GROWTH RATES OF PSEUDOPEDINELLA PYRIFORME (CHRYSOPHYCEAE) IN RESPONSE TO 75 COMBINATIONS OF LIGHT,TEMPERATURE AND SALINITY1

Clonal cultures were established of single cells of Pseudopedinella pyriforme Carter isolated from the Chesapeake Bay. Axenic cultures were incubated at 5, 10 and 15 C; in 2 1/2, 5, 7 1/2, 10 and 15‰; and at 143, 285, 428, 571 and 714 μW/cm² white light. Maintainable growth rates were calculated from absorbance readings taken for 3 to 5 days from acclimated cultures. The maximum number of doublings per day was 0.9 at 2 1/2‰ salinity, 571 μW/ cm² and 15 C. A salinity of 5 ‰ produced the fastest growth at most light intensities up to 428μW/cm² and temperatures of 5, 10 and 15 C.     

Effect of liver cell coat acid mucopolysaccharide on the appearance of density-dependent inhibition in hepatoma cell growth.

Yoshida ascites hepatoma 66 (AH 66) cells grown in monolayer cultures show a lack of density-dependent inhibition of growth. When acid mucopolysaccharide (AMPS) isolated from rat liver cell coats is added to growing cultures at concentrations of 50–100 μg/ml, AH 66 cell cultures became markedly inhibited and exhibited density-dependent inhibition of growth at a cell density of 19 × 10⁴ cells/cm². Inhibition reached 84% below control levels. Inhibition is a density-related phenomenon since cells at densities below 19×10⁴ cells/cm² do not exhibit inhibition of growth. AH 66 cells inhibited in the plateau state are capable of resuming growth when AMPS is removed from the cultures. When AMPS is added at a concentration of 0.5 μg/ml, growth of tumor cells is promoted. Promotion reaches 78% above control levels. It is suggested that AMPS may play an important part in the regulation of cellular proliferation.     

Effect of dibutyryl cyclic AMP on the restoration of contact inhibition in tumor cells and its relationship to cell density and the cell cycle

KB cell cultures exposed to 10⁻⁴ M dibutyryl cyclic AMP were significantly inhibited and exhibited contact inhibition of growth at cell densities of 8 × 10⁴/cm² irrespective of the initial plating density. Control cultures reached densities of 2.5 × 10⁵/cm². Inhibition of growth did not occur in KB cells when the density was below 1 × 10⁴ cells/cm². When dibutyryl cyclic AMP was removed from KB cells in the contact-inhibited state, growth resumed with DNA synthesis beginning in about 6 h. Labeled metaphases increased rapidly after 22 h without the appearance of an early rise in unlabeled metaphases. This suggests that the inhibitory effect of dibutyryl cyclic AMP is on the G1 phase of the cell cycle.     

Murine mesenchymal stem cell isolated and expanded in low and high density culture system: surface antigen expression and osteogenic culture mineralization

Marrow culture from mice has been reported to be overgrown by non-mesenchymal cells. In almost all protocols for isolation of murine mesenchymal stem cells (MSCs), high density culture systems have been employed. Since MSCs are colonogenic cells, the initiating cell seeding density may have significant impact on their cultures. This subject was explored in this study. For this purpose, the bone marrow cells from NMRI mice were plated at 2.5 × 10⁶ cells/cm² and upon confluency were reseeded as either low density (50 cells/cm²) or high density (8 × 10⁴ cells/cm²) cultures. The cells were expanded through an additional subculture and the passage 2 cells as a product of two culture systems were statistically compared with respect to their surface antigen profiles and osteogenic culture mineralization. While low density culture grew with multiple colony formation, there were no distinct colonies in high density cultures. In contrast to high density cultures, passage 2 cells from low density system possessed typical homogenous fibroblastic morphology. Some cells from high density system but not the low density cultures expressed hematopoietic and endothelial cell markers including CD135, CD34, CD31, and Vcam surface antigens. Furthermore, osteogenic cultures from low density system displayed significantly more mineralization than those from high density system. Taken together, it seems that low density culture system resulted in more purified MSC culture than its counterpart as high density culture system.     

