Physical and nutritional factors in gel culture of mammalian cells

Summary The growth of human glioma cells, cultured as spherical colonies in agarose gel, stopped after about 10 days for both large and small colonies apparently due to an increased osmolality in the gel. When osmolality was kept under control by addition of distilled water, growth continued. However, a continuous increase in the population-doubling period, similar for both large and small colonies, then was observed. The increase persisted although excess amounts of nutrition were added. When the cells were cultured in liquid suspension above a thin layer of agarose gel and most of the medium was repeatedly changed, the colonies continued to grow beyond the limits in gel culture. HeLa and hamster embryonic lung cell colonies showed a growth pattern in agarose gel similar to the glioma cells. The results imply that the osmolality must be kept under precise control to prevent growth inhibition. However, it seems difficult to ascertain optimal growth in gel culture for more than about 2 weeks probably because of the accumulation of toxic products. The work was supported financially by the Swedish Cancer Society and the Swedish Natural Science Research Council.     

Effects of bacterial agarase on agarose gel in cell culture

Summary Bacterial agarase, concentrated and purified from culture filtrate of agar-degrading bacteria, has been used to clean cells cultured in soft agarose from gel residues. The enzyme also has been used to liquefy the gel directly in the dishes to facilitate the removal of cells. The surfaces of glioma cells from agarase-treated colonies could not be distinguished in the scanning electron microscope from surfaces of cells which had never been in contact with agarose or agarase. This implies that most agarose residues had been removed, and also that the treatment did not seriously alter the cell surfaces. The influence of the agarase treatment also was tested by comparison of the mitotic index and the incorporation of [³H]thymidine in agarase-treated and untreated cells. No effects of the treatment could be seen in these tests. The work has been supported by the Swedish Cancer Society and the Swedish Natural Science Research Council.     

Stimulation of myeloid colony growth from peripheral blood by medium with an initially low osmolality

The clonal growth of myeloid colonies from peripheral blood was maximal when cultures were established with an initial osmolality of 220 mosmol/kg which increased during incubation as a result of partial drying. When osmolality was stabilized by secondary humidification, the optimum osmolality was 270 mosmol/kg, but growth was always two- to fivefold less than similar cultures established at low osmolality and incubated on an open shelf. Cultures established at 270 mosmol/kg or above were statistically similar whether or not drying was eliminated. Maximum colonies were apparent after 14 days incubation under both conditions; addition of conditioned medium did not alter the pattern of growth. The greater sensitivity of cultures established at 220 mosmol/kg is advantageous when assaying circulating progenitors in pathological conditions where a low number of granulocyte/macrophage colony-forming units is common.     

GROWTH AND ORGANIZED DEVELOPMENT OF CULTURED CELLS. V. THE GROWTH OF COLONIES FROM FREE CELLS ON NUTRIENT AGAR

When angiosperm cells are cultured in a liquid medium they may grow in the form of free, single cells and form small to large groups of cells. This has been shown in earlier papers. This paper deals with the growth of strains of cells (Daucus carota and Haplopappus gracilis cells were used), washed and filtered free from the larger groups, on nutrient agar media in Petri dishes, thus simulating familiar microbiological technique. Each discrete member of a suspension is referred to as a “unit.” On the order of 1–10% of the separate units of a suspension may be induced to grow into viable colonies, depending on the strain and the conditions employed. Whereas at least 30% of the free single carrot cells were shown to be capable of division, only up to about 4% continued their growth to form macroscopically visible colonies when they were widely dispersed. Coconut milk promotes the growth of carrot cells into colonies. Both coconut milk and napthaleneacetic acid, which interact synergistically, arc required for the growth of Haplopappus cells. Various techniques which affect viability (the frequency with which units grow into colonies) were investigated. The viability of carrot units was found (1) to increase with their density on the plates; (2) to decrease upon washing the suspensions prior to plating; (3) to increase with increasing initial size; and (4) to decrease to a vanishingly low value in rigorously filtered suspensions which consist principally of single cells, although the single cells were found to grow with appreciable frequency when the larger units were also present; and (5) to increase dramatically (100-fold) when a rigorously filtered suspension was plated on a medium upon which pieces of growing cultured tissue were placed. Thus, the induction of growth in free cells is enhanced, even in an environment nutritionally optimal for the growth of the larger cell masses, by as yet unknown factors which are contributed by the cells themselves, or by adjacent cells or groups of cells. It is suggested that within a group of cells growing in culture, and perhaps also in the organized growing regions of intact plants, the dividing cells are nourished or stimulated by adjacent but less frequently dividing cells. The implications of these results are discussed.     

