High Initial Cell Density Research Articles

Transport limitation of chlorine disinfection of Pseudomonas aeruginosa entrapped in alginate beads

An artificial biofilm system consisting of Pseudomonas aeruginosa entrapped in alginate and agarose beads was used to demonstrate transport limitation of the rate of disinfection of entrapped bacteria by chlorine. Alginate gel beads with or without entrapped bacteria consumed chlorine. The specific rate of chlorine consumption increased with increasing cell loading in the gel beads and decreased with increasing bead radius. The value of an observable modulus comparing the rates of reaction and diffusion ranged from less than 0.1 to 8 depending on the bead radius and cell density. The observable modulus was largest for large (3-mm-diameter) beads with high cell loading (1.8 x 10(9) cfu/cm(3)) and smallest for small beads (0.5 mm diameter) with no cells added. A chlorine microelectrode was used to measure chlorine concentration profiles in agarose beads (3.0 mm diameter). Chlorine fully penetrated cell-free agarose beads rapidly; the concentration of chlorine at the bead center reached 50% of the bulk concentration within approximately 10 min after immersion in chlorine solution. When alginate and bacteria were incorporated into an agarose bead, pronounced chlorine concentration gradients persisted within the gel bead. Chlorine did gradually penetrate the bead, but at a greatly retarded rate; the time to reach 50% of the bulk concentration at the bead center was approximately 46 h. The overall rate of disinfection of entrapped bacteria was strongly dependent on cell density and bead radius. Small beads with low initial cell loading (0.5 mm diameter, 1.1 x 10(7) cfu/cm(3)) experienced rapid killing; viable cells could not be detected (<1.6 x 10(5) cfu/cm(3)) after 15 min of treatment in 2.5 mg/L chlorine. In contrast, the number of viable cells in larger beads with a higher initial cell density (3.0 mm diameter, 2.2 x 10(9) cfu/cm(3)) decreased only about 20% after 6 h of treatment in the same solution. Spatially nonuniform killing of bacteria within the beads was demonstrated by measuring the transient release of viable cells during dissolution of the beads. Bacteria were killed preferentially near the bead surface. Experimental results were consistent with transport limitation of the penetration of chlorine into the artificial biofilm arising from a reaction-diffusion interaction. The methods reported here provide tools for diagnosing the mechanism of biofilm resistance to reactive antimicrobial agents in such applications as the treatment of drinking and cooling waters.

Production of human hematopoietic progenitors in a clinical-scale stirred suspension bioreactor

Ex vivo expanded primitive hematopoietic cells can be utilized in bone marrow transplantation therapies to treat patients suffering from various cancers and hematopoietic malignancies. A high initial cell density (106 cells/mL) and the supplement of soluble factors secreted by stromal feeders in combination with growth-promoting (interleukin-3 and stem cell factor) and growth-inhibiting (macrophage-inflammatory protein-1α) cytokines resulted in high, long-term expansions (17-fold over a 14-day culture period) of human hematopoietic progenitors in a stirred suspension bioreactor. This study demonstrated that a transplantable dosage of human hematopoietic progenitor cells (8.1 ± 1.3 × 106 colony forming unit-granulocyte/macrophage) can be generated from approximately 10 mL of bone marrow aspirate in a 14-day culture using a 250 mL suspension bioreactor system. © Rapid Science Ltd. 1998

Development of resting membrane potentials in differentiating murine neuroblastoma cells (N1E-115) evaluated by flow cytometry.

With the aid of a voltage-sensitive oxonol dye, flow cytometry was used to measure relative changes in resting membrane potential (V(m)) and forward angle light scatter (FALS) profiles of a differentiating/differentiated murine neuroblastoma cell line (N1E-115). Electrophysiological differentiation was characterized by V(m) establishment. The (V(m))-time profile was found to be seed cell concentration-dependent for cell densities of less than 2 × 10(4) cells/cm(2). At higher initial cell densities, under differentiating culture conditions, V(m) development commenced on day 2 and reached a steady-state on day 12. The relative distribution of differentiated cells between low and high FALS has been proposed as a potential culture electrophysiological differentiation state index. These experiments offer a general methodology to characterize cultured excitable cells of nervous system origin, with respect to electrophysiological differentiation. This information is valuable in studies employing neuroblastoma cells as in vitro screening models for safety/hazard evaluation and/or risk assessment of therapeutical and industrial chemicals under development.

