Temperature sensitivity on proliferation and morphologic alteration of human esophageal carcinoma cells in culture

Summary As basic studies of hyperthermia and hypothermia on malignant tumor, the kinetics of proliferative activity, the morphologic changes in the two cell lines, SGF-3 and SGF-5, established in our department after the change of culture temperature were examined. The results obtained were: a) A significant difference was found in the sensitivity to temperatures between the two cell lines originated from human esophageal squamous cell carcinoma. The temperature range allowing cultured cell to proliferate were from 31° to 39° C in SGF-3 and from 29° to 41° C in SGF-5. b) Minor difference occurred in the results between the two cell lines examined during the recovery of proliferative activity, but no proliferative activity was discovered after the cells were exposed to 42° C for 72 h. Two cell lines resumed their proliferation after having been exposed to 27° or 28° C for 72 h. c) Morphologic changes of the cell lines cultured at high temperature were cytoplasmic vacuolation and cell aggregation by phase contrast microscope and the increase of heterochromatin, the decrease of granular formation in nucleoli, and nucleolar vacuolation by transmission electron microscopy (TEM). At low temperatures the changes observed included cytoplasmic ballooning and circumnuclear halo formation by phase contrast microscope, and the increase of heterochromatin, nucleolar segregation, swelling of mitochondria, and dilatation of rough endoplasmic reticulum (rER) by TEM.     

Response of plasma and tissue levels of enteroglucagon immunoreactivity to intestinal resection,lactation and hyperphagia

Elevation of plasma enteroglucagon has been described in the presence of intestinal hyperplasia in both animals and man. In order to investigate whether enteroglucagon plays a role in the stimulation of small intestinal growth, fasting plasma and small intestinal tissue levels of enteroglucagon immunoreactivity were measured in control rats and in 3 different rat models of intestinal adaptation: 5 weeks after proximal and distal small bowel resection, on the 12th day of lactation and following 5 weeks of cold acclimation induced hyperphagia. Plasma enteroglucagon levels increased significantly from the control value of 89±(SEM) 13.1 fmol/ml to 147±13.6 after proximal resection (p < 0.005) and to 207±32.6 following 5 weeks of hyperphagia (p < 0.001). During lactation ileal tissue enteroglucagon increased by 183% from 58.1±8.6 pmol/g tissue in controls to 163.9±20.4 (p < 0.001). Ileal tissue enteroglucagon levels were also elevated by 60% following proximal resection (p < 0.005) and by 91% following the hyperphagia of cold-acclimation (p < 0.005). In contrast, jejunal tissue enteroglucagon levels of cold-acclimated rats were only increased by 55% when compared to the control value of 27±2.9 pmol/g tissue (p < 0.02). These results confirm that in the rat, ileal enteroglucagon levels are significantly greater than those found in the jejunum (p < 0.005). Enteroglucagon levels were also significantly elevated in the 2 groups of rats with hyperphagia, namely the lactating and cold-acclimated groups. The data lend further support to the proposal that enteroglucagon may play a trophic role in producing intestinal growth.     

Variation of Telomerase Activity and Morphology in Porcine Mesenchymal Stem Cells and Fibroblasts during Prolonged in vitro Culture

The purpose of this study was to examine the telomerase activity, population doubling time (PDT), morphological alterations, and the cell cycle status with activity of senescence-associated-ß-galactosidase in porcine mesenchymal stem cells (MSCs) and fibroblasts during an extended in vitro culture. MSCs and fibroblasts were isolated from bone marrow and ear skin of a miniature pig, respectively, and cultured up to 20 passages. The analysis was carried out in MSCs and fibroblasts at 1, 5, 10, 15, and 20 passages. Relative telomerase activity (RTA) levels were significantly (P < 0.05) higher in MSCs than in fibroblasts at all the passages. The PDT and cellular size slightly increased in MSCs at later passages. In contrast, fibroblasts had significantly (P < 0.05) increased PDT and cellular size, and the morphology revealed senescent-like abnormal type after passage 10. Further, the high incidence of ß-galactosidase stained cells was observed in fibroblasts compared to that of MSCs at passage 15, and cell cycle stage at G0 / G1 phase was significantly (P < 0.05) increased in the fibroblasts at 15 and 20 passages compared to that of MSCs. Based on these observations, we concluded that porcine MSCs possessed more tolerance against senescence and aging compared to fibroblasts following prolonged in vitro culture.     