Effects of retinoic acid on the growth and morphology of hamster tracheal epithelial cells in primary culture

Hamster tracheal epithelial cells were grown in primary culture on a collagen gel substrate in hormone-supplemented serum-free Ham's F12 medium with 10(-8) M retinoic acid (RA+), or without retinoic acid (RA-). On days 1 and 2, the colonies were composed of large (secretory) cells and lesser numbers of small (basal) cells; ciliated cells were rare. At these times, cell number, thymidine incorporation, and total labelling indices (small and large cells, combined) were similar in RA+ and RA- cultures, but the large cells became flat in RA- medium on day 2. On days 3-5, thymidine incorporation and total labelling indices were less in RA- than RA+ cultures, and on days 4-6, cell numbers were decreased in RA- cultures. On day 3, the large cells of the RA- colonies had flattened further and clusters of small basal cells had formed. On day 4, the RA+ colonies were composed of densely-packed cuboidal secretory cells, small basal cells were inconspicuous; the total labelling index was about 27%. The RA- colonies were composed of large flat secretory cells and numerous small basal cells which were clustered in groups; the total labelling index was about 7%. Since large and small cells could be discriminated by size in RA- colonies, a labelling index was generated based on cell size. On days 2, 3 and 4, the labelling index of the small basal cells in the RA- colonies was 44%, 43% and 24% respectively, whereas the labelling index of the large secretory cells fell rapidly over the same period (56%, 14% and 2%). On days 5 and 6, the cuboidal secretory cells in the RA+ cultures had differentiated further and the cells were stratified focally. Some new ciliated cells had formed on day 6. In RA- cultures, mucous granules were not observed in the large flat cells and ciliated cells were not seen. The total labelling indices were 11% and 0.35% in RA+ cultures, and 0.5% and 0.25% in RA- cultures on days 5 and 6, respectively. The study shows that the target cell for vitamin A in the hamster tracheal epithelium is the secretory (mucous) cell. When retinoic acid was deficient, the secretory cells flattened and their capacity to divide was greatly diminished. Since the basal cells continued to replicate when the secretory cells did not, the population density of the basal cells increased disproportionally, which could be interpreted erroneously as a "basal cell hyperplasia".(ABSTRACT TRUNCATED AT 400 WORDS)     

Long-term maintenance of multilineal hemopoiesis in collagen gel culture.

We examined the long-term maintenance of multilineal hemopoiesis in a collagen gel culture of mouse bone marrow cells. When cells were inoculated into the gel, stromal cells formed foci that were composed of sinusoidlike capillary structures, fibroblastic cells, adipocytes and macrophages. Many small hemopoietic foci similar to granulocyte-macrophage colonies (CFU-GM) appeared within a week and disappeared after two weeks. Several large hemopoietic foci appeared after two to three weeks of culture, without a second challenge of marrow cells. These large hemopoietic foci were composed mainly of myeloid cells. Megakaryocytes and mast cells were also observed. When erythropoietin (EPO) was added to the culture at the beginning, the erythroid focus appeared after 3 weeks and the number of megakaryocytes was greater than that in the culture without EPO. However, when EPO was added to the cultures after 6 or 12 weeks, erythroid cells appeared after 1 week and the number of megakaryocytes increased. This hemopoiesis lasted more than 6 months.     

