Effect of different fetal bovine serum concentrations on the replicative life span of cultured chick cells

Summary The effect of Eagle's minimal essential medium, containing different fetal bovine serum (FBS) concentrations, on the proliferation and replicative life span of cultured chick cells has been studied. Our results showed that the rate of chick cell proliferation and the cell density at stationary phase increased as a function of serum concentration between 5 and 30% FBS. The replicative life span of cultured chick cells was dependent on the FBS concentration between 5 and 20% in a medium volume of 0.20 ml/cm². The maximum replicative life span of chick cells was obtained by serially propagating cells in a medium volume of 0.20 ml/cm² containing 20 or 30% FBS, or, alternatively, in 0.53 ml/cm² containing 10, 20 or 30% FBS. Cells grown in medium containing 5% serum had a calendar life span of 35 days, whereas cells propagated in medium containing higher serum concentrations had a calendar life span of 50 days. These results reenforce the concept that, although the kinetics of cell population aging can be affected by the culture medium composition, the aging of cells in culture is controlled by alterations within the cell. This work was supported by IIT Research Institute.     

Stimulation of WI-38 cell cycle transit: Effect of serum concentration and cell density

Flow microfluorometry has been used to characterize the effects of serum concentration and cell density on the initiation of cell cycle transit of stationary phase (G₀) human diploid fibroblasts (strain WI-38). The concentration of serum used to stimulate these cultures had no effect on the time cells began appearing in S (the DNA synthetic period), nor on the synchrony with which they moved around the cell cycle. However, as the serum concentration increased, the fraction of the stationary phase population released from G₀ increased. Cell density modulated the ability of serum to stimulate cell cycle traverse. For example, at a cell density of 1.81 × 10⁴ cells/cm², 78% of the population was sensitive to serum stimulation; whereas, when the density was increased to 7.25 × 10⁴ cells/cm², only 27% of the population could be stimulated. This effect of cell density on the serum response is not simply the result of changing the ratio of serum concentration to cell density, but appears to reflect a true modulation of the population's sensitivity to serum stimulation. These results are consistent with the interpretation that the primary action of serum is to determine the transition of cells from a non-cycling G₀ state to a cycling state and that cell density determines the proportion of the population capable of undergoing this transition.     

Giardia lamblia: Evaluation of roller bottle cultivation

A modified “outside-in” roller bottle with a high ratio of surface area to volume was used to cultivate Giardia lamblia. Yields were high, more so when bottles were rotated at 6 rph (9.3 ± 4.0 × 10⁸ trophozoites/bottle) than at 12 rph (4.2 ± 1.9 × 10⁸ trophozoites/bottle). The method was more efficient than stationary tube culture with respect to utilization of culture medium; trophozoite concentration after roller bottle culture (1.7 ± 0.8 × 10⁶ trophozoites/ml) was significantly higher (by a factor of 2.8) than concentrations obtained from stationary tube culture (0.6 ± 0.4 × 10⁶ trophozoites/ml, P < 0.002). Increased yields from roller bottle culture were not accounted for by a reduction in mean trophozoite generation time (roller culture, 10.7 ± 1.2 hr; stationary tube culture, 10.3 ± 0.6 hr) but may be related to prolongation of the period of log phase growth or increased trophozoite survival. Trophozoite yields expressed per unit surface area were significantly higher from roller bottle culture (7.2 ± 3.1 × 10⁵ trophozoites/cm²) than from stationary tubes (1.9 ± 1.0 × 10⁵ trophozoites/cm², P < 0.002). Attempts to cultivate G. lamblia in spin culture using polystyrene beads (Biosilon) as a microcarrier were unsuccessful, trophozoite growth being inhibited rather than promoted. Roller bottle culture of G. lamblia, however, is efficient, economical, and less laborious than stationary tube culture, particularly when more than 10⁸ trophozoites are required.     