CELL DEATH, SURVIVAL AND PROLIFERATION IN TETRAHYMENA THERMOPHILA. EFFECTS OF INSULIN, SODIUM NITROPRUSSIDE, 8-BROMO CYCLIC GMP, N G-METHYL-L-ARGININE AND METHYLENE BLUE

Cells of the ciliate Tetrahymena thermophilaproduce compounds that act as autocrine (paracrine) survival and/or growth factors. 8-Bromo cyclic GMP, sodium nitroprusside, hemin, protoporphyrin IX, human recombinant and bovine insulin were tested for their ability to substitute for the cell-produced factors and stimulate cell survival and proliferation. The cells were inoculated into conical flasks in a nutritionally complete, chemically defined medium at known cell densities from 5 to 5000 cells/ml. In unsupplemented medium cells at 5 to 500 cells/ml (‘low initial cell density cultures’) died within 8 h, whereas cells at 1000 and 5000 cells/ml (‘high initial cell density cultures’) proliferated with lag phases lasting for up to 4 h. In the presence of insulin compounds, hemin, protoporphyrin IX, or 8-bromo cyclic GMP, cells also proliferated at all low initial cell densities. Sodium nitroprusside was effective over two separate concentration ranges: at the nanomolar levels as well at low pico- to femtomolar levels. At initial population densities of up to 50 cells/ml the cells at both concentrations of sodium nitroprusside survived about 4-fold longer than the controls. At 500 initial cells/ml, cells at thehigh concentrations of sodium nitroprusside survived about 4-fold longer than those of the control cultures; they proliferated in the low concentrations of sodium nitroprusside. Concentrations of hemin, too low to have any effects on their own, had synergistic effects with sodium nitroprusside. N G-methyl-L-arginine inhibited proliferation at high initial cell densities. This inhibitory action was reduced by high concentrations of L-arginine, protoporphyrin IX, sodium nitroprusside, or 8-bromo cGMP, but not by insulin. Methylene blue inhibited cell proliferation at high initial cell densities. This inhibition was circumvented by addition of 8-bromo cGMP. The findings that insulin-related material may be released from Tetrahymenaand that insulin and sodium nitroprusside increase intracellular cGMP in these cells are discussed in relation to the presented results. Together these observations suggest that cGMP is responsible for supporting cell survival in Tetrahymenaand switching the cells into their proliferative mode, and that cell-produced signal molecules and insulin stimulate an NO-dependent guanylate cyclase into producing cGMP.

Biomass relationship to growth and phosphate uptake of Pseudomonas fluorescens, Escherichia coli and Acinetobacter radioresistens in mixed liquor medium.

The ability of Pseudomonas fluorescens, Escherichia coli and Acinetobacter radioresistens to remove phosphate during growth was related to the initial biomass as well as to growth stages and bacterial species. Phosphate was removed by these bacteria under favourable conditions as well as under unfavourable conditions of growth. Experiments showed a relationship between a high initial cell density and phosphate uptake. More phosphate was released than removed when low initial cell densities (10(2)-10(5) cells ml-1) were used. At a high initial biomass concentration (10(8) cells ml-1), phosphate was removed during the lag phase and during logarithmic growth by P. fluorescens. Escherichia coli, at high initial biomass concentrations (10(7) cells ml-1), accumulated most of the phosphate during the first hour of the lag phase and/or during logarithmic growth and in some cases removed a small quantity of phosphate during the stationary growth phase. Acinetobacter radioresistens, at high initial cell densities (10(6), 10(7) cells ml-1) removed most of phosphate during the first hour of the lag phase and some phosphate during the stationary growth phase. Pseudomonas fluorescens removed phosphate more than A. radioresistens and E. coli with specific average ranges from 3.00-28.50 mg L-1 compared to average ranges of 4.92-17.14 mg L-1 for A. radioresistens and to average ranges of 0.50-8.50 mg L-1 for E. coli.

Effect of inoculum level of xylitol production from rice straw hemicellulose hydrolysate by Candida guilliermondii.

The effect of inoculum level on xylitol production by Candida guilliermondii was evaluated in a rice straw hemicellulose hydrolysate. High initial cell density did not show a positive effect in this bioconversion since increasing the initial cell density from 0.67 g L-1 to 2.41 g L-1 decreased both the rate of xylose utilization and xylitol accumulation. The maximum xylitol yield (0.71 g g-1) and volumetric productivity (0.56 g L-1 h-1) were reached with an inoculum level of 0.9 g L-1. These results show that under appropriate inoculum conditions rice straw hemicellulose hydrolysate can be converted into xylitol by the yeast C. guilliermondii with efficiency values as high as 77% of the theoretical maximum.