Early chick embryonic cells can form clones in agarose cultures

Early chick embryonic cells prior to the formation of the primitive streak, have been cultured in a two-layer soft-agarose system. Single, primary cells when grown in this system were capable of producing colonies ranging in size from 30 to 100 cells. The plating efficiency varied between 1 and 5% and the colonies remained viable for about 2 weeks. We believe this is the first report of normal, non-passaged cells which show anchorage-independent growth properties by forming colonies in a standard agarose culture in the absence of additional factors. The importance of being able to use normal monoclonal embryonic cell populations in studying early developmental processes is also discussed.     

Quantification of transformation efficiency using a new method for clonal growth and selection of axenic Dictyostelium cells

A new method for clonal growth of Dictyostelium axenic amoebae has been developed. Cells are plated in growth medium containing 1% ultra-low gelling temperature agarose. Cells grow normally in the agarose and form colonies up to several millimeters in diameter. When the colonies have grown to a sufficient size, they begin multicellular development. Pseudoplasmodia are formed, migrate to the surface of the agar, and then undergo fruiting body formation. Cells can be removed from the soft agarose colonies with a toothpick or by picking spores from the fruiting bodies. This method should be useful for drug, auxotrophic, and temperature selections where clonal maintenance of axenic colonies is important. This method has been used in combination with a selection for resistance to G418 to isolate independent colonies following DNA-mediated transformation. Several parameters in the calcium phosphate and electroporation transformation protocols have been optimized and the transformation frequency quantified. Independent transformed colonies are obtained at a frequency of 1 in 10(4) to 1 in 10(5) cells when integrating plasmids are introduced using calcium phosphate coprecipitation. The frequency is about tenfold higher when extrachromosomal shuttle vectors are introduced into cells.     

Transformed growth phenotype of mouse mammary epithelium in primary culture induced by specific fetal mesenchymes.

When mesenchyme from fetal mammary or salivary gland is implanted into adult mouse mammary gland, adjacent epithelium responds with intense hyperplasia. The hyperplastic cells are more vulnerable than are non-stimulated cells to transformation in vivo by a chemical carcinogen or by mammary tumor virus. This system provides a potentially useful model for determining how stroma contributes to mammary tumorigenesis. We have developed co-culture systems and used them to investigate in more detail the nature of the signal produced by the mesenchyme cells. Monolayers of mesenchyme cells were prepared on tissue-culture wells. The mesenchyme cells were trapped on the surface by a thin overlay of agarose. Primary mammary epithelial cells were cultured atop this barrier layer, either as organoids in collagen gels for assessment of anchorage-dependent growth, or as single-cell dispersions in soft agarose for assessment of anchorage-independent growth. Our procedures for assay of anchorage-independent growth allow us for the first time to detect and measure this transformation-defining characteristic in non-immortalized mammary epithelial cells in primary culture. Fetal mammary fat pad precursor tissue and fetal salivary mesenchyme both stimulated anchorage-dependent growth of mammary epithelium, with cell number increasing as much as fifteenfold during a 6-day culture period. These same fetal tissues also stimulated anchorage-independent growth of the mammary epithelial cells, with colony-forming efficiencies of up to 40% in co-cultures with salivary mesenchyme. No colonies formed in the absence of mesenchyme. Cells of colonies contained keratin, which indicates that the colonies grew from epithelial cells and not from a contaminant of another cell type. When co-cultured epithelial cells were subsequently re-cultured in the absence of mesenchyme, they lost their ability to grow independent of anchorage. No colonies grew in co-cultures with fetal cells from heart, kidney, or lung, which is consistent with the lack of stimulation by these tissues in the mammary gland in vivo. A tumor promoter, 12-O-tetradecanoylphorbol acetate (TPA), also caused anchorage-independent growth of the dispersed mammary epithelial cells. Culture medium conditioned by primary or early-passage salivary mesenchyme cells was capable of stimulating growth under both anchorage-dependent and anchorage-independent conditions, confirming that these effects are mediated by a paracrine factor. The results indicate that stimulatory fetal mesenchymes produce soluble molecules that act analogously to transforming growth factors.     