Low temperature effects on growth and actin cytoskeleton organisation in suspension cells of winter oilseed rape

Rhodamine-phalloidin staining of winter oilseed rape suspension cells revealed that the structure of actin cytoskeleton changes with the phase of cell growth. In small, 4-day-old cells, entering the exponential phase of growth, a dense and uniformly distributed cortical microfilament networks was seen. In six-day-old vacuolated cells, which reached the stationary phase of growth, the actin cytoskeleton was composed of thicker microfilament cables in irregular arrangements. In cells acclimated in cold for 7 days a dense, uniformly distributed and cortical microfilament network was still seen. The fine microfilament network was sensitive to extracellular freezing since the structures underwent depolymerization at −3 °C (in the presence of extracellular ice), both in non-acclimated and cold-acclimated cells. The thicker transvacuolar cables in cells of the stationary growth phase resisted freezing to −7 °C. Acclimation of suspensions at 2 °C resulted in slowing down growth of cells and in the increased freezing tolerance of cells as indicated by a decrease of LT₅₀ from −11 °C to −17.5^o or to −25 °C when determined 7 or 20 days after the beginning of the cold treatment, respectively. Freezing tolerance of non-acclimated cells decreased from −11 °C to −8 °C during subculture, showing a transient increase to −17 °C on the day 6. Results indicate that the arrangement of actin microfilaments and their sensitivity to freezing-induced depolymerization depends on the phase of cell growth rather than on cell acclimation status. Possible mechanisms involved in the freezing-induced depolymerization of actin microfilaments are discussed. This revised version was published online in June 2006 with corrections to the Cover Date.     

Effect of thermal stress on HSP70 expression in dermal fibroblast of zebu (Tharparkar) and crossbred (Karan-Fries) cattle

The present studies were conducted to investigate the difference response of dermal fibroblasts to heat stress in Tharparkar and Karan-Fries cattle. Skin is the most important environmental interface providing a protective envelope to animals. In skin, dermal fibroblasts are the most regular cell constituent of dermis that is crucial for temperature homeostasis. The study aimed to examine the reactive oxygen species (ROS) formation, cytotoxicity (%) and heat shock protein 70 (HSP70) genes expression in dermal fibroblast of Tharparkar and Karan-Fries cattle and to assess whether resistance of dermal fibroblast to heat stress is breed specific. Dermal fibroblasts from ear pinna of Tharparkar and Karan-Fries cattle were exposed at 25 °C, 37 °C, 40 °C and 44 °C for 3 h to measure the ROS, cytotoxicity (%) and HSP 70 (HSPA1A, HSPA2 and HSPA8) genes’ expression. The results showed that ROS formation at low temperature (25 °C) decreased in both breeds as compared to control (37 °C) and the differences were significant (P<0.0001). Heat stress at 40 °C did not increase ROS formation significantly in Tharparkar but increased significantly (P<0.001) in Karan-Fries cattle. The overall cytotoxicity (%) was also found to be significantly different (P<0.001) between Tharparkar and Karan-Fries cattle, and on exposure to different temperatures (P<0.001). The cytotoxicity (%) in dermal fibroblast cells of Karan-fries cows was more than Tharparkar. The expression studies indicated that all HSP70 genes (HSPA8, HSPA1A and HSPA2) were up-regulated at different temperatures in both breeds. In Tharparkar, the relative mRNA expression of HSPA8 gene was higher but HSPA1A and HSPA2 genes were low as compared to Karan-Fries cattle. At 40 and 44 °C, the relative expressions of inducible HSP 70 genes (HSPA1A and HSPA2) were higher in Karan-Fries than Tharparkar. In summary, dermal fibroblast resistance to heat shock differed between breeds. Dermal fibroblasts of Tharparkar were observed to be more heat tolerant than crossbred Karan-Fries cattle. The study concludes that zebu cattle (Tharparkar) dermal fibroblasts are more adapted to tropical climatic condition than crossbreed cattle (Karan-Fries). Differences exist in dermal fibroblasts of heat adapted and non-adapted cattle.     