Simulation of oxygen concentration profile in calcium alginate gel beads entrapping microbes during biological phenol degradation

Numerical simulation was carried out to elucidate the effects of gel-bead diameter, initial cell density, initial phenol concentration, and oxygen concentration in the bulk liquid on the change in the oxygen concentration profile in a gel bead during biological aerobic phenol degradation with activated sludge entrapped in the calcium alginate gel bead. The results of simulation show that oxygen-limiting occurs in the central parts of the gel beads under most conditions examined in this study. After phenol degradation starts, the oxygen concentration in the beads is found to be immediately reduced and slightly increased instantly, and then slowly decreased linearly to a minimum value as the degradation proceeds. Finally, the oxygen concentration rapidly recovers to the same level of the bulk liquid. For small-sized gel beads, at low initial cell densities no oxygen-limiting is found to occur even in the core of the gel beads due to the small mass transfer resistance for oxygen diffusion, low oxygen consumption rate, or both. The critical gel-bead diameter for freedom from oxygen depletion is about 1 mm. At high phenol concentrations, oxygen may penetrate to the central parts of the gel beads as a result of reduction of the oxygen consumption rate by substrate inhibition. The effectiveness factors for small-sized cell-loaded gel beads were found to be unity at relatively high phenol concentrations in the bulk liquid and to decrease with increasing cell density. Further, it is found that at higher initial cell densities or for large-sized gel beads a high cell density layer will be formed around the surface of the gel beads during phenol degradation periods.

Fibroblastic cell cycling in collagen gels.

Quiescent C3H10T1/2 mouse fibroblasts resume DNA synthesis and proliferation following incubation in medium containing fresh serum both when grown in monolayer and when grown in a collagen matrix. We observed that the rate of DNA synthesis is reduced at high initial cell densities and low initial collagen concentrations. In a collagen matrix, fibroblasts contract the matrix causing an increase in cell density and collagen concentration. We studied the chronological relationship between the kinetics of DNA synthesis and the collagen matrix contraction. The rate of collagen collection per cell changes in time, dependent on initial cell and collagen concentration. The kinetics of the collagen collection showed a positive correlation with the kinetics of DNA synthesis, 16 h later.

Dynamics of plasmid transfer on surfaces

A protocol was developed to study the dynamics of growth and plasmid transfer in surface populations of bacteria. This method allows for quantitative estimates of cell population densities over time, as well as microscopic observations of colony growth and interactions. Using this 'surface slide system' (SSS), the dynamics of the plasmid R1 and its permanently derepressed mutant R1drd19 in surface cultures of Escherichia coli K12 was examined. In surface culture, the stationary-phase cell densities were constant over a wide range of initial cell density (= colony density) and comparable to those obtained in liquid culture. For high initial cell densities, where the cells formed a confluent layer at stationary phase, the kinetics of growth and plasmid transfer was similar to that obtained in liquid culture, and the relative yields of R1drd19 and R1 transconjugants were similar in the two habitats. In surface culture, however, R1drd19 transconjugant yield was profoundly affected, and R1 transfer to a lesser extent, by colony density. In contrast, liquid matings were virtually unaffected by initial cell density. The transfer advantage of the permanently depressed over the repressed plasmid was much less apparent for lower colony densities. I propose a hypothesis for plasmid transfer between colonies that explains these observations as a consequence of the geometry of the surface habitat and the effect of transitory derepression of the synthesis of pili.

Batch fermentations with a mixed culture of lactic acid bacteria immobilized separately in ?-carrageenan locust bean gum gel beads

Streptococcus salivarius subsp. thermophilus and Lactobacillus delbrueckii subsp. Bulgaricus were immobilized separately in κ-carrageenan-locust bean gum gel beads. The beads were prepared by a dispersion process in a two-phase system (water in oil) and two ranges of bead diameter selected by sieving (0.5–1.0 mm and 1.0–2.0 mm). Fermentations with the two strains were conducted in bench bioreactors in a supplemented whey permeate medium. Free and entrapped cells (two ranges of bead diameter and two levels of initial bead cell load) were grown in mixed culture, and carbohydrate utilization, acid production and cell growth or cell release rate measured. Fermentation rates were influenced by bead diameter and initial cell load of the beads. Beads with high initial cell density increased fermentation rates compared to low cell density beads or free cells. Smaller diameter beads (0.5–1.0 mm) showed a stable tendency (not statistically significant p a > 0.05) towards higher cell release rates, lactose utilization, galactose accumulation and lactic acid production than did larger diameter beads (1.0–2.0 mm). Immobilization of S. salivarius subsp. thermophilus and L. delbrueckii subsp. bulgaricus in separate beads did not seem to affect protocooperation during batch fermentation, and allowed for high cell release rates into the medium.

