (DO), multiplicity of infection (MOI), nutrients consumption, and metabolites production. The medium surface oxygen transfer was shown to be higher in shaker bottles than in spinner ones, which was in direct correlation to the higher cell density obtained. Best quantitative performances of PIBs production were obtained with a SF900II medium volume/shaker-bottle volume ratio of 15% and MOI of 0.5 to 1 performed at a cell concentration at infection (CCI) of 1 to 2.5×10⁶ cells/ml in a medium containing enough glucose and glutamine. Upon infection, a decrease in the cell multiplication was observed to be dependent on the MOI used, and the μX at the exponential growth phase in infected and non-infected cultures were, respectively, of 0.2832 and 0.3914 (day⁻¹). The glucose consumption and lactate production were higher in the infected cultures (μGlucose and μLactate of, respectively, 0.0248 and 0.0089×10⁻⁸ g/cell×day in infected cultures and 0.0151 and 0.0046×10⁻⁸ g/cell×day in non infected ones). The glutamine consumption did not differ in both cultures (μGlutamine of 0.0034 and 0.0037×10⁻⁸ g/cell×day in, respectively, infected and non infected cultures). When a virus MOI of 0.1 to 1 was used for infection, a higher concentration of PIBs/ml was obtained. This was in direct correlation to a higher cell concentration present in these cultures, where a decrease in cell multiplication due to virus infection is minimized. When a MOI of 1 was used, a more effective decrease in cell multiplication was observed and a lower concentration of PIBs/ml was obtained, but with the best performance of PIBs/cell. Correlations between MOI and CCI indicate that a MOI 0.1 to 1.4 and a CCI of 10⁶ to 2×10⁶ cells/ml led to the best PIBs production performances. The virulence of PIBs produced in cultures infected at low or high MOI showed comparable DL₅₀. Culture and infection in scaling-up conditions, performed in a bioreactor, were shown to provide the cells with a better environment and be capable of potentially improving the shaker-Schott findings. For an accurate qualitative control of PIB virulence, hemolymph from AgMNPV infected Anticarsia gemmatalis was used as starting material for passages in Sf9 cells. These led to a loss of virulence among the PIBs with an increase in the DL₅₀. The loss of virulence was accompanied by a loss in budded virus titer, a decreased number of PIBs produced and an altered DNA restriction pattern, suggesting the generation of defective interference particles (DIPs). Transmission electron microscopy (TEM) studies revealed that after cell passages, PIBs lacking virions were progressively synthesized. The study described here point out the biological constraints and bioprocess issues for the preparation of AgMNPV PIBs for biological control.     

Inhibitory effect of gonadotrophin-releasing hormone (GnRH) on rat granulosa cell deoxyribonucleic acid synthesis

Gonadotropin-releasing hormone (GnRH) has been found to be expressed within the ovary and to modulate cell differentiation in ovarian cells. In the present study we have analyzed the influence of GnRH on DNA synthesis in rat granulosa cells. Cells were obtained from immature DES-treated rats and cultured in defined medium (DMEM:F12) containing combinations of FSH, estradiol, and transforming growth factor-β (TGF-β), both in the presence and absence of GnRH. A GnRH analog, Leuprolide (GnRHa), caused a dose-dependent inhibition of ³H-thymidine incorporation in cells cultured in the presence of FSH (20 ng/ml) and TGFβ (2.5 ng/ml), at concentrations as low as 5 × 10⁻¹¹ M. Similarly, a complete inhibition of hormonally stimulated DNA synthesis were observed with another analog (Buserelin, ED₅₀ = 1.58 ± 0.22 × 10⁻¹⁰ M) and native GnRH (ED₅₀ = 1.4 ± 0.3 × 10⁻⁶ M). A competitive antagonist of GnRH (Antide) was used to neutralize the GnRH agonist effects. Antide 10⁻⁸ M could prevent the inhibition elicited by 10⁻⁷ M of Leuprolide. These results suggest that GnRH may play a role in the regulation of rat granulosa cell proliferation during follicular development. Mol. Reprod. Dev. 47: 170–174, 1997. © 1997 Wiley-Liss, Inc.     

Potassium permanganate–acridine yellow chemiluminescence system for the determination of fluvoxamine,isoniazid and ceftriaxone

Based on the oxidation of acridine yellow by permanganate in basic medium, a new chemiluminescence system was developed for the sensitive determination of some important drugs. The remarkable inhibiting effect of fluvoxamine, ceftriaxone and isoniazid on this reaction was applied to their detection. A possible mechanism was proposed for this system based on chemiluminescence emission wavelengths and experimental observations. Under optimum conditions, calibration graphs were obtained for 1 × 10^?9 to 1 × 10^?6 mol/L of fluvoxamine; 2 × 10^?8 to 8 × 10^?6 mol/L of ceftriaxone and 5 × 10^?8 to 4 × 10^?5 mol/L of isoniazid. This proposed method was satisfactorily used in the determination of these drugs in pharmaceutical samples and human urine and serum. Copyright © 2014 John Wiley & Sons, Ltd.     