Evidence that sprouty 2 is necessary for sarcoma formation by H-Ras oncogene-transformed human fibroblasts

Sprouty 2 (Spry2) acts as an inhibitor of receptor tyrosine kinase signaling in various cellular contexts. Interestingly, Spry2 also prevents the c-Cbl-induced degradation of epidermal growth factor receptor (EGFR). We compared human fibroblasts malignantly transformed by overexpression of H-Ras(V12) oncogene to their nontransformed parental cells and found that the malignant cells express a high level of Spry2. These cells also exhibited an increase in the level of EGFR compared with their precursor cells. We found that intact EGFR was required if H-Ras-transformed cells were to grow in the absence of exogenous growth factors or form large colonies in agarose. When we decreased expression of Spry2, using a Spry2-specific shRNA, the H-Ras(V12)-transformed fibroblasts could no longer form large colonies in agarose, grow in reduced levels of serum, or form tumors in athymic mice. The level of active H-Ras in these cells remained unaltered. A similar, but less pronounced, effect in tumor formation was observed when Spry2 was down-regulated in human patient-derived fibrosarcoma cell lines. In H-Ras-transformed cells Spry2 sustained the level and the downstream signaling activity of EGFR. In the parental, non-H-Ras-transformed fibroblasts, expression of Spry2 resulted in the inhibition of H-Ras and ERK activation, suggesting that the positive effect of Spry2 in tumor formation is specific to H-Ras transformation. Co-immunoprecipitation studies with H-Ras-transformed cells revealed that Spry2 and H-Ras interact and that H-Ras interacts with Spry2-binding partners, c-Cbl and CIN85, in a Spry2-dependent manner. These data show that Spry2 plays a critical role in the ability of H-Ras-transformed cells to form tumors in athymic mice.     

Proliferative effects of purified granulocyte colony-stimulating factor (G-CSF) on normal mouse hemopoietic cells

When granulocyte colony-stimulating factor (G-CSF), purified to homogeneity from mouse lung-conditioned medium, was added to agar cultures of mouse bone marrcw cells, it stimulated the formation of small numbers of granulocytic colonies. At high concentrations of G-CSF, a small proportion of macrophage and granulocyte-macrophage colonies also developed. G-CSF stimulated colony formation by highly enriched progenitor cell populations obtained by fractionation of mouse fetal liver cells using a fluorescence-activated cell sorter, indicating that G-CSF probably acts directly on target progenitor cells. Granulocytic colonies stimulated by G-CSF were small and uniform in size, and at 7 days of culture were composed of highly differentiated cells. Studies using clonal transfer and the delayed addition of other regulators showed that G-CSF could directly stimulate the initial proliferation of a large proportion of the granulocvte-macrophage progenitors in adult marrow and also the survival and/or proliferation of some multipotential, erythroid, and eosinophil progenitors in fetal liver. However, G-CSF was unable to sustain continued proliferation of these cells to result in colony formation. When G-CSF was mixed with purified granulocyte-macrophage colony-stimulating factor (GM-CSF) or macrophage colony-stimulating factor (M-CSF), the combination stimulated the formation by adult marrow cells of more granulocyte-macrophage colonies than either stimulus alone and an overall size increase in all colonies. G-CSF behaves as a predominantly granulopoietic stimulating factor but has some capacity to stimulate the initial proliferation of the same wide range of progenitor cells as that stimulated by GM-CSF.     

Low incidence of tumourigenicity in agarose colonies from spontaneous murine mammary tumours

In these experiments individual colonies growing in agarose seeded with monocellular suspensions from freshly disaggregated naturally-occurring mouse mammary tumours, induced by the murine mammary tumour virus (MMTV), were reimplanted into mammary fat pads of virus-free mice. It was found that only a small proportion of these colonies generated tumours and that the implantation of multiple colonies in each site did not result in disproportionate, synergistic, increase in tumour takes. It was also observed that the proportion of colonies which were tumourigenic on reimplantation differed for each donor tumour and represented only a small fraction of the total cell population (0.001%-0.1%). However, this value was significantly higher in tumours which produced large numbers of deposits in lung colony assays following i.v. injections, than in tumours of low pulmonary colonisation potential. A point of particular interest was that tumours derived from agarose colonies of spontaneously metastatic donor tumours were substantially more spontaneously metastatic themselves than those from nonmetastatic donors, indicating that this property is heritable through numerous cell divisions, manipulations in vitro and transplantation procedures. From these results it is concluded that measurement of clonogenicity in agar is useful as an index of the capability of a tumour to propagate itself and to colonise new sites, but that individual agarose colonies are not all the progeny of potentially immortal stem cells.     