Additive Effects of Alcohols, Their Acidic By-Products, and Temperature on the Yeast Pachysolen tannophilus

The effects of alcohols on the growth and fermentation of the yeast Pachysolen tannophilus were investigated at both 30 and 35 degrees C. Addition of alcohols to the culture medium decreased both the growth rate and the final cell yield in a dose-dependent manner, and this decrease was more severe at 35 degrees C. The concentration for 50% growth rate inhibition decreased as the chain length of the alcohol increased. In fermentations using a high initial cell density, production of acids was always observed when the medium was supplemented with alcohols. Supplementation of the culture medium with a short-chain alcohol plus the corresponding acid was shown to exert an additive deleterious effect on fermentation, and this effect increased with temperature. Production of acids was associated with the presence of alcohol dehydrogenase activity in cell extracts.

THE ROLE OF UNICELLS IN THE POLYMORPHIC SCENEDESMUS ARMATUS (CHLOROPHYCEAE)1

ABSTRACTThe UTEX 2193 strain of Scenedesmus armatus (Chod.) Chod, when cultured in any of several media (whether natural or artificial, concentrated or dilute) produced a variety of colonial morphologies as well as a unicell population. Morphological expression was related to culture ape. When the initial cell density was just a feu1 hundred cells per mL. the culture first produced a unicell population, then spiny colonies, and as stationary phase was approached, spine‐less colonies.Two classes of spiny colonies were detected. Type I colonies had elongate cells with the terminal cells shorter than median cells. Spines were longer than cell length. The wider, oval, grainy cells of Type II colonies were uniform m length. Spines were shorter and thicker than those on Type I colonies. Only Type I colonies produced unicells: the latter appeared as two morphs. The smaller unicell was obovoid with four delicate spines: the larger had ovate cells bearing four thicker spines.Control of unicell development in all media was achieved by carefully monitoring colony type and cell number used for the inoculum. A unicellular population developed in batch culture in defined media, both concentrated and dilute, when the initial cell density (either Type I or Type II colonies) was low (below 1000 cells‐mL−1), as well as in synchronous cultures. With higher initial cell densities, e.g. 2 × 104 cells·mL−1, the inoculum had to contain Type I colonies to produce unicells. Unicells were also produced in water from Agronomy Pond, where the strain originated. We discuss the role of unicell populations in the distribution of Scenedesmus.

A link between cell movement and gene expression argues that motility is required for cell-cell signaling during fruiting body development.

Nonmotile mutants of Myxococcus xanthus (Myxobacterales) failed to execute the morphogenetic movements required to shape a fruiting body. In addition, nonmotile mutants produced very few spores when plated for fruiting body development at cell densities appropriate for wild-type cells. At higher initial cell densities, the proportion of nonmotile cells that sporulate increased, indicating that one important function of motility in fruiting body development is to increase the local cell density. However, even at 10 times normal cell density, nonmotile cells sporulated at only 1% the wild-type level. This sporulation deficiency of nonmotile mutants accompanies an altered pattern of gene expression, monitored by using transcriptional fusions of lacZ to genes expressed at specific times during fruiting body development. Motility was not required for normal expression of five lac fusions that are expressed within the first 6 hr of fruiting-body development. However, the levels of expression from five lac fusions to later-expressed genes were reduced or abolished in nonmotile strains. beta-Galactosidase expression in these late Tn5 lac insertions was increased, and fruiting body development occurred in certain nonmotile strains that can be stimulated to move when mixed with a donor strain. This shows that motility itself is required because the stimulated cells are nonmotile genotypically. The nonmotile mutations had the same effect on developmental beta-galactosidase expression from these 10 lac fusions as an insertion mutation in the csg (formerly spoC) gene. csg mutants have a cell-cell interaction defect that blocks fruiting body development at approximately 6 hr. The similarity in the pattern of developmental expression of motility mutants and csg mutants suggests that motility is required for this csg-mediated cell-cell interaction.