Cell susceptibility to apoptosis by glutamine deprivation and rescue: Survival and apoptotic death in cultured lymphoma-leukemia cell lines

Human leukemia/lymphoma cells maintained in culture medium without provision of fresh nutrients lose viability and die by a process resembling apoptosis within a few days. Upon incubation in an FCS-supplemented RPMI 1640 medium containing 2 mM L-glutamine CEM, Namalwa, HL-60 and U937 cells, seeded at initial densities of 0.2 to 1 × 10⁶ cells/ml, ceased growing within 3–5 days and progressively entered an apoptotic pathway, as assessed by nucleosomal DNA fragmentation and morphology. Both the major energy-source nutrients in the medium, glucose and glutamine, became rapidly exhausted during the incubation. Further studies were performed using CEM cells. Incubation in glutamine-free or glucose-free medium renewed every 24 h showed that glutamine deprivation is associated with cell death by apoptosis independent of energetic failure, whereas glucose deprivation is followed by rapid loss of mitochondrial function with sharp drop of intracellular ATP and cell death by necrosis. A 12–24 h incubation in glutamine-depleted medium was required to direct the cells toward the apoptotic pathway. Growth arrest followed by apoptotic death was detected in CEM cells when medium glutamine concentration remained below 0.3–0.4 mM for at least 24 h, but a reinstatement of medium glutamine to 2 mM within this period rescued the cells from growth arrest and death. © 1996 Wiley-Liss, Inc.     

Accelerated Biodegradation of High and Low Concentrations of p-Nitrophenol (PNP) by Bacterial Inoculation in Industrial Wastewater: The Role of Inoculum Size on Acclimation Period

The effect of inoculum size on the acclimation period and rate and extent of p-nitrophenol (PNP) degradation at high (1–10 mg/L) and low (26 μg/L) concentrations for two bacteria was determined in defined media as well as industrial wastewater. Increased inoculum size did not affect the acclimation period of either bacterium at high (1–10 mg/L) PNP concentrations. At low PNP concentrations (26 μg/L), the two bacteria behaved differently. The acclimation period was shortened and both the rate and extent of mineralization of PNP were enhanced by increasing the Corynebacterium sp. inoculum size from 3 × 10⁵ to 3 × 10⁶ cells/ml. Addition of phosphate or elimination of predators also reduced the acclimation period. Conversely, increasing the inoculum size from 3 × 10⁵ to 5 × 10⁶ cells/ml of Pseudomonas putida lengthened the acclimation period and reduced both the rate and extent of mineralization. It is suggested that, in a given environment, the success of an introduced species to enhance the degradation of a chemical depends upon (i) concentration of the chemical, (ii) selection of an appropriate microorganism, and (iii) utilization of a suitable inoculum size. Received: 1 April 1996 / Accepted: 6 May 1996     

Cell metabolism and medium perfusion in VERO cell cultures on microcarriers in a bioreactor

Parameters of VERO cell growth and metabolism were studied in cultures performed on microcarriers (MCs) using a bioreactor with a working capacity of 3.7?l. Kinetic studies of VERO cell growth in batch, semi-batch and perfusion cultures using concentrations of 2 and 10?mg/ml of MCs showed that a high concentration of MCs (10?mg/ml) and the use of medium perfusion allowed the attainment of higher final yields of VERO cells (6?×?10⁶ cells/ml after 10 days of culture). Perfusion also allowed better use of MCs as indicated by the observation of about 100% of MCs totally covered by cells and the appearance of multilayered cells on 64% of MCs after 13 days of VERO cell culture with 2?mg/ml of MCs. Concerning the concentration of nutrients in the cultures, the medium perfusion was able to sustain suitable levels of galactose and glutamine, which quickly decreased after 4 days in batch cultures. The air inlet in the batch cultures was capable of eliminating the NH₄ ⁺ which accumulated in the medium culture. Lactate accumulated during the first days of culture but then was utilized by the cells and decreased along the culture time. The optimization of VERO cell cultures on microcarriers as indicated by the concentration of MCs, medium perfusion and air inlet is discussed.     