In vitro cloning of tumor stem cells in semi-solid media containing agar and agarose

Summary The formation of tumor stem cell colonies in vitro has been studied by comparing the growth of three mouse teratocarcinoma derived cell lines and one human teratocarcinoma derived cell line in semi-solid media containing either agar or agarose. We show that agaroses should be used in higher concentrations than agar to obtain comparable results. The maximum number of colonies were obtained in agarose over a broader range of concentrations (1%–4% for SeaPrep and 0.5%–2% for SeaPlaque agarose) than in agar, which allowed anchorage-independent growth of tumor cells only over a narrow concentration range (0.3%–0.5%). Overall, the preparation of media containing agarose was less cumbersome than preparation of agar-containing media, primarily because agaroses gelled more slowly and remained liquid in the physiological temperature range. Furthermore, the transfer of colonies from semi-solid media containing agarose to solid surface tissue culture dishes was much more efficient than the transfer of colonies from agar. The stock solutions of SeaPrep agarose could be kept ready for use for extended periods of time. All these features show that the low melting point agarose has considerable advantages over agar for preparation of semi-solid media for anchorage-independent tumor cell growth.     

An In Vitro Expansion System for Generation of Human iPS Cell-Derived Hepatic Progenitor-Like Cells Exhibiting a Bipotent Differentiation Potential

Hepatoblasts, hepatic stem/progenitor cells in liver development, have a high proliferative potential and the ability to differentiate into both hepatocytes and cholangiocytes. In regenerative medicine and drug screening for the treatment of severe liver diseases, human induced pluripotent stem (iPS) cell-derived mature functional hepatocytes are considered to be a potentially good cell source. However, induction of proliferation of these cells is difficult ex vivo. To circumvent this problem, we generated hepatic progenitor-like cells from human iPS cells using serial cytokine treatments in vitro. Highly proliferative hepatic progenitor-like cells were purified by fluorescence-activated cell sorting using antibodies against CD13 and CD133 that are known cell surface markers of hepatic stem/progenitor cells in fetal and adult mouse livers. When the purified CD13^highCD133⁺ cells were cultured at a low density with feeder cells in the presence of suitable growth factors and signaling inhibitors (ALK inhibitor A-83-01 and ROCK inhibitor Y-27632), individual cells gave rise to relatively large colonies. These colonies consisted of two types of cells expressing hepatocytic marker genes (hepatocyte nuclear factor 4α and α-fetoprotein) and a cholangiocytic marker gene (cytokeratin 7), and continued to proliferate over long periods of time. In a spheroid formation assay, these cells were found to express genes required for mature liver function, such as cytochrome P450 enzymes, and secrete albumin. When these cells were cultured in a suitable extracellular matrix gel, they eventually formed a cholangiocytic cyst-like structure with epithelial polarity, suggesting that human iPS cell-derived hepatic progenitor-like cells have a bipotent differentiation ability. Collectively these data indicate that this novel procedure using an in vitro expansion system is useful for not only liver regeneration but also for the determination of molecular mechanisms that regulate liver development.     

