Kinetics of cell death of frozen-thawed human embryonic stem cell colonies is reversibly slowed down by exposure to low temperature

A major challenge in the widespread application of hES (human embryonic stem) cells in clinical therapy and basic scientific research is the development of efficient cryopreservation protocols. Conventional slow-cooling protocols utilizing standard cryoprotectant concentrations i.e. 10% (v/v) DMSO, yield extremely low survival rates of less than 5% as reported by previous studies. This study characterized cell death in frozen-thawed hES colonies that were cryopreserved under standard conditions. Surprisingly, our results showed that immediately after post-thaw washing, the overwhelming majority of hES cells were viable (approximately 98%), as assessed by the trypan blue exclusion test. However, when the freshly thawed hES colonies were placed in a 37 degrees C incubator, there was a gradual reduction in cell viability over time. The kinetics of cell death was drastically slowed down by keeping the freshly thawed hES colonies at 4 degrees C, with more than 90% of cells remaining viable after 90 min of incubation at 4 degrees C. This effect was reversible upon re-exposing the cells to physiological temperatures. The vast majority of low temperature-exposed hES colonies gradually underwent cell death upon incubation for a further 90 min at 37 degrees C. Hence, our observations would strongly suggest involvement of a self-induced apoptotic mechanism, as opposed to cellular necrosis arising from cryoinjury.     

Interaction of Yeast Phosphoglyceromutase with Nucleotides

Moderate cell growth occurred after a long lag phase of about 100 hr when oxygen-sensitive hydrogen bacterium N34 was cultivated chemoautotrophically under 40% O₂. A decrease in cell growth or viable count was not observed during the lag phase. These cells grown under 40 % O₂ were oxygen-resistant because when used as inocula for fresh 40 % O₂-culture, the growth lag period was less than 10 hr. Nine oxygen-sensitive colonies developed from a single oxygen-sensitive cell respectively. When these colonies were inoculated into 40% O₂-culture, they showed an almost equal lag period and growth rate. These results suggest that cell growth in 40% O₂-culture inoculated with oxygen-sensitive strain N34 occurred not by selection of oxygen-resistant variants which might preexist but by adaptation of very oxygen-sensitive cells to high oxygen tension. Oxygen-resistance thus developed was maintained after successive subcultures under 10% O₂ for more than one year.     

The role of tissue transglutaminase in the germinal vesicle breakdown of mouse oocytes

Calcium/calmodulin-dependent protein kinase II (CaMKII) is a widely distributed protein kinase that regulates numerous physiological functions. Inhibitors of CaMKII are useful tools for investigating the CaMKII functions. Here we identified a novel CaMKII inhibitor protein (CaM-KIIN) from the human dendritic cell cDNA library by large-scale random sequencing. Human CaM-KIIN contains 79 amino acids, which shares 98% identity and 98% positives with rat CaMKII inhibitor protein beta and 65% identity and 78% positives with rat CaMKII inhibitor alpha. Human CaM-KIIN mRNA expression was detectable in various tissues and cell lines by Northern blot and RT-PCR. To investigate its biological functions, full-length human CaM-KIIN was overexpressed in colon adenocarcinoma LoVo cells. When expressed in LoVo cells, it could inhibit cell proliferation, block cell growth, and decrease the viable cell number. These results characterize a potential cellular inhibitor protein of CaMKII that plays an important role in the regulation of cell growth.     

Filterability of Streptococcal L-Forms

The filterability of the broth-grown stable L-form derived from Streptococcus faecium F24 was tested by filtration under the influence of varying amounts of applied pressure. A decrease in the pore size of the filter resulted in a corresponding decrease in viable count, but no major effect was noted due to the different pressures applied. Serial filtration of a deoxyribonuclease-treated L-form culture in the mid-logarithmic phase of growth resulted in recovery of viable L-forms from the 0.45-mum filtrate but not from the 0.22-mum filtrate. It is possible that disruption of the L-form bodies with release of small viable elements had occurred. Protoplasts, diluted in an osmotic stabilizer, were filtered similarly; L-forms could be grown from the filtrate passing through the filters of 0.45 mum or greater. Filtration of the parent streptococci gave rise to streptococcal colonies from the 1.2-mum filtrate only.     

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.     