Large-scale production of Erythroid Differentiation Factor (EDF) by gene-engineered Chinese hamster ovary (CHO) cells in suspension culture

From Chinese hamster ovary (CHO) cells which were producing Erythroid Differentiation Factor (EDF) in a culture medium, anchorage-independent cells, named as CHO-SPN, were produced by repeated cultivating in a suspension system. The growth time and maximum cell density of the CHO-SPN cells were 48 h and 7.8×10⁵ viable cells/ml. CHO-SPN cells accumulated 8,000 units/ml (corresponding to 4 mg/ml) of EDF in 4d. After 20 cycles of culture, CHO-SPN cells still possessed the same EDF productivity and the same growth kinetics. Furthermore, in an appropriate dissolved oxygen concentration and pH controlled culture system, the growth time and cell density became 24 h and 1×10⁶ viable cells/ml. The critical level of dissolved oxygen for cell growth was 0.015 atm. The maximum oxygen demand was 3.3×10⁻⁹ mole of O₂/ml/min.Fetal bovine serum (FBS) was indispensable for cell growth. However, a FBS-free medium (ASF201) was available for maintenance of the CHO-SPN cells, and EDF production occurred in the same medium.     

Induction of apogamy inEquisetum arvense

InEquisetum arvense, apogamous sporophytes were produced on medium containing 5×10^?6–5×10^?8 g/ml kinetin. NAA, IAA, GA₃, glucose and saccharose were ineffective for the induction of apogamy. On medium containing 5×10^?7–5×10^?8 g/ml kinetin, the gametophytes passed into sporophytic structures directly. On medium containing 5×10^?6 g/ml kinetin, some gametophytes passed into sporophytic structures directly, and others became a callus-like cell mass from which an apogamoun shoot arose. The results of the morphological observations on them were reported and compared with the sexually produced sporophyes. The apogamous sporophytes induced by 5×10^?7 g/ml kinetin were haploid in their nuclear phase and some of those induced by 5×10^?6 g/ml kinetin had a tendency to become diploid.     

Development of a chemically defined platform fed-batch culture media for monoclonal antibody-producing CHO cell lines with optimized choline content

A chemically defined platform basal medium and feed media were developed using a single Chinese hamster ovary (CHO) cell line that produces a monoclonal antibody (mAb). Cell line A, which showed a peak viable cell density of 5.9 × 10⁶ cells/mL and a final mAb titer of 0.5 g/L in batch culture, was selected for the platform media development. Stoichiometrically balanced feed media were developed using glucose as an indicator of cell metabolism to determine the feed rates of all other nutrients. A fed-batch culture of cell line A using the platform fed-batch medium yielded a 6.4 g/L mAb titer, which was 12-fold higher than that of the batch culture. To examine the applicability of the platform basal medium and feed media, three other cell lines (A16, B, and C) that produce mAbs were cultured using the platform fed-batch medium, and they yielded mAb titers of 8.4, 3.3, and 6.2 g/L, respectively. The peak viable cell densities of the three cell lines ranged from 1.3 × 10⁷ to 1.8 × 10⁷ cells/mL. These results show that the nutritionally balanced fed-batch medium and feeds worked well for other cell lines. During the medium development, we found that choline limitation caused a lower cell viability, a lower mAb titer, a higher mAb aggregate content, and a higher mannose-5 content. The optimal choline chloride to glucose ratio for the CHO cell fed-batch culture was determined. Our platform basal medium and feed media will shorten the medium-development time for mAb-producing cell lines.     

Development of a serum-free medium for dihydrofolate reductase-deficient chinese hamster ovary cells (DG44) using a statistical design: Beneficial effect of weaning of cells

Summary To develop serum-free (SF) medium for dihydrofolate reductase-deficient Chinese hamster ovary cells (DG44), a statistical optimization approach based on a Plackett-Burman design was adopted. DG44 cells which were normally maintained in 10% serum medium were gradually weaned to 0.5% serum medium to increase the probability of successful growth in SF medium. A basal medium was prepared by supplementing Dulbecco’s modified Eagle’s medium and Ham’s nutrient mixture F12 with hypoxanthine (10 mg/l) and thymidine (10 mg/l). Twenty-eight different supplements were selected as variables on the basis of their growth-promoting abilities. From statistical analysis, leucine, tryptophan, lysine, proline, histidine, hydrocortisone, ethanolamine, and phosphatidylcholine were identified as important components showing positive effects on cell growth. A new SF medium (SF-DG44) was formulated by supplementing the basal medium with these components. When the weaned cells were inoculated at 1.0 × 10⁵ cells/ml, a maximum viable cell concentration of 6.4×10⁵ cells/ml was achieved in SF-DG44 medium. In contrast, when the unweaned cells were used, a concentration of only 4.1×10⁵ cells/ml was reached under the same culture conditions, indicating that weaning of cells improves cell growth in SF medium. In summary, we found that development of a novel SF medium for DG44 cells was facilitated using a Plackett-Burman design technique and weaning of cells.     