The pattern of density dependent growth inhibition in murine fibroblasts

Observations on the pattern of nuclear incorporation of ³H-TdR in long term (8-day) and short term (3-day) 3T3 cultures with local cell densities between 0.2 × 10⁴ and 6.2 × 10⁴ cells/cm² are reported. Contrary to a number of previous studies our observations indicate that density dependent inhibition is exhibited in relatively sparse cultures, commencing at 0.5 × 10⁴ cells/cm². Various possible mechanisms which could have caused the observed pattern of density-dependent regression in labelling index are discussed.     

Microcalorimetric investigations of the metabolism of yeasts

Summary Microcalorimetric experiments on growth ofSaccharomyces under oxygen and nitrogen pressure between 0 and 10 kp/cm² are described. Within this range there are no alterations of the metabolism by the pressureper se but increased cell volume and a pronounced number of cells are observed. With nitrogen the enthalpy change amounts to a value of 130 cal/g glucose invariable with pressure. For oxygen a maximum heat evolution of 650 cal/g glucose is found in stirred cultures at the minimum pressure of almost 0 kp/cm². With rising O₂ pressure one observes a strong repression of heat flux which drops to a minimum value at 2 kp/cm². This repression is overcome by substrate concentrations less than 2 mg/ml. In unstirred cultures exposed to oxygen pressure the growth is determined by the geometrical and temporal distribution of cells and oxygen in the vessels. The calorimetric data are discussed in view of the mean volume and the dry weight of the cells.Herrn Prof. Dr. W. Stein zum 60. Geburtstag gewidmet.     

Morphological transformation of C3H/10T1/2 cells subcultured at low cell densities

Morphological transformation in $\text{C3H/10T}\text{1}\text{2}$ cells treated with varied concentrations of benzo (α) pyrene (BP) was measured following subculture at low cell densities. Subconfluent cultures exposed to BP were allowed to grow to confluence, trypsinized, and reseeded at cell densities ranging from 5 to 2,300 surviving cells/cm². These secondary cultures were incubated for 8 to 9 weeks, stained, and examined for evidence of morphological transformation. BP-treated cells reseeded in virtual isolation in microwells (approx. 5 surviving cells/cm²) transformed at frequencies up to 14.5%. At these low initial cell densities, transformation frequency did not demonstrate a significant dependence on BP concentration. However, BP-treated cells reseeded at higher densities (11 to 2,300 surviving cells/cm²) showed both density-dependent transformation frequencies and BP-concentration dependence of transformation. As reported previously (Haber et al., Cancer Res. 37 1644, 1977), the subculturing of treated cells did not affect the BP-concentration dependence of focus formation in the $\text{C3H/10T}\text{1}\text{2}$ transformation assay. Cell density-dependent suppression of morphological transformation has now been observed over a wide range of BP concentration. We suggest that this phenomenon is associated with colony interactions and consider various possible mechanisms of BP involvement.     

Biogenesis of plasma lipoproteins in rat hepatoma McA-RH7777: Importance of diffusion-mediated events during cell growth