Cryoprotectant removal temperature as a factor in the survival of frozen rice and sugarcane cells

Removal of cryoprotective additives through use of a room temperature (22 °C) washing step, instead of 0 °C, was found to improve the recovery of sugarcane suspension culture and rice callus tissues. Cultured cells were cryoprotected by gradual addition of a mixture of polyethylene glycol, glucose, and DMSO (PGD) to a final concentration of 10%-8%-10%, w/v, respectively, added at either 0 or 22 °C. After a programmed slow freezing of the cells, they were thawed rapidly and the cryoprotectants were gradually diluted and washed out using a 22 or 0 °C washing medium. Viability of suspension cultured sugarcane cells protected with PGD was greatly diminished when a cold washing solution was used, whether the cells had been frozen (?23 °C) or not. Two mutant lines of rice callus when frozen to ?196 °C in PGD and thawed showed less growth than unfrozen cells, but their growth was improved by washing the thawed cells with a 22 °C solution. With all cultures tested, the addition of PGD at 0 °C and post-thaw washing out at 22 °C gave improved survival. Particularly with the rice lines, optimizing the addition and washing procedures allowed culture survival of liquid nitrogen freezing not otherwise attained.     

Hypothermia downregulates inflammation but enhances IL-6 secretion by stimulated endothelial cells

Hypothermia is a standard method for organ protection during cardiac surgery in children. However, the mechanisms of hypothermia-induced cell protection have not yet been clearly established. Therefore, the aim of our studies was to elucidate molecular effects of clinically relevant mild and deep hypothermia on endothelial cells. The endothelium plays a pivotal role in the interaction between blood cells and actively participates in complex inflammatory events. We isolated primary human umbilical vein endothelial cells (HUVEC) and investigated cell viability, proliferation and inflammatory characteristics after TNF-α stimulation under mild (32 °C) and deep (17 °C) hypothermia in comparison to normothermia (37 °C). As a protective mechanism of endothelial cells kept under hypothermic conditions we found a significant upregulation of the antiapoptotic protein Bcl-2, resulting in the same cell viability under hypothermic conditions. Unexpectedly we demonstrated significantly higher IL-6 release after 6 h of mild hypothermia. In contrast, hypothermia diminished inflammatory chemokines such as IL-8, MCP-1 and COX-2 protein expression which could lead to reduced leukocyte recruitment under hypothermia. Underlying mechanisms of this downregulation were found to be reduced ERK 1/2 phosphorylation and incomplete IκB-α degradation resulting in reduced NFκB-dependent proinflammatory gene expression. The upregulation of Bcl-2 protein and the higher IL-6 release after 6 h of mild hypothermia are new and interesting cellular mechanisms of hypothermia in endothelial cell biology. Both factors may play a major role as cell protective mechanisms in hypothermia.     

Survival of human lymphoblastoid cells after DNA damage measured by growth in microtiter wells