Growth and Immunologic Studies on the Culture Forms of Trypanosoma lewisi*

SYNOPSIS. Culture forms of Trypanosoma lewisi grown at 27 C in a diphasic blood agar medium resemble in structure the stage found in the invertebrate host. Cultures inoculated with approximately 1 × 10⁶ trypanosomes/ml attain maximum populations of 2–7 × 10⁷ organisms/ml after 5–6 days of incubation. The stationary phase persists 6–15 days. The decline of the population is of relatively long duration with approximately 1 × 10⁶ viable organisms/ml present after 90 days. Variations in growth were attributed to the preparation of defibrinated heated rabbit blood incorporated into the culture medium. With inocula of 3.0 × 10⁵ trypanosomes/ml there was a lag in growth not observed with larger inocula. Trypanosomes incubated at elevated temperatures had altered growth curves compared to organisms at 27 C. Agitation of cultures did not affect the growth or stationary phases, but hastened the population decline. Heated and unheated 5% (v/v) normal rat serum incorporated in the liquid phase of the medium altered the growth of the organisms. Heated serum caused a decrease in the population and an extended lag phase. The effects on growth were more marked with unheated serum suggesting that both heat-stable and labile components affect growth. Antisera from rats injected with live culture forms included in the liquid phase inhibited, while antisera from rats 24 days after infection with the blood stream forms had no effect on the growth of the culture forms. Antisera from rabbits immunized with sonicates of culture forms also altered the growth of the organisms in culture. Rabbit antisera prepared by immunization with sonicates of dividing and non-dividing blood stream forms had no effect on the in vitro growth. Antisera from animals immunized with rat blood and culture medium were also without effect. The immunologic implications of the data are considered and discussed.     

Effect of different fetal bovine serum concentrations on the replicative life span of cultured chick cells

The effect of Eagle's minimal essential medium, containing different fetal bovine serum (FBS) concentrations, on the proliferation and replicative life span of cultured chick cells has been studied. Our results showed that the rate of chick cell proliferation and the cell density at stationary phase increased as a function of serum concentration between 5 and 30% FBS. The replicative life span of cultured chick cells was dependent on the FBS concentration between 5 and 20% in a medium volume of 0.20 ml/cm2. The maximum replicative life span of chick cells was obtained by serially propagating cells in a medium volume of 0.20 ml/cm2 containing 20 or 30% FBS, or, alternatively, in 0.53 ml/cm2 containing 10, 20 or 30% FBS. Cells grown in medium containing 5% serum had a calendar life span of 35 days, whereas cells propagated in medium containing higher serum concentrations had a calendar life span of 50 days. These results reenforce the concept that, although the kinetics of cell population aging can be affected by the culture medium composition, the aging of cells in culture is controlled by alterations within the cell.     

MITOTIC AND NONMITOTIC MULTIPLE-LAYERED PERFUSION CULTURES

Cell types in addition to those previously described (Kruse et al. 1963. J. Nat. Cancer Inst. 31:109; Kruse and Miedema. 1965. J. Cell Biol. 27:273) were found to form multiple-layered cultures by perfusion-culture technique. Dense populations containing 43 x 10⁶ embryonic rat muscle (NF-ER) cells, 23 x 10⁶ diploid human tonsillar (NF-JAM) cells, 77 x 10⁶ human pleural effusion isolate (RPMI 2650) cells, 35 x 10⁶ embryonic diploid human lung (Flow 2000) cells, 21 x 10⁶ bovine lung (FB4BM) cells, 108 x 10⁶ bat lung (Tb1Lu) cells, and 81 x 10⁶ SV-40 virus-transformed embryonic diploid human lung (WI-38VA13A) cells were obtained in 6–14 days from dilute inocula in T-60 or T-75 flasks; these were equivalent to about 4, 3, 3, 4, 2, 4, and eight monolayers, respectively. Perfusion of an NF-ER culture for 6 wk with medium plus 10% whole calf serum yielded a cell density equivalent to 12 monolayers (140 x 10⁶ cells per T-75 flask). This culture exhibited random labeling of nuclei from bottom to top after pulsing for 90 min with thymidine-³H. Medium plus 0.1% serum maintained NF-JAM cultures at constant viable cell numbers with virtual absence of thymidine-³H labeling. Similar results were obtained with WI-38 cultures, but WI-38VA13A cells continued active DNA synthesis and mitosis in medium with 0.1% serum to form 16–20 layers of cells (191–239 x 10⁶ cells per T-75 flask) in 27 days. WI-38VA13A cells ceased proliferation and became nonviable rapidly in serumless medium.     