Normal mouse serum contains peptides which induce fibroblasts to grow in soft agar

The untransformed mouse fibroblast cells NIH/3T3, C3H/10T1/2, and rat NRK cells do not grow in soft agar in medium supplemented with 10% fetal calf serum. When fetal calf serum in the growth medium was supplemented with less than 1% of sera from mice or other vertebrates, however, these cells responded, forming large colonies. The morphology of soft agar colonies was a function of the treated cell type. In the presence of 10% serum from C57BL/6 mice, NRK cells grew to smooth-surfaced spherical colonies, while NIH/3T3 colonies showed individual round cells on their surface and C3H/10T1/2 cells grew as extended cells forming columns of end to end connected fibroblasts. Mus Musculus Castaneus-Epithelial (MMC- E) cells were not stimulated to grow in soft agar under these conditions. The major fibroblast colony-inducing factor (F-CIF) was partially purified from mouse serum by acid/ethanol-extraction, gel permeation chromatography, and reverse-phase high-pressure liquid chromatography. F-CIF is a polypeptide, which does not compete for binding to epidermal growth factor (EGF) receptors, but stimulates normal fibroblasts to form small colonies in semisolid medium and very large colonies in the presence of added EGF (2 ng/ml). In contrast to unfractionated mouse serum, purified F-CIF did not induce C3H/10T1/2 cells to grow in soft agar, suggesting that serum contains additional cell type-specific agar growth-stimulating activities.     

The characteristics of an anchorage-independent clonal agar assay for primary explanted bovine granulosa cells

Normal, primary explanted, bovine granulosa cells grow reproducibly in agar culture as anchorage-independent clones. Epidermal growth factor (EGF) and rat erythrocytes are effective stimulators of colony formation, and when both are added to the culture medium at optimal concentrations, there is an enhancement of colony numbers and colony size, indicative of an independent, and operationally additive, mode of action for the two factors. The ability of cells propagated from agar clones to secrete progesterone, and to augment progesterone secretion 4-fold in the presence of 1 mM dbcAMP is proof that colonies originate from and are composed of functional granulosa cells. Maximal colony numbers are present at day 10 of incubation, and colony forming cells undergo self-renewal as assessed by the ability of cells from primary colonies to reclone in agar. Absolute cloning efficiency, however, is dependent on a number of factors. Inherent variability exists in cloning efficiency of granulosa cells from individual follicles. Quantitative and qualitative clonal growth was improved at an osmolality of less than 300 mOsm when compared with higher osmolalities. Cl-1 medium and the alpha modification of Eagle's medium were equally effective in supporting agar clonogenic growth, whereas both Ham's F12 and NCTC 135 media exhibited poor clonogenic growth supporting properties. The substitution of agarose for agar did not affect colony numbers but colonies grown in the presence of agarose tended to be smaller and more uniform in size.     

Cyclic AMP in relation to proliferation of the epidermal cell: a new view.

Four agents known to increase the level of cellular cAMP by different means (cholera toxin, dibutyryl cAMP, methyl isobutyl xanthine and isoproterenol) increase the growth of colonies of cultured human epidermal cells and of keratinocytes derived from other stratified squamous epithelia. This effect is due to an increase in the overall rate of cell proliferation in the colonies. When added to cultures under hitherto optimum conditions for epidermal cell growth [in the presence of supporting 3T3 cells and epidermal growth factor (EGF)], most of the agents exert an effect of considerable magnitude, the toxin being the most potent. Since the toxin exerts an effect in the absence of supporting 3T3 cells, it must be able to act directly on the keratinocytes. It can also act in the absence of ECF and of medium conditioned by 3T3 cells, although proliferation is greatest when supporting 3T3 cells and EGF are present. The increased proliferation in the presence of the toxin is associated with an increased proportion of small cells known to include the multiplying fraction. The use of toxin makes the cultivation of keratinocytes from epidermis and other stratified squamous epithelia much easier and prolong the culture life of the cells. Whether cell proliferation in the intact epidermis is regulated through agents affecting cAMP (in a direction opposite to that suggested by much of the earlier literature) remains to be elucidated, but the existence of such a mechanism in cultured cells suggests that it may function in the intact epithelium.     