Survival of cells in suspension culture after treatment with damaging agents is usually measured by extrapolation from growth curves or by growth of colonies in soft agar. We have developed a survival assay which measures the ability of small numbers of cells to initiate microscopic cultures in wells of microtiter plates without agar or feeder layers. Suitable human lymphoblastoid lines were obtained by selection of rapidly growing cultures from microtiter wells in which <200 cells were inoculated in 0.2 ml RPMI 1640 medium and incubated at 37° with 5% CO₂ at 95% relative humidity. Survival after damage was measured by inoculating groups of 24 microtiter wells with appropriate serial dilutions of cells. The wells were examined microscopically at intervals and scored for evidence of cell proliferation. Survival was calculated with the Poisson formula on the basis of the fraction of wells in which cells were not proliferating. Survival did not change appeciably after 2–3 weeks incubation. Survival measured by the microtiter-well assay was found to be similar to survival measured by extrapolation from growth curves after damaging the cells with bleomycin or with 8-methoxypsoralen plus long-wavelength ultraviolet radiation. The microtiter-well assay affords a simple, accurate measure of cell survival in human lymphoblastoid cells with suitable growth ability.     

Calcium uptake by isolated rat intestinal cells

Intestinal cells were isolated by a combination of mechanical and enzymatic means, and their calcium uptake was assayed by a rapid filtration procedure. Calcium uptake was a time- and concentration-dependent process that was markedly elevated at 25 and 37°C, as compared to 0°C. Cells isolated from rat duodenum exhibited higher uptakes than cells from jejunum, which in turn took up more calcium than cells from the ileurn. Duodenal cells from vitamin D-deficient animals took up less calcium than cells from vitamin D-replete cells. In vivo vitamin D repletion with 1,25-dihydroxyvitamin D₃ raised calcium uptake by duodenal cells from treated animals toward that of cells from replete rats. Furthermore, calcium uptake by duodenal cells from vitamin D-deficient animals approximated that of ileal cells from replete rats. These findings with isolated cells parallel prior findings of tissue calcium transport and suggest that cellular calcium uptake may be related to the saturable component of intestinal calcium absorption. Isolated intestinal cells may therefore constitute one experimental model for the study of transcellular calcium transport.     

Cell preservation in a programmed cooling machine: the effect of variations in supercooling

When cells are cryopreserved in programmed cooling machines, they supercool to a variable and uncontrolled extent. Experiments were carried out with three cell-types (human peripheral lymphocytes, Chinese hamster lung fibroblasts, and mouse lymphoma cells) to determine whether there was any effect of supercooling on cell survival. Samples were cooled at 1 °C min^?1 in the presence of 12% v/v dimethyl sulphoxide (Me₂SO) to ?100 °C, and then thawed rapidly in a 37 °C water bath. There was no correlation between the extent of supercooling or the maximum cooling rate after freezing and cell survival, but the time taken for the sample temperature to return to the temperature at which freezing occurred did influence the survival of the two tissue culture cell lines. These results are interpreted on the basis of current theories according to which cells require sufficient time to lose water as they cool in order to avoid subsquent intracellular freezing, but must be cooled sufficiently rapidly to minimise solution effects. It is concluded that the variations in supercooling that occur in programmed cooling machines present no particular difficulties, providing appropriate cooling rates are chosen.     

The effect of membrane lipid perturbers on survival of mammalian cells to cold

Butylated hydroxytoluene (BHT), an antioxidant and common food additive, is an organic soluble molecule which modifies the properties of lipid bilayers and biological membranes. Adamantane and its derivatives, although structurally quite different, have similar effects on membranes. When Chinese hamster lung cells (V79) were pretreated with 0.1 mm BHT before exposure to +5 °C for up to 12 days in suspension culture or attached to plastic, significant protection, as assessed by colony survival, was observed compared to controls. No protection was observed when the cells were exposed to +20 °C. Use of adamantane or 2-adamantanone in serum-free medium in suspension culture at +5 °C showed protection of cells as good as, or better than, that provided by the addition of serum to control cells. Experiments with synchronized cells indicated that BHT protected cells in all phases of the cell cycle against the effects of exposure to +5 °C. However, the protection was greatest in G₁ and early S phase. Exposure of cells containing BHT to +20 °C resulted in no preferential protection in any phase of the cell cycle.     