DNA-binding proteins in young and senescent normal human fibroblasts

DNA-binding proteins (DBP) from normal human diploid cells, strain WI38, were isolated by DNA-cellulose chromatography using undenatured calf thymus DNA. The DBP in the 0.15 M NaCl eluate were fractionated by polyacrylamide gel electrophoresis. Comparisons of the amounts of the DBP in different cell populations were made by labelling the cells with either ³H- or ¹⁴C-amino acid precursors for 40 h prior to pooling the cells for co-isolation of their DBP. When WI38 cells in the replicative and stationary phases were compared, five proteins, P5b (87 000 D), P6a (50 000 D), P8 (33 000 D), P9 (28 000 D) and P10 (25 000 D) were labelled to a greater extent in the replicating cells and two proteins, P5c (72 000 D) and P12 (18 000 D) were labelled to a greater extent in the stationary phase cells. In addition, several high molecular weight DBP, partially characterized as collagen and protocollagen, were preferentially labelled in the stationary phase cells. Stationary phase senescent WI38 cells at or near the end of their in vitro lifespan characteristically showed an increased proportion of protein component P8 (33 000 D) relative to stationary phase WI38 cells at early population doubling levels. Further characterization of WI38-P8 showed that it binds preferentially to single-stranded DNA and amounts to greater than 1% of the total soluble protein in young cells in growth phase. Thus WI38-P8 appears to be comparable to the P8 protein studied by Tsai & Green [27] in mouse 3T6 and human SB cells. The component which is increased in senescent or terminal phase non-dividing cell populations is judged to be the P8 protein by its position in SDS-gels and its preferential binding to single-stranded DNA.     

Cultivation of fall armyworm ovary cells in simulated microgravity

Summary A methodology is presented to culture Fall Armyworm Ovary cells in simulated microgravity using a novel bioreactor developed by NASA, the High-Aspect Ratio Vessel. In this vessel, the growth and metabolic profile for these insect cells were profoundly different than those obtained in shaker-flask culture. Specifically, stationary phase in the NASA vessel was extended from 24 h to at least 7 d while cell concentration and viability remained in excess of 1 × 10⁷ viable cells/ml and 90%, respectively. Measurements of glucose utilization, lactate production, ammonia production, and pH change indicate that simulated microgravity had a twofold effect on cell metabolism. Fewer nutrients were consumed and fewer wastes were produced in stationary phase by as much as a factor of 4 over that achieved in shaker culture. Those nutrients that were consumed in the NASA vessel were directed along different metabolic pathways as evidenced by an extreme shift in glucose utilization from consumption to production in lag phase and a decrease in yield coefficients by one half in stationary phase. These changes reflect a reduction in hydrodynamic forces from over 1 dyne/cm² in shaker culture to under 0.5 dyne/cm² in the NASA vessel. These results suggest that cultivation of insect cells in simulated microgravity may reduce production costs of cell-derived biologicals by extending production time and reducing medium requirements.     

Control by cell interaction of phosphate uptake in 3T3 cells.

Phosphate uptake by monolayers of 3T3 cell decreases when the cultures enter the stationary phase, even when incubated in fresh medium containing 10% serum. However, SV 3T3 cultures retain a high rate of phosphate uptake when the cells reach saturation densities.We have observed that 3T3 cells grown to stationary phase in monolayers and then trypsinized and incubated in suspension, display an increase in phosphate uptake when the cell concentration is decreased from 10⁶ cells/ml to 10⁵ cells/ml. Where the cell concentration is further reduced from 10⁵ cells/ml to 2.5 × 10⁴ cells/ml there is no further increase in the rate of phosphate uptake. We observed, on the contrary, a small decrease.The “concentration effect” (the decrease of phosphate uptake when the cell concentration increases from 10⁵ to 10⁶ cells/ml) is larger when cells originate from a culture in stationary phase than when they originate from a culture in log phase.The “concentration effect” may be observed 10 min after cell incubation but is larger after a lag time of 40 min incubation.Differences in the “concentration effect” may be noted between 3T3 and SV 3T3 cells. In SV 3T3 cells no significant variations of phosphate uptake were observed when the cell concentration was changed. Thus, differences between phosphate uptake in 3T3 and SV 3T3 cells are large when cells are incubated at high concentrations or at high densities and small when they are incubated at low concentrations or at low densities.The “concentration effect” in 3T3 cells supports the assumption that interactions between cells cause the decrease of phosphate metabolism in dense culture. Diffusion of an inhibitor into the medium remains the more plausible explanation of the data.     

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.     