Continuous flow cultures of a HeLa cell line as a basis for a steady supply of Rubella virus

A HeLa cell line was propagated in semicontinuous suspension culture, 85 liters final volume, and in continuous flow culture with a volume of 300 ml. or 5 liters in an autoclavable medium to which 8% calf serum had been added. A medium containing 0.1% Methocel and 2% calf serum was also tested. Maximum productivity was obtained at a dilution rate of 0.33 day^?1 with a cell density of about 1.0 × 10⁶ cells/ml. The same cell line was also infected with Rubella virus and the production of virus was followed at the 5-liter cultivation level.     

New Culture Medium for Maintenance of Tsetse Tissues and Growth of Trypanosomatids*

SYNOPSIS. A new culture medium (SM), based on the amino-acid composition of tsetse hemolymph and containing fetal bovine serum, was designed for the maintenance of tsetse organs and the cultivation of various trypanosomatids. For optimum growth 20% (v/v) serum was required. The medium supported prolonged peristalsis of the alimentary tract and salivary glands of pre-emerged Glossina morsitans morsitans. In established cultures, derived from bloodstream forms of pleomorphic Trypanosoma brucei brucei and Trypanosoma brucei rhodesiense strains, inocula of ～ 10⁶ procyclics/ml yielded 4–5 × 10⁷ organisms/ml after 4 or 5 days of incubation at 28 C. Bloodstream forms of a cloned monomorphic T. b. brucei strain were also able to transform into procyclics, which, however, multiplied at a lower rate, with maximum yields of ～ 2 × 10⁷ after 5 days. Cultures of Trypanosoma congolense and of a nearly monomorphic Trypanosoma brucei gambiense strains could be established in SM medium only in the presence of tsetse alimentary tract. The procyclic trypomastigotes of these species, adapted to SM medium and able to grow in it without Glossina organs, gave maximum populations of ～ 4.5 × 10⁷ cells/ml. Promastigotes of Leishmania donovani, cultivated routinely in a diphasic Table's medium, multiplied actively upon being transferred into SM medium, producing yields of ～ 4 × 10⁷ cells/ml.     

Large-Scale Temperature-Induced Synchrony of Cell Division in Euglena gracilis*

SYNOPSIS. Temperature-induced patterns of synchronous cell division and cell size were obtained with Euglena gracilis. The alga was cultured in a glutamate-sucrose medium in 6-liter quantities. Synchrony was induced by non-lethal shifts of temperature between 14.5 and 28.5 C. Three liters of cells containing 2 × 10⁶ cells/ml were harvested in each 24-hour cycle.     

Growth ofChlamydia psittaci strain menigopneumonitis in mouse L cells cultivated in a defined medium in spinner cultures

Summary L cells were grown in spinner cultures in a defined medium consisting of Waymouth medium MB752/1 (19) supplemented with 2 mg of fatty acid-free bovine serum albumin (BSA) per ml and 5 μg of oleate per ml (WO₅ medium). Growth in WO₅ medium was comparable to spinner L cell growth in two serum-containing media. The optimal concentration of oleate in the WO medium was 5 to 10 μg per ml. The use of 20 to 80 μg of oleate per ml of medium resulted in lower peak populations and earlier declines in viable cell counts. Cell death occurred rapidly in WO₁₆₀ medium. Cell growth in WO medium containing 5 to 80 μg of oleate per ml was well above the level of growth observed when no oleate was present in the medium. Since the total lipid and fatty acid compositions of the BSA used in this study have been characterized by the authors, the WO medium may be considered a defined medium. L cells have been continuously maintained in spinner cultures in WO₅ medium for over 50 passages with no major variation in the growth pattern. A 1000-fold increase inChlamydia psittaci strain meningopneumonitis, with a peak titer of 9.3×10⁷ plaque-forming units per ml, was observed when the chlamydial agents were grown in spinner L cells in WO₅ medium. This investigation was supported by Public Health Service Research Grant HE 08214 from the Program Projects Branch, Extramural Programs, National Heart and Lung Institute; The World Health Organization; and The Hormel Foundation.     

Characterization of proliferation and differentiation of opossum kidney cells in a serum-free defined medium

Summary Proliferation and differentiation of opossum kidney cells in a serum-free defined medium was investigated and compared to that under conditions in which fetal bovine serum FBS (10%) was employed. Monolayers were grown in Dulbecco's modified Eagle's medium-Ham's F12 nutrient mixture containing insulin (10 μg/ml), bovine serum albumin fraction V (1 mg/ml) and fetuin (1 mg/ml). Cells in serum-free medium seeded at 1×10⁴ per cm² grew to confluency within 6 to 8 d and formed hemicysts or domes at a frequency equivalent to those in serum-containing medium. Electron microscopy of cultures grown in serum-free medium revealed polarized monolayers with the presence of microvilli and tight junctions. The differentiated characteristics, including sodium-dependent phosphate transport, the inhibition of this transport by parathyroid hormone (PTH), and the generation of cyclic AMP in response to PTH, were preserved in opossum kidney cells grown in serum-free medium.