Summary Cultured McA-RH7777 rat hepatoma cells actively synthesize and secrete plasma lipoproteins. However, synthesis of [¹⁴C]triglyceride declines monotonically throughout the early growth period and remains low in postconfluent cultures; and net secretion of [¹⁴C]triglyceride is 10-fold more efficient in logarithmically growing cultures than in postconfluent cultures. Secretion of apolipoproteins associated with very low density and low density lipoproteins is selectively reduced in postconfluent cultures. The temporal reductions in [¹⁴C]triglyceride production are related more strongly to increasing cell concentration (cells/cm³ medium) than to increasing cell density (cells/cm² growth surface). We have allowed cells to grow either retained within small circular corrals or unrestricted in culture dishes. When seeded at equal density (10⁴ cells/cm²) but at one-fifth the cell concentration, corralled cells synthesize twice as much [¹⁴C]triglyceride per cell after 2 and 4 d, and are 10 times as efficient in [¹⁴C]triglyceride secretion by 6 d of growth, as noncorralled cells. When seeded at equal cell concentration (10⁵ cells/dish) but at 5 times the cell density, corralled cells are only 20% less efficient at [¹⁴C]triglyceride synthesis and secretion than noncorralled cells. Conditioned medium depresses synthesis and secretion efficiency of [¹⁴C]triglyceride. Orotic acid exposure also inhibits synthesis of [¹⁴C]triglyceride and secretion of certain [³⁵S]apolipoproteins in early cultures, but it has no significant effect on late cultures. We conclude that diffusion-mediated events are important regulators of triglyceride and apolipoprotein production in growing rat hepatoma cells, but that events associated with formation of cell-to-cell contacts play a minor role in regulation of plasma lipoprotein biogenesis. This research was supported by grants to R. H. from the American Heart Association (85-805), from NHLBI (15062, Specialized Center of Research in Atherosclerosis), and from the Louis Block Fund. S. T. was supported as an American Heart Association Medical Student Research Fellow. S. T. and H. S. are recipients of predoctoral training grants from the National Institutes of Health, Bethesda, MD (HL 723711, HD 07009). This work has been presented elsewhere in preliminary form (14, 48, 49).     

Light and calcium interactions in chlorella inhibited by sodium chloride

Analysis of NaCl toxicity in Chlorella sorokiniana showed decreased growth rates, increased dry weight per cell, increased intracellular Na⁺ and Cl⁻, more total chlorophyll per cell, a decreased chlorophyll a to chlorophyll b ratio, increased rates of O₂ evolution, and decreased rates of CO₂ fixation when the extracellular concentration of NaCl was increased from zero to 0.3 m. Cultures did not grow at concentrations greater than 0.3 m NaCl unless 10 mm calcium salts were present. Inclusion of that concentration of Ca²⁺ extended the tolerance to 0.5 m NaCl before growth stopped. Increasing the light intensity from 1.2 to 9.4 mw/cm² increased growth rates for cultures in 0.10 to 0.45 m NaCl. At 14 mw/cm² added Ca²⁺ reduced growth rates of cultures in 0.3 m NaCl compared to controls without added Ca²⁺. Maximal growth rates for cultures in NaCl media were achieved by addition of 10 mm CaSO₄ and maintenance of the light intensity at 9.4 mw/cm². The maximal growth rate of the organism was 9.6 doublings/day achieved at 2.7 mw/cm² for control cultures. In 0.3 m NaCl the growth rate was 4.3 doublings/day at 2.7 mw/cm² and 8.2 doublings/day at 9.4 mw/cm² with 10 mm CaSO₄ added.     

The effect of choleragen and epidermal growth factor on proliferation and maturation in vitro of human ectocervical cells

Summary The role of choleragen (CT) and epidermal growth factor (EGF) has been examined in relation to the control of growth and differentiation of adult human cervical epithelial (HCE) cells derived from the ectocervix. Cervical biopsies derived from hysterectomy specimens were trypsin disaggregated and HCE cells were plated at 5×10³/cm² in the presence of 2×10⁴/cm² lethally irradiated Swiss 3T3 fibroblasts. Cultures were grown in Liebovitz medium supplemented with 10% fetal bovine serum and hydrocortisone. Epidermal growth factor at 10 ng/ml and choleragen at 10⁻¹⁰ M were added to cultures either singly or in combination. DNA replication in these cultures was measured autoradiographically after exposing cells to tritiated thymidine for 2 h. Differentiation was assessed histochemically by determining glycogen accumulation using the periodic acid Schiff technique. Choleragen increased colony plating efficiency by at least a factor of two but had no effect on colony size Epidermal growth factor did not increase plating efficiency but did increase colony size. In EGF treated colonies DNA replication occurred throughout the colony compared to CT treated colonies in which replication was restricted to the periphery. In the absence of EGF, population doublings achieved in culture did not exceed 32 and glycogen accumulation was evident in cells early in culture life. Colonies treated with EGF exhibited glycogen accumulation late in culture life and the EGF treated cells achieved at least 50 population doublings in culture. The results are discussed in relation to the role of EGF and choleragen on cell differentiation.     