The serum growth and survival requirements of SV40-transformed 3T3 cells

Summary Simian virus 40-transformed 3T3 cells are dependent on serum for survival and growth. This growth activity can be separated on a pH 2 Sephadex G100 column into two fractions: a high molecular weight activity and a low molecular weight substance that has recently been characterized as containing as its major agent, biotin. To replace the remainder of the serum requirement, hormones and other growth factors were tested. Both insulin at high, nonphysiological concentrations (200 to 500 ng/ml) and transferrin (5×10⁻⁸ M) stimulate the growth rate in low serum medium (0.3% v/v bovine calf serum DME) individually and, when added together, are nearly as growth enhancing as 10% serum. The need for the residual serum in this medium can be eliminated by the use of crystalline trypsin during trypsinization. Under these serum-free conditions, biotin and transferrin supplementation provide for moderately good growth (20 to 30 hr population doubling time, 1×10⁶ cells/3.2-cm dish final cell density). Insulin addition further stimulates the growth rate (16 to 20 hr) and the final density (1.5×10⁶ cells). Although the protein growth factors, EGF (0.5 to 1.0 ng/ml) and FGF (4 to 10 ng/ml), also appear to enhance growth individually and additively, their effects are slight and very variable. Nevertheless, the complete serum-free medium (DME supplemented with biotin, transferrin, insulin, EGF and FGF) yields growth comparable but still inferior to 10% serum supplementation (14-versus 12-hr population doubling time, 1 to 2×10⁶ versus 2 to 3×10⁶ cells final cell density). This work was supported by NIH Grant CA 20040.     

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.     

Cell growth optimization in microcarrier culture

Summary Three monkey kidney cell lines and primary chicken embryo cells were grown in microcarrier culture. The carrier support was DEAE-Sephadex gel beads at low anion exchange capacity prepared according to a protocol developed at the Massachusetts Institute of Technology. The growth rate of the cells and the final cell density in microcarrier culture was dependent on the concentration of the beads in culture and on the size of the initial cell inoculum. A bead concentration of 1.0 to 2.0 mg of beads/ml of tissue culture medium and a cell inoculum of 20,000 cells/cm² of bead surface appeared to be optimal. The efficiency of the microcarrier culture system was compared to that of stationary and roller bottle cultures. Stationary flasks gave cell densities about twofold higher than maximal densities in roller bottles and about threefold and twofold higher than cell densities in microcarrier culture at a bead concentration of 2.5 and 1.0 mg/ml, respectively. In terms of cell yield per millitier of tissue culture medium, the microcarrier culture was superior to roller bottle and stationary cultures. An advantage of the microcarrier culture system is its suitability for a scale up into large volume production units.     

Changes in membrane transport function in G0 and G1 cells

Confluent quiescent monolayers of aneuploid and euploid cells in culture can be stimulated to proliferate by appropriate nutritional changes. In confluent monolayers of WI-38 human diploid fibroblasts the uptake of cycloleucine is increased three hours after these cells are stimulated to proliferate by a change of medium plus 10% serum. No changes in the uptake of cycloleucine are observed in logarithmically-growing WI-38 cells exposed to fresh medium plus 10% serum, or in WI-38 confluent monolayers in which the conditioned medium has been replaced by fresh medium with 0.3% serum (a change that does not cause stimulation of cellular proliferation in WI-38 cells). In 3T6 cells in the stationary phase stimulated to proliferate by nutritional changes, there is a prompt increase in the uptake of cycloleucine, within one hour after stimulation of cell proliferation. Similar results were obtained with stationary 2RA cells which are SV-40 transformed WI-38 fibroblasts. In addition, chromatin template activity which is known to increase in the early stages after stimulation of confluent WI-38 cells, was unchanged in confluent 3T6 or 2RA cells stimulated to proliferate. These results show that at least two of the very early biochemical events occurring in response to stimulation of cell proliferation are different in WI-38 diploid cells and in aneuploid 2RA or 3T6 cells. It is proposed that WI-38 cells in the stationary phase are arrested in the G₀ phase of the cell cycle, while 2RA and 3T6 cells are arrested in the G₁ phase.     

Growth regulation of WI38 cells in a serum-free medium

A serum-free growth medium composed of MCDB-104 supplemented with platelet-derived growth factor (PDGF) (3 μg/ml), epidermal growth factor (EGF) (100 ng/ml), insulin (INS) (5 μg/ml), transferrin (TRS) (5 μg/ml), and dexamethasone (DEX) (55 ng/ml) supports the proliferation of WI38 cells at a rate and to a density similar to that of medium supplemented with 10% fetal bovine serum (FBS). EGF exerts its effect at moderate cell densities while PDGF appears to modulate cell proliferation at high densities. Cells seeded into EGF-, INS-, TRS-, and DEX-supplemented medium enter S phase approx. 3 h earlier than cells seeded into PDGF-, EGF-, INS-, TRS- and DEX-supplemented medium or FBS-supplemented medium.     