Culture of soybean mesophyll protoplasts in alginate beads

Mesophyll protoplasts were isolated from leaves of 10 day old aseptically grown soybean seedlings, or from surface disinfested leaves of 3 week old plants grown in environmental chambers. The protoplasts were encapsulated in 2mm diameter Ca alginate beads. Immobilized protoplasts were induced to divide by culturing in shaker flasks containing an actively growing soybean cell suspension. The feeder cell suspension supported the division of protoplasts independent of the protoplast density in the Ca alginate beads. At day 18 after encapsulation, the alginate matrix was dissolved, releasing viable callus colonies. The feeder cell suspension obviated plating of protoplasts at high density which is usually required for subsequent cell division and colony development. Since the protoplasts were embedded at low density, the cell colonies were derived from single cells.     

Brood cell size of <Emphasis Type="Italic">Apis</Emphasis> <Emphasis Type="Italic">mellifera</Emphasis> modifies the reproductive behavior of <Emphasis Type="Italic">Varroa</Emphasis> <Emphasis Type="Italic">destructor</Emphasis>

We undertook a field study to determine whether comb cell size affects the reproductive behavior of Varroa destructor under natural conditions. We examined the effect of brood cell width on the reproductive behavior of V. destructor in honey bee colonies, under natural conditions. Drone and worker brood combs were sampled from 11 colonies of Apis mellifera. A Pearson correlation test and a Tukey test were used to determine whether mite reproduction rate varied with brood cell width. Generalized additive model analysis showed that infestation rate increased positively and linearly with the width of worker and drone cells. The reproduction rate for viable mother mites was 0.96 viable female descendants per original invading female. No significant correlation was observed between brood cell width and number of offspring of V. destructor. Infertile mother mites were more frequent in narrower brood cells.     

In vitro culture of tobacco protoplasts: Use of feeder techniques to support division of cells plated at low densities

Summary Tobacco protoplasts obtained from leaf mesophyll cells and suspended in agar nutrient medium divided and produced colonies only when plated at high densities, above 10⁴ cells per ml. At such densities coalescence of the expanding colonies occurred at high frequency. Nondividing X-irradiated protoplasts used as feeder cells supported division of viable protoplasts plated at densities as low as 10² cells per ml. The feeder cell technique should thus facilitate the application of screening procedures for the isolation of colonies originating from single mutated cells occurring in a suspended population.     

Tetrazolium staining by optical scanning overestimates colony size and number of colonies counted

We measured the effect that staining with 2-(P-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride (INT) had on the number and size distribution of tumor colonies counted using an optical image analyzer (FAS II). Staining increased the number of tumor colonies counted. By using opaque tumor cells or pigmented melanoma cells and measuring colony growth kinetics, we demonstrated that the use of INT staining to assist in counting tumor colonies artificially increased the size of viable tumor cell aggregates by adding a red precipitate to the outside surface of the cells. Laboratories that are using the INT method for drug screening are probably measuring colonies down to and below 42 microns in diameter. These small colonies could result from as few as one or two divisions. Thus, potentially useful drugs may be missed in the screen because of the presence of abortive colonies: i.e., lethally damaged cells completing only one or two divisions.     

Studies on the biological role of DNA methylation: inhibition of methylation and maturation of the bacteriophage phichi174 by nicotinamide.

Nicotinamide was found to be a potent inhibitor of DNA methylation in vivo without interfering with protein or DNA synthesis. The inhibition of DNA methylation in a phage-infected cell resulted in a parallel decrease in the production of viable virus particles. In vitro experiments revealed that nicotinamide inhibits DNA methylase activity in a competitive fashion with respect to S-adenosylmethionine and non-competitively with respect to DNA. These results were interpreted to mean that DNA methylation is an essential step in the process of maturation of the bacteriophage phichi174.     

The effect of sodium chloride concentration and pH on the growth of Salmonella typhimurium colonies on solid medium

The growth of Salmonella typhimurium colonies on a model food system (agar solidified culture medium) was followed. Colony radius, determined using computer image analysis (IA) techniques, and viable cell number per colony were measured as indices of colony growth, and the effect of [NaCl] (0.5–3.5% (w/v)) and pH (7.0–5.0) on colony growth at 30°C was observed; colonies were point inoculated from serial dilutions. Colony growth (between 13 and 26 h after inoculation) was linear when expressed in terms of radius, and exponential when expressed in terms of viable cell number per colony. Overall, both increasing the [NaCl] and decreasing the pH had little effect on colony growth, other than to delay the onset of linear radial growth. Initial specific growth rate (μ) ranged from 0.73 to 0.87 h⁻¹. Thin films of agar medium on microscope slides allowed the growth of microcolonies to be observed after just 4 h incubation. A greater understanding of the growth kinetics of bacterial colonies, and the effects of environment on such data, may enable better control of foodborne bacterial pathogens, and consequently an improvement in food product safety.     