Predicting Aggregation Kinetics of DU 145 Prostate Cancer Cells in Liquid-Overlay Culture

The predictive capacity of a novel population-balance model to simulate aggregation kinetics of attachment-dependent cells at the resolution of one-cell increments has been evaluated. Using spheroid assembly of DU 145 human prostate cancer cells as a representative system, the mathematical model proved to be robust in simulating aggregation over a 5-fold range of surface densities from 5×10³ to 2.5×10⁴ cells/cm² with a single matrix of rate constants. For cultures at 1×10⁵ cells/cm², more than 75% of simulated aggregate concentrations are within the standard deviation of measured concentrations. For the two extreme densities, at least two-thirds of model predictions are within 35% of the mean for experimental data. Error in model predictions is attributed to uncertainty in measurements and intrinsic changes in aggregation. The model has application to the rational design of spheroids in tissue engineering and bioseparation processes in pharmaceutical manufacturing.     

Induction of polyamine limitation in Chinese hamster ovary cells by α-methylornithine

Chinese hamster ovary (CHO) cells in culture were limited for polyamines through the use of α-methylornithine (αMO), a competitive inhibitor of ornithine decarboxylase. Initial exposure of the cells to the inhibitor caused growth rate and intracellular polyamine content to decline continuously. Reseeding the αMO-treated cells into medium containing the inhibitor resulted in steady-state (exponential) growth at cell densities below 5 × 10³ cells/cm², at a rate approximately twofold slower than untreated cells. Under these conditions, putrescine and spermidine were undetectable and spermine remained relatively constant at a level approximately half that found in untreated cells. Addition of exogenous putrescine elevated the polyamine content and stimulated the growth of αMO-treated cultures. Thus, growth rate correlated with polyamine content in the αMO-treated cells. The growth of reseeded. αMO-treated cells became nonexponential at a density (5 × 10³ cells/cm²) far below that at which untreated cells departed from exponential growth (1 × 10⁵ cells/cm²). Medium obtained from high density, αMO-treated cultures inhibited the growth of cells at low density in the presence of αMO. Doubling the concentration of the defined components of conditioned medium did not markedly affect its capacity to inhibit growth. However, dialysis completely removed the inhibitory activity from conditioned medium. The results imply that a low molecular weight inhibitor of growth is produced by polyamine-limited cells. This is a variable that must be controlled in studies with polyamine-limited animal cells. Morphological studies indicated that subcellular organelles, including mitochondria, were largely unaffected by treatment with αMO. The maintenance of mitochondrial integrity in the presence of αMO demonstrates that the swelling of mitochondria observed previously in cells treated with methylglyoxal bis(guanylhydrazone) was not due to polyamine limitation. αMO-treated cells did, however, accumulate numerous cytoplasmic vacuoles. The identity of these vacuoles and their relationship to cellular physiology is not yet understood.     

DNA synthesis and cell proliferation in C6 glioma and primary glial cells exposed to a 836.55 MHz modulated radiofrequency field