Effect of hyper-osmotic stress on alanine content of Leishmania major promastigotes

Earlier studies showed that Leishmania major promastigotes are sensitive to osmotic conditions. A reduction in osmolality caused the cells to shorten and to rapidly release most of their large internal pool of alanine. In this study some effects of hyper-osmotic stress were examined. An increase in osmolality of the culture medium from 308 to 625 mOsm/kg caused only a small decrease in growth rate. When cells grown in the usual culture medium (308 mOsm/kg) were washed, resuspended in iso-osmotic buffer, and subjected to acute hyper-osmotic stress by addition of mannitol, the alanine content increased even in the absence of exogenous substrate. Promastigotes, depleted of alanine by a 5-min exposure to hypo-osmotic conditions, also synthesized alanine when resuspended in iso-osmotic buffer. Washed cells resuspended in iso-osmotic buffer consume their internal pool of alanine under aerobic conditions. Rates of consumption decreased on addition of mannitol, becoming zero at about 440 mOsm/kg. At higher osmolalities, alanine synthesis occurred. To estimate whether proteolysis could account for alanine synthesis in the absence of exogenous substrate, cells that had been grown with [1-14C]leucine were washed and resuspended under hypo-, iso-, and hyper-osmotic conditions and the amounts of 14CO2 and 14C-labelled peptides released in 1 h were measured. Little proteolysis occurred under these conditions, but the possibility that proteolysis was the source of the alanine increase, observed in response to hyper-osmotic stress, cannot be ruled out.     

Volume changes of an endothelial cell monolayer on exposure to anisotonic media

The osmotic process plays an important role in controlling the distribution of water across cell membranes and thus the cell volume. A system was designed to detect the volume changes of an endothelial cell monolayer when cells were exposed to media with altered osmolalities. Electrodes housed in a flow chamber measured the resistance of ionic media flowing over a cultured cell layer. Assuming the cell membrane acts as an electrical insulator, volume changes of the cell layer can be calculated from the corresponding changes in chamber resistance. The media used in the experiments had osmolalities in the range 120-630 mmol/kg. When cells were exposed to hypertonic media, there was rapid shrinkage with an approximate 30% reduction in total cell volume for a twofold increase in osmolality. On exposure to hypotonic media, the cells initially swelled with an approximate 20% volume increase for a decrease in osmolality by half. With sustained exposure to low osmolality media, there was a gradual and partial return of cell volume towards isotonic values that started 10 minutes after and was complete within 30 minutes of the osmolality alteration. This finding suggests regulatory volume decrease (RVD); however, no regulatory volume increase (RVI) was observed with the continued exposure to hypertonic media over 45 minutes.     

Effect of Hyper-Osmotic Stress On Alanine Content of Leishmania Major Promastigotes

ABSTRACT Earlier studies showed that Leishmania major promastigotes are sensitive to osmotic conditions. A reduction in osmolality caused the cells to shorten and to rapidly release most of their large internal pool of alanine. In this study some effects of hyper-osmotic stress were examined. an increase in osmolality of the culture medium from 308 to 625 mOsm/kg caused only a small decrease in growth rate. When cells grown in the usual culture medium (308 mOsm/kg) were washed, resuspended in iso-osmotic buffer, and subjected to acute hyper-osmotic stress by addition of mannitol, the alanine content increased even in the absence of exogenous substrate. Promastigotes, depleted of alanine by a 5-min exposure to hypo-osmotic conditions, also synthesized alanine when resuspended in iso-osmotic buffer. Washed cells resuspended in iso-osmotic buffer consume their internal pool of alanine under aerobic conditions, Rates of consumption decreased on addition of mannitol, becoming zero at about 440 mOsm/kg. At higher osmolalities, alanine synthesis occurred. to estimate whether proteolysis could account for alanine synthesis in the absence of exogenous substrate, cells that had been grown with [1-¹⁴C]leucine were washed and resuspended under hypo-, iso-, and hyper-osmotic conditions and the amounts of ¹⁴CO₂ and ¹⁴C-labelled peptides released in 1 h were measured. Little proteolysis occurred under these conditions, but the possibility that proteolysis was the source of the alanine increase, observed in response to hyper-osmotic stress, cannot be ruled out.