The effect of temperature on the growth,development and survival of Macropetasma africanus (Balss) (Penaeoidea:Penaeidae) larvae reared in the laboratory

Macropetasma africanus (Balss) has been successfully spawned and its larvae reared under controlled laboratory conditions. The relationship between egg number (E) and female total length (L) was E = 18.59 L^2.11. An experiment was designed to test the effect of temperature on larval development, survival and growth. Temperature effected larval development time, from 13–15 days at 25°C, to 25 days at 15°C (nauplius 1 to post-larva). Mortality was low for the naupliar stages at 25, 22 and 18°C, while at 15°C only 52% of the larvae reached nauplius 6. Mortality was highest from nauplius 6 to protozoea 1 (17, 21, and 18% at 25, 22, and 18°C, respectively), but decreased considerably for all temperatures once the mysis stage was reached. Overall survival rates from nauplius 1 to post-larva decreased with decreasing temperature (65, 54, 48, and 39% at 25, 22, 18, and 15°C respectively). Temperature also significantly affected larval growth. At 25°C mean total length was significantly (P < 0.05) larger than at 15°C (protozoea 2 to post-larva), while from protozoea 3 to post-larva total length differences were significantly different (P < 0.05) between 18 and 25°C. M. africanus has a major spawning peak in summer, suggesting that there may be a selective advantage to reproducing during the warmer months.     

Detection of naturally expressed receptors for gastrin-releasing peptide and tachykinins using cyanine 3-labelled neuropeptides

Summary Peptides labelled with the fluorophore cyanine 3 were used to study naturally expressed neuropeptide receptors by confocal microscopy in continuous cell lines, primary cultures, and unfixed tissue. Swiss 3T3 fibroblasts bound cyanine 3-gastrin-releasing peptide at 4°C, and internalized the peptide after 10 min at 37°C. Internalization was specific, since it was blocked by incubation with unlabelled peptide. Primary cultures of myenteric neurons of the guinea pig incubated with cyanine 3-substance P at 4°C had specific surface labelling. After 30 s at 37°C, the peptide was internalized into vesicles in both the soma and neurites. Direct observation of live neurons showed movement of fluorescent vesicles to a perinuclear region after 30 min. Endocytosis was associated with a loss of surface binding sites. Unfixed whole mounts of guinea pig and rat ileum were incubated with cyanine 3-neurokinin A at 4°C. After 5 min at 37°C, Cy3-neurokinin A was specifically internalized in neurons and smooth muscle cells. After 30 min, a perinuclear labelling occurred in some cells. Labelling in rat neurons was diminished by the NK3-R antagonist SR142801. Thus, cyanine 3-neuropeptides are valuable tools to study expression and endocytosis of naturally expressed receptors.     

Effects of temperature on eggs and larvae of Anisakis simplex sensu stricto and Anisakis pegreffii (Nematoda: Anisakidae) and its possible role on their geographic distributions

Effects of temperature on development of eggs, recently hatched larvae and L3 larvae of the marine parasitic nematodes Anisakis simplex sensu stricto (s.s.) and A. pegreffii were examined in vitro. The eggs of A. simplex s.s. hatched at 3–25 °C and those of A. pegreffii hatched at 3–27 °C. Days before hatching varied between 2 days at 25 °C and 35–36 days at 3 °C in A. simplex s.s. and between 2 and 3 days at 27 °C and 65 days at 3 °C in A. pegreffii. Hatching rates of A. simplex s.s. were maintained high at temperatures between 3 and 25 °C but decreased to 0% at 27 °C. In contrast, those of A. pegreffii were lowest particularly at 3 °C, but also at 27 °C. The mean 50% survivals of hatched larvae ranged from 5.3 days at 25 °C to 82.3 days at 9 °C in A. simplex s.s., while in A. pegreffii it ranged from 1.2 days at 27 °C to 77.2 days at 9 °C. L3 larvae of A. pegreffii exhibited higher survival rates and activity than those of A. simplex s.s., particularly at 20 and 25 °C. These results suggest that the early stages of A. simplex s.s. are more adapted to lower temperatures whereas those of A. pegreffii are more tolerant to warm environments, which may correspond to their distribution patterns in Japan and Europe.     