We have tested the hypothesis that modulated radiofrequency (RF) fields may act as a tumor-promoting agent by altering DNA synthesis, leading to increased cell proliferation. In vitro tissue cultures of transformed and normal rat glial cells were exposed to an 836.55 MHz, packet-modulated RF field at three power densities: 0.09, 0.9, and 9 mW/cm², resulting in specific absorption rates (SARs) ranging from 0.15 to 59 μW/g. TEM-mode transmission-line cells were powered by a prototype time-domain multiple-access (TDMA) transmitter that conforms to the North American digital cellular telephone standard. One sham and one energized TEM cell were placed in standard incubators maintained at 37 °C and 5% CO₂. DNA synthesis experiments at 0.59–59 μW/g SAR were performed on log-phase and serum-starved semiquiescent cultures after 24 h exposure. Cell growth at 0.15–15 μW/g SAR was determined by cell counts of log-phase cultures on days 0, 1, 5, 7, 9, 12, and 14 of a 2 week protocol. Results from the DNA synthesis assays differed for the two cell types. Sham-exposed and RF-exposed cultures of primary rat glial cells showed no significant differences for either log-phase or serum-starved condition. C₆ glioma cells exposed to RF at 5.9 μW/g SAR (0.9 mW/cm²) exhibited small (20–40%) significant increases in 38% of [³H]thymidine incorporation experiments. Growth curves of sham and RF-exposed cultures showed no differences in either normal or transformed glial cells at any of the power densities tested. Cell doubling times of C₆ glioma cells [sham (21.9 ± 1.4 h) vs. field (22.7 ± 3.2 h)] also demonstrated no significant differences that could be attributed to altered DNA synthesis rates. Under these conditions, this modulated RF field did not increase cell proliferation of normal or transformed cultures of glial origin. Bioelectromagnetics 18:230–236, 1997. © 1997 Wiley-Liss, Inc.     

Anchorage-dependent mammalian cell culture using polyurethane foam as a new substratum for cell attachment

Summary Anchorage-dependent mammalian cells were cultivated at high cell density in a novel culture system using polyurethane foam (PUF) as a substratum for cell attachment. PUF has a macroporous structure giving a high surface area to volume ratio. Monkey kidney cells (Vero) and Chinese hamster ovary cells (CHO-K1) attached to the internal surface of PUF and grew to a high cell density (1.04 × 10⁸ cells/ cm³ PUF and 3.5 × 10⁷ cells/ cm³ PUF, respectively) in PUF stationary cultures. In addition, we have designed a PUF-particle packed-bed culture system for high density mass cell culture. A maximum cell density of 2.4 × 10⁷ cells/cm³ culture vessel volume was obtained in a packed-bed culture of Vero cells. Offprint requests to: K. Funatsu     

Tissue plasminogen activator (t-PA) production by a high density culture of weakly adherent human embryonic kidney cells using a polyurethane-foam packed-bed culture system

Weakly adherent cells of the 293 line attached well to the internal surface of polyurethane foam (PUF) and grew to the high density of 6.83 × 10⁷ cells/cm³ PUF in stationary culture. The maximum productivity of tissue plasminogen activator (t-PA) was 0.158 IU/10⁶ cells per day. The productivity decreased at the stationary phase of cell growth, so we designed a PUF-plate packed-bed culture system for high density culture and continuous production of t-PA. A maximal cell density of 3.24 × 10⁷ cells/cm³ PUF and a t-PA productivity of 0.326 IU/10⁶cells per day were obtained in 25-day perfusion cultures. Although the cell density decreased to half that in PUF stationary culture, the t-PA productivity increased twofold and was maintained for 25 culture days.     

Buoyant Densities and Dry-Matter Contents of Microorganisms: Conversion of a Measured Biovolume into Biomass

Several isolates of bacteria and fungi from soil, together with cells released directly from soil, were studied with respect to buoyant density and dry weight. The specific volume (cubic centimeters per gram) of wet cells as measured in density gradients of colloidal silica was correlated with the percent dry weight of the cells and found to be in general agreement with calculations based on the partial specific volume of major cell components. The buoyant density of pure bacterial cultures ranged from 1.035 to 1.093 g/cm³, and their dry-matter content ranged from 12 to 33% (wt/wt). Average values proposed for the conversion of bacterial biovolume into biomass dry weight are 1.09 g/cm³ and 30% dry matter. Fungal hyphae had buoyant densities ranging from 1.08 to 1.11 g/cm³, and their dry-matter content ranged from 18 to 25% (wt/wt). Average values proposed for the conversion of hyphal biovolume into biomass dry weight are 1.09 g/cm³ and 21% dry matter. Three of the bacterial isolates were found to have cell capsules. The calculated buoyant density and percent dry weight of these capsules varied from 1.029 g/cm³ and 7% dry weight to 1.084 g/cm³ and 44% dry weight. The majority of the fungi were found to produce large amounts of extracellular material when grown in liquid cultures. This material was not produced when the fungi were grown on either sterile spruce needles or membrane filters on an agar surface. Fungal hyphae in litter were shown to be free from extracellular materials.     