Acidity, aluminium and multiplication of Rhizobium trifolii: Effects of initial inoculum density and growth phase

Factors influencing the response of Rhizobium strains to acidity and aluminium were studied in an arabinose-galactose-glutamate liquid medium. Rhizobium trifolii strains HP3 and BEL 1192 multiplied at the same rate at pH S.S. BEL1192 multiplied at a slower rate at pH 4.5, and all produced visible turbidity from a low initial cell density. HP3 multiplied at pH 4.5 but produced no turbidity. The pH value of the medium increased when cell densities reached 10 6-10 7 cfu ml −1. Diauxic growth responses occurred, only at pH 4.5, and HP3 demonstrated a population effect at pH 4.5 with visible turbidity produced from high initial cell densities. 50 μm Al caused a decrease in numbers of BEL 1192 at pH4.5 and HP3 at pH 5.5 irrespective of initial cell densities. 50 μ m Al was more toxic to HP3 cells in log phase than in stationary phase.

Evidence for sequential appearance of cartilage matrix proteins in developing mouse limbs and in cultures of mouse mesenchymal cells

Abstract The initiation of synthesis and the accumulation of four cartilage matrix proteins (type II collagen and three noncollagenous proteins, one of M r 148, one of M r 59, and an oligometric protein of M r above 500 with 100-kDa subunits, respectively) were studied in developing mouse limbs and in cultures of limb bud mesenchyme by means of immunolocalization. On day 13 of gestation, type II collagen was observed throughout the entire humerus, whereas the 148-kDa protein was localized only in the central portion. Neither the 100-kDa-subunit protein nor the 59-kDa protein could be demonstrated in the humerus at that stage. On day 14½, type II collagen and the 148-kDa protein were codistributed throughout the humerus. The 100-kDa-subunit protein was detectable in the periphery of the humerus, whereas little 59-kDa protein could yet be demonstrated. On day 18, all four proteins being studied could be detected immunologically in the developing mouse humerus. They differed in immunolocalization. Type II collagen, the 148-kDa protein, and the 100-kDa-subunit protein were codistributed throughout the distal and proximal parts of the cartilage. However, the 148-kDa protein could no longer be detected immunochemically in the outermost part of the cartilage in the proximal shoulder joint. The 148-kDa protein codistributed with type II collagen and the 100-kDa-subunit protein in the distal cartilaginous region, where joint development was less advanced. On the other hand, the 59-kDa protein was not demonstrated directly within the hyaline cartilaginous structures, but surrounded the entire structure. This protein was also present in the same part of the proximal joint region as that in which the 148-kDa protein was not detected. To develop an in vitro model for studies of skeletogenesis, mesenchymal cells prepared from mouse limb buds were cultured as micromass cultures at high initial cell density to favor chondrogenesis. On day 3 of culture, type II collagen was the only protein that could be detected immunochemically in the cultures, whereas on day 6 the 148-kDa protein was demonstrated, and a few chondrocytes in the central portion of each cartilaginous nodule were associated with the 100-kDa-subunit protein. The 59-kDa protein could not yet be immunochemically detected. On day 14, type II collagen, the 148-kDa protein, and the 100-kDa-subunit protein could be detected throughout the cartilaginous nodules, whereas the 59-kDa protein was distributed in tissues surrounding each nodule. On day 55, the distribution of the four cartilage matrix proteins was the same on day 14, except that the 59-kDa protein was also demonstrated near some of the chondrocytes. In conclusion, the four cartilage matrix proteins being studied appeared in sequence. The order of appearance in the developing mouse humerus was identical to that in cultures of mesenchymal cells from the early mouse limb bud. These results indicate that the mesenchymal cell culture system can be used as an in vitro model for studies for skeletogenesis.

Internal standards for survival: increasing the accuracy for human cell mutation assays

In mutation and cell transformation assays, it has long been recognized that the common practice of using different numbers of cells on dishes with or without selective conditions creates a source of bias in mutant fraction determination. This is simply because colony formation may be enhanced or suppressed at higher initial cell densities, depending on the assay and agent tested. We propose a solution that consists of the inclusion of an experimentally distinguishable population of cells as an internal standard for colony-forming ability at the high cell density required for detection of rare variants. This method is found to be highly satisfactory for use in measuring mutation to 6-thioguanine resistance in a diploid human B lymphoblast line. For treatment with anti-2,3-dihydroxy-1,10 b-epoxy-1,2,3-trihydrofluoranthene (FDE), N-methyl- N′-nitro- N-nitrosoguanidine (MNNG), and 4-nitroquinoline-oxide and 4-nitroquinoline-oxide (4NQO), the calculated induced mutant fractions using the internal-standard method were significantly lower than those calculated using the conventional low-density-plating efficiency method. The results of these experiments and our analysis lead us to conclude that this approach is applicable to all single cell mutation of transformation assays and is necessary feature of assays in which an accurate knowledge of the fraction of rare variants is required.