The growth requirements of BHK-21 cells in serum-free culture

A serum-free medium for serial culture of baby hamster kidney cell line 21 (BHK-21) as container-adherent cells was developed. The medium is a 1:1 (v/v) mixture of Dulbecco's modified Eagle's medium and Ham's F-12 medium supplemented with fibroblast growth factor, fibronectin, insulin, oleic acid (preincubated with fatty-acid-free bovine serum albumin as carrier), and transferrin. The fibronectin was required for cell adherence, the other factors for optimal cell multiplication. When cell input was greater than about 1,900 cells/cm², this serum-free medium supported cell multiplication at a rate approximately equal to the rate in medium with 10% serum. At lower cell input, growth in the serum-free medium was poor unless it was supplemented with serum-free medium which had been conditioned by BHK-21 cells. The conditioned medium contained a factor(s) which enabled or stimulated cell multiplication.     

Electrotransformation of intact cells ofBrevibacterium flavum MJ-233

Summary Electroporation allowed transformation of intact cells ofBrevibacterium flavum MJ-233. The two plasmids used for electroporation were pCRY2 (6.3 kilobases) and pCRY3 (8.2 kilobases). Both plasmids contain the chloramphenicol-resistance gene and the autonomous replication origin inB. flavum MJ-233. The efficiency of electrotransformation was optimal with cells harvested at the middle log phase of growth, and was imporved by the addition of 1.0U/ml of penicillin G to the culture medium. The optimum yield of transformants per g DNA was 5×10⁴ when the cell suspension was pulsed at a cell density of 1×10¹⁰/ml and at a DNA amount of 1.0g.     

Development of a submerged-liquid sporulation medium for the potential smartweed bioherbicide Septoria polygonorum

Submerged culture experiments were conducted in three phases to determine the optimal medium for rapidly producing conidia of the fungal bioherbicide Septoria polygonorum. In phase I, 47 crude carbon sources were evaluated to determine which would support sporulation. Under the conditions tested, pea brine (5–10% v/v) provided best conidiation. In phase II, a fractional factorial design was utilized to screen 38 different medium adjuncts in combination with pea brine for improved sporulation. MgSO₄ was the only factor that resulted in a significant improvement. In phase III, a central composite design with response surface methodology was used to optimize concentrations of these critical factors. The model predicted optimal sporulation in a medium composed of 8.88% v/v pea brine+0.1 molar MgSO₄ with an expected titer of 1.78×10⁸ conidia/ml. Actual mean titer attained with the model-derived medium was 1.15×10⁸ conidia/ml. No significant difference was observed in virulence of conidia produced on agar vs. the model-derived (liquid) medium.     

High cell density perfusion culture of mouse-human hybridomas

Summary Five mouse-human hybridomas, H2, H3, V1, V2 and V6 cells secreting anti-virus human monoclonal immunoglobulin G (IgG) were cultured in serum-free media at high density in a settling perfusion culture vessel with an inner cell sedimentation zone. The H2, H3 and V6 cells reached a density of 10⁷ cells/ml in 0.5% (w/v) BSA-ITES-eRDF (see Materials and methods). The H2 cells reached only 6.8 × 10⁶ cells/ml in the absence of bovine serum albumin (BSA), but the addition of 0.2% (w/v) Pluronic F68 increased the maximum cell density to 1.1 × 10⁷ cells/ml, which was the same level as in BSA including medium. On the other hand, Pluronic F68 showed no stimulative effect on the growth of H2 cells in static culture. Pluronic F68 also increased the maximum cell density of V2 cells from 4.6 × 10⁶ cells/ml to 6.9 × 10⁶ cells/ml even in the presence of 0.5% (w/v) BSA.     

Growth of Fish Cell Lines on Microcarriers

Microcarrier beads were evaluated as substrates for the propagation of five anchorage-dependent fish cell lines. Growth of rainbow trout gonad (RTG-2) and Atlantic salmon cells was limited on microcarriers maintained in suspension. However, stationary microcarriers were suitable substrates for the growth of RTG-2, AS, Chinook salmon embryo (CHSE-214), and fathead minnow cells. Cell yields ranged from 2 × 10⁶ to 2.9 × 10⁶ cells per ml, representing 7- to 10-fold increases over the initial cell concentrations. The yield of new RTG-2 cells per unit volume of growth medium was 2.8 times greater in microcarrier cultures than in standard monolayer cultures. Northern pike cells failed to grow on microcarriers. Yields of infectious pancreatic necrosis virus propagated in microcarrier cultures of RTG-2 cells were more than twice the yields in standard monolayer cultures. The greater economy of microcarrier cultures in terms of growth vessel and medium requirements holds great promise for the large-scale production of anchorage-dependent fish cell cultures and fish viruses.