Capacity for tumor cell implantation as a function of in vitro cell density

Implantation properties of two melanoma cell lines, line 26 (low rate of implantation) and line 37 (high rate of implantation) were studied as a function of the cell density of the cells grown in monolayer in vitro. Sparse cultures (collected at a density of 0.8 × 10³ cells cm^?2) of line 37 produced 7.7 times as many lung tumor foci as those of line 26. Confluent cultures (collected at a density of 40 × 10³ cells cm^?2) resulted in greater numbers of tumor foci for both cell lines, but line 37 produced only 3.1 times as many tumor foci as did line 26 cells. Thus the high implantation line (37) has a much greater ability to implant when grown in the sparse state and injected than the low implantation line (26), but both lines have high implantation rates when injected as confluent cells.     

High cell density perfusion cultures of anchorage-dependent Vero cells in a depth filter perfusion system

A depth filter perfusion system (DFPS) with polypropylene fibers had been demonstrated to support high density cultures of anchorage-independent hybridoma cells. The DFPS provides advantages of high surface-to-volume ratio of 450–600 cm²/cm³, low cost set-up, easy operation and scale-up. To test the feasibility of using DFPS for high density cultures of anchorage-dependent cells, Vero cells were cultivated in the DFPS. Gelatin coating on polypropylene fibers in the DFPS was necessary to promote cell attachment and growth. Dissolved oxygen (DO) concentrations could be controlled by sparging air into the reservoir vessel through a filter sparger. When DO concentration was controlled above 40% of air saturation in the DFPS with 40 m pore size, the maximum cell concentration as estimated on specific lactate production rate, was 3.81×10⁷ cells/ml of the total reactor volume. This viable cell concentration is approximately 18 times higher than that obtained in a T-flask batch culture. Taken together, the results obtained here showed the potential of DFPS for high-density cultures of anchorage-dependent cells.     

Initial Cell Seeding Density Influences Pancreatic Endocrine Development During in vitro Differentiation of Human Embryonic Stem Cells

Human embryonic stem cells (hESCs) have the ability to form cells derived from all three germ layers, and as such have received significant attention as a possible source for insulin-secreting pancreatic beta-cells for diabetes treatment. While considerable advances have been made in generating hESC-derived insulin-producing cells, to date in vitro-derived glucose-responsive beta-cells have remained an elusive goal. With the objective of increasing the in vitro formation of pancreatic endocrine cells, we examined the effect of varying initial cell seeding density from 1.3 x 10⁴ cells/cm² to 5.3 x 10⁴ cells/cm² followed by a 21-day pancreatic endocrine differentiation protocol. Low density-seeded cells were found to be biased toward the G2/M phases of the cell cycle and failed to efficiently differentiate into SOX17-CXCR4 co-positive definitive endoderm cells leaving increased numbers of OCT4 positive cells in day 4 cultures. Moderate density cultures effectively formed definitive endoderm and progressed to express PDX1 in approximately 20% of the culture. High density cultures contained approximately double the numbers of PDX1 positive pancreatic progenitor cells and also showed increased expression of MNX1, PTF1a, NGN3, ARX, and PAX4 compared to cultures seeded at moderate density. The cultures seeded at high density displayed increased formation of polyhormonal pancreatic endocrine cell populations co-expressing insulin, glucagon and somatostatin. The maturation process giving rise to these endocrine cell populations followed the expected cascade of pancreatic progenitor marker (PDX1 and MNX1) expression, followed by pancreatic endocrine specification marker expression (BRN4, PAX4, ARX, NEUROD1, NKX6.1 and NKX2.2) and then pancreatic hormone expression (insulin, glucagon and somatostatin). Taken together these data suggest that initial cell seeding density plays an important role in both germ layer specification and pancreatic progenitor commitment, which precedes pancreatic endocrine cell formation. This work highlights the need to examine standard culture variables such as seeding density when optimizing hESC differentiation protocols.