Importance of culture conditions during the morula-to-blastocyst period on capacity of inner cell-mass cells of bovine blastocysts for establishment of self-renewing pluripotent cells

The hypothesis was tested that the pluripotency of the inner cell mass (ICM) of the bovine embryo is enhanced by the glycogen synthase kinase-3β inhibitor CHIR99021 and the MAPK1 and MAPK3 inhibitor PD032591. Treatment with the two inhibitors from Days 6 to 8 after insemination increased blastocyst steady state concentrations of mRNA for NANOG (P < 0.05) and SOX2 (P = 0.055) and tended to decrease (P = 0.09) expression of GATA6. To evaluate pluripotency, the inner cell mass was isolated by immunosurgery at Day 8, seeded on a feeder layer of bovine embryonic fibroblasts, and cultured in the presence of the inhibitors. Ten of 52 (19%) ICM from control embryos had primary outgrowth formation vs. 23 of 50 (46%) of the ICM from embryos cultured with inhibitors (P < 0.01). For ICM outgrowths from embryos cultured without inhibitors, colonies either did not persist through Passage 2 or became differentiated. In contrast, for the inhibitor group, four colonies survived beyond Passage 2, and one line persisted for 19 passages. This cell line possessed alkaline phosphatase activity, expressed several genes characteristically expressed in pluripotent cells, and differentiated into embryoid bodies when cultured in the absence of the signal transduction inhibitors and the feeder layer. Propagation of the cells was difficult due to slow growth and inefficiency in survival through each passage. In conclusion, exposure to inhibitors during the morula-blastocyst transition facilitated formation of self-renewing pluripotent cell lines from bovine blastocysts.     

DNA topoisomerase inhibitors block erythropoiesis and delay hemoglobinization in vitro

To examine the importance of topological constraints on DNA during erythroid development, we measured the effects of camptothecin and teniposide, two tumoricidal agents which are also specific inhibitors of type I and type II topoisomerases respectively, on the formation of hematopoietic colonies by cultured human bone marrow cells. When added to bone marrow culture, each inhibitor alone impairs the formation of early BFU-E-derived colonies, late CFU-E-derived colonies and mixed hematopoietic (CFU-GEMM-derived) colonies by up to 100%. Inhibition of colony formation is directly related to the time of inhibitor addition and the inhibitor concentration tested. Although either inhibitor alone reduces colony formation by 90%, when added together at a submaximal concentration, camptothecin and teniposide exert a synergistic suppressive effect. Furthermore, addition of topoisomerase inhibitors to culture impairs hemoglobinization of colony erythroblasts in a time-dependent fashion. In contrast to the effects of topoisomerase inhibitors, the antiproliferative agent aphidicolin reduces erythroid colony number and size without altering hemoglobinization of colony erythroblasts. Since neither topoisomerase inhibitor alters the morphology of cultured cells, the capacity of cells to exclude trypan blue or the potential to form erythroid colonies through the interval required for the first progenitor cell division, it is unlikely that camptothecin or teniposide are cytotoxic to hematopoietic cells. Human mononuclear cells enriched in bone marrow lymphocytes and nucleated erythroblasts from both human and mouse sources release DNA into the detergent soluble fraction. Release requires functional topoisomerases and is altered by acute exposure to topoisomerase inhibitors. Our results suggest that topoisomerases are critical not only to proliferation but also to differentiation of human marrow erythroid progenitor cells and stem cells in culture.     

Regulation of scatter factor/hepatocyte growth factor responses by Ras, Rac, and Rho in MDCK cells.

Scatter factor/hepatocyte growth factor (SF/HGF) stimulates the motility of epithelial cells, initially inducing centrifugal spreading of cell colonies followed by disruption of cell-cell junctions and subsequent cell scattering. These responses are accompanied by changes in the actin cytoskeleton, including increased membrane ruffling and lamellipodium extension, disappearance of peripheral actin bundles at the edges of colonies, and an overall decrease in stress fibers. The roles of the small GTP-binding proteins Ras, Rac, and Rho in regulating responses to SF/HGF were investigated by microinjection. Inhibition of endogenous Ras proteins prevented SF/HGF-induced actin reorganization, spreading, and scattering, whereas microinjection of activated H-Ras protein stimulated spreading and actin reorganization but not scattering. When a dominant inhibitor of Rac was injected, SF/HGF- and Ras-induced spreading and actin reorganization were prevented, although activated Rac alone did not stimulate either response. Microinjection of activated Rho inhibited spreading and scattering, while inhibition of Rho function led to the disappearance of stress fibers and peripheral bundles but did not prevent SF/HGF-induced motility. We conclude that Ras and Rac act downstream of the SF/HGF receptor p190Met to mediate cell spreading but that an additional signal is required to induce scattering.     

