Human ES cells: starting culture from frozen cells

Here we demonstrate how our lab begins a HuES human embryonic stem cell line culture from a frozen stock. First, a one to two day old ten cm plate of approximately one (to two) million irradiated mouse embryonic fibroblast feeder cells is rinsed with HuES media to remove residual serum and cell debris, and then HuES media added and left to equilibrate in the cell culture incubator. A frozen vial of cells from long term liquid nitrogen storage or a -80 C freezer is sourced and quickly submerged in a 37 C water bath for quick thawing. Cells in freezing media are then removed from the vial and placed in a large volume of HuES media. The large volume of HuES media facilitates removal of excess serum and DMSO, which can cause HuES human embryonic stem cells to differentiate. Cells are gently spun out of suspension, and then re-suspended in a small volume of fresh HuES media that is then used to seed the MEF plate. It is considered important to seed the MEF plate by gently adding the HuES cells in a drop wise fashion to evenly disperse them throughout the plate. The newly established HuES culture plate is returned to the incubator for 48 hrs before media is replaced, then is fed every 24 hours thereafter.     

Passaging HuES human embryonic stem cell-lines with trypsin

In this video we demonstrate how our lab routinely passages HuES human embryonic stem cell lines with trypsin. Human embryonic stem cells are artifacts of cell culture, and tend to acquire karyotypic abnormalities with high population doublings. Proper passaging is essential for maintaining a healthy, undifferentiated, karyotypically normal HuES human embryonic stem cell culture. First, an expanding culture is washed in PBS to remove residual media and cell debris, then cells are overlaid with a minimal volume of warm 0.05% Trypsin-EDTA. Trypsin is left on the cells for up to five minutes, then cells are gently dislodged with a 2mL serological pipette. The cell suspension is collected and mixed with a large volume of HuES media, then cells are collected by gentle centrifugation. The