Prolonged Ex vivo expansion and differentiation of naïve murine CD43− B splenocytes

Ex vivo expansion of naive primary B cells is still a challenge, yet would open new possibilities for in vitro studies of the immune response or the production of monoclonal antibodies. In our hands, unstimulated murine B cells did not expand in significant numbers, while culture viability decreased rapidly within a few days. Activation mimicking in vivo stimulation through either T cell‐independent or T‐cell dependent signaling, led to several division cycles, albeit accompanied by irreversible differentiation. By co‐culturing B cells under moderate hypothermia (30°C) on live feeder fibroblasts expressing recombinant CD40 ligand (CD154) and by repeatedly transferring cultured B cells to new feeder cell cultures, we could extend the growth of primary mouse B cells compared to cultures maintained at 37°C. B cells under these conditions showed an activated phenotype as shown by the presence of AID and IRF4, two factors required for IgH class switch recombination in antigen‐activated B cells. In contrast to cells cultured at 37°C, B cells under hyperthermia did surprisingly not differentiate into Blimp‐1 expressing plasmablasts. Thus, the repeated batch process under hyperthermic conditions represents a first step towards the development of a continuous cultivation system for the expansion of primary B cells. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:978–989, 2016     

Establishment,growth kinetics,and susceptibility to AcMNPV of heat tolerant lepidopteran cell lines

Lepidopteran heat-tolerant (ht) cell lines have been obtained with sf-9, sf-21 and several Bombyx cells. They have a distinct karyotype, membrane lipid composition, morphology and growth kinetics from the parental cell lines. In this paper, we report the development of ht cell lines from other insect species and examination of their growth characteristics and virus susceptibility. Adaptation of cell lines sf-9, BTI-TN-5B1-4 (High5) and BTI-TN-MG1 (MG1) to 33°C and 35°C was carried out by shifting the culture temperature between 28°C and higher temperatures by a gradual stepwise increase in temperature. The process of adaption to a higher culture temperature was accomplished over a period of 2 months. The cell lines with the temperature adaption were designated as sf9-ht33, sf9-ht35, High5-ht33, High5-ht35, MG1-ht33, MG1-ht35. These cell lines have been subcultured over 70 passages. Adaption to high temperatures was confirmed by a constant population doubling time with individual cell lines. The population doubling time of heat adapted cell lines were 1–4 h less than these of parental cell lines. Cell shapes did not show obvious change, however, the cell size of sf9-ht cells was enlarged and those of High5 and MG1 ht cells were reduced after heat adaption. When the cell lines were infected with Autographa californica nuclear polyhedrosis virus (AcMNPV) at 28°C, 33°C, 35°C and 37°C, production of budded virus and occlusion bodies in each cell line was optimum at its own adapted temperature.     

Role of serum on cryopreservation and subsequent viability of mouse bone marrow hemopoietic stem cells

Normal mouse marrow cells were frozen in an automatically controlled freezer at a cooling rate of 1 °C/min to ?40 °C and 7 °C/ min to ?100 °C using dimethylsulfoxide as a cryoprotective agent. The freezing solution contained in addition either 10% homologous serum or 10% fetal calf serum. Control samples were frozen with serum-free medium. After thawing, stepwise dilution, and washing, the cells were counted, checked for CFU-s content, and cultured in Millipore diffusion chambers for 2 and 7 days.HS resulted in a recovery of 59.7% nucleated cells and 100.5% CFU-s whereas FCS and serum-free medium resulted in 59.8 and 34.7% nucleated cells and 24.5 and 18.2% CFU-s, respectively. After 2 days of culture, D.C. data showed a correlation with the CFU-s results. After 7 days of culture, no significant difference was observed between the three groups. The results of these experiments indicate that HS is required for an optimal stem cell cryopreservation and that a 2-day D.C. culture is a reliable assay system for transplantable hemopoietic tissue.