An investigation of the diffusion-limited growth of animal cells around single hollow fibers

To compare modeling with experimental data of cell growth surrounding individual fibers, the growth profiles of hybridoma cells in the extracapillary space of single hollow fiber bioreactors were examined. Agarose was provided in the extracapillary space to provide support and minimize convection. By sacrificing bioreactors at various time intervals, the growth profiles of cells surrounding a single hollow fiber could be monitored with increasing time. Using photomicroscopy and viable staining, areas of viable and nonviable cell growth were examined at various stages of development ranging from initial seeding to stable growth conditions. Cells were found to act as nucleation sites for the growth of individual colonies within the agarose. Profiles at stable growth conditions resulted in a thick cell mass near the surface of the fiber wall followed by cell colonies of decreasing size with increasing radial distance. A simplified theoretical model for cell growth was developed using mass balance equations for substrate penetration into individual cell colonies as well as away from the wall of a single fiber. The resulting profiles derived from theory were compared with experiments and found to be in good agreement for entering oxygen concentrations of 5% and 20%. (c) 1992 John Wiley & Sons, Inc.     

Viability and virulençe of Escherichia coli suspended by membrane chamber in semitropical ocean water

Abstract Escherichia coli H10407 was suspended in seawater (38.5‰ salinity) contained in membrane chambers (0.4-μm polycarbonate membrane) incubated in situ at 25°C in Nixon's Harbor, South Bimini, Bahamas. Although colonies of E. coli could not be cultured after 13 h post chamber inoculation, the number of fluorescent-antibody staining cells remained constant. Direct viable counts revealed that viable cells were present, even though the cell suspension was not culturable on the media tested. After exposure to seawater for 112 h, cells were concentrated by centrifugation and introduced into ligated rabbit ileal loops. E. coli H10407 proved viable for recovery from inoculated loops and was confirmed by detection of characteristic plasmid bands. Results indicate that enteric pathogens remain viable in seawater long after they cease to be cultivable on laboratory media.     

Isolation and culture of protoplasts from cotyledons of Pinus coulteri D. Don.

A protocol was developed for the isolation and culture of protoplasts from the cotyledons of seedlings of Pinus coulteri D. Don. Incubation of cotyledon pieces in a mixture consisting of cellulase Onozuka R10 2%, Pectolyase Y-23 0.1%, mannitol 10%, CaCl₂ 500 mg/l and other macro and micro-nutrients yielded viable protoplasts. After 24 hours of culture in a complex nutrient medium, the protoplasts regenerated new cell walls and the first divisions were observed within 7–10 days. Small cell colonies were formed within 15–20 days, but these started to accumulate phenolics and no further growth of the colonies was observed.     

Loss of viability during freeze–thaw of intact and adherent human embryonic stem cells with conventional slow-cooling protocols is predominantly due to␣apoptosis rather than cellular necrosis

Summary A major challenge in the widespread application of human embryonic stem (hES) cells in clinical therapy and basic scientific research is the development of efficient cryopreservation protocols. Conventional slow-cooling protocols utilizing standard cryoprotectant concentrations i.e. 10% (v/v) DMSO, yield extremely low survival rates of <5% as reported by previous studies. This study characterized cell death within frozen–thawed hES colonies that were cryopreserved under standard conditions. Surprisingly, our results showed that immediately after post-thaw washing, the overwhelming majority of hES cells were viable (≈98%), as assessed by the trypan blue exclusion test. However, when the freshly-thawed hES colonies were incubated within a 37 °C incubator, there was observed to be a gradual reduction in cell viability over time. The kinetics of cell death was drastically slowed-down by keeping the freshly-thawed hES colonies at 4 °C, with >90% of cells remaining viable after 90 min of incubation at 4 °C. This effect was reversible upon re-exposing the cells to physiological temperature. The vast majority of low temperature-exposed hES colonies gradually underwent cell death upon incubation for a further 90 min at 37 °C. Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end-labeling (TUNEL) assay confirmed apoptosis-induced nuclear DNA fragmentation in frozen–thawed hES cells after incubation at 37 °C for 90 min. Expression of active caspase-3 enzyme, which is another prominent marker of apoptosis, was confirmed by immunocytochemical staining, while transmission electron microscopy showed typical ultrastructural features of apoptosis such as chromatin condensation and margination to the nuclear membrane. Hence, our results demonstrated that apoptosis instead of cellular necrosis, is the major mechanism of the loss of viability of cryopreserved hES cells during freeze–thawing with conventional slow-cooling protocols.