Carbohydrate Fermentation by Streptococcus cremoris and Streptococcus lactis Growing in Agar Gels

When lactic streptococci were embedded in agar gels and incubated at 30 degrees C, the end products of carbohydrate fermentation depended on the initial cell density, which determined the subsequent distribution and size of colonies in the gel. With high initial cell densities, microcolonies formed close together and lactose and glucose were converted almost entirely to lactate. However, inoculation with a small number of cells, which then grew to form widely spaced and comparatively large colonies, resulted in up to 30% diversion of end product, usually to formate, ethanol, and acetate. In these "low-colony-density" gel cultures, the initial rate of fermentation was exponential and only lactate was formed. However, this rate then became linear and fermentation became progressively more heterolactic. Streptococcus lactis ML(8) was the only strain among the 10 tested which remained homolactic. Incubation at temperatures either above or below the optimum for growth and metabolism decreased the diversion to end products other than lactate. The change from homo- to heterolactic fermentation appears to be caused by carbohydrate depletion in the vicinity of the colony, so that fermentation is then limited by the diffusion of substrate. Growth of cells on gel surfaces exposed to air resulted in up to 40% diversion of end product from lactate, mainly to CO(2), acetoin, 2,3-butanediol, and acetate. Six of the 12 Streptococcus cremoris strains tested remained homolactic under these aerobic conditions, whereas all 8 of the S. lactis strains tested, including ML(8), were heterolactic.

Cytotoxicity of metronidazole (Flagyl) and misonidazole (Ro-07-0582): enhancement by lactate.

The cytotoxic activity of metronidazole (Flagyl) and misonidazole (MISO) to hypoxic Chinese hamster ovary (CHO) cells suspended in standard medium in sealed vials and in gassed spinner flasks has been investigated. Flagyl (5 mM) was only cytotoxic at high initial cell densities. However, when lactate (20 mM) was included in the medium the cytotoxicity of Flagyl at low cell densities was considerable, and similar to that of misonidazole under the same conditions. The relevance of this "lactate effect" to in vivo systems, and the possible mechanisms involved, are discussed.

Nutritional requirements of protoplast-derived, haploid tobacco cells grown at low cell densities in liquid medium

Preliminary attempts to define a completely synthetic medium able to support divisions of haploid tobacco mesophyll protoplasts at low initial densities have failed. High protoplast concentrations together with large amounts of naphtaleneacetic acid in the medium (3 mg l(-1) NAA) were required for maximal induction of protoplast division. However, cell suspensions derived from haploid protoplasts after four days of preculture at high initial cell densities could be diluted to densities as low as 1-4 cells ml(-1), provided the concentration of NAA in the medium was lowered to below 0.3 mg l(-1). The optimal NAA supply for low cell density growth was affected by the nature of the nitrogen source.A simple minimal medium which supports the growth of these haploid cells with a plating efficiency of 30-40%, independent of the cell density in the range of 1-4 to 3·10(4) cells ml(-1), has been established. In this medium inositol was the only vitamin stringently required for growth.Growth of cells at low densities was also possible in a medium initially containing 3 mg l(-1) NAA, provided it was conditioned by the growth of protoplasts at high densities. Preliminary experiments with [(14)C]NAA showed that the amount of free NAA remaining in the medium after preincubation at high densities was drastically reduced. Simultaneously, NAA conjugates accumulated in the medium. The implications of these results are discussed.

EXPERIMENTAL MANIPULATION OF ALTERNATIVE PATHWAYS OF DIFFERENTIATION IN CULTURES OF EMBRYONIC CHICK NEURAL RETINA.

Embryonic chick neural retina cells can transdifferentiate during long-term cell culture into either pigmented epithelium or lens fibres. We have found that some culture conditions influence the choice between these pathways. Pigment cell development is promoted by low initial cell densities and by the use of a medium based on Earle's salt formulation rather than Hank's, while lens fibre development is encouraged by high initial cell densities and by folding the cell sheet into multilayered regions. Some differences in in vitro cell properties of neural retina are reported for two genotypes previously found to exhibit differences in in vitro cell properties of lens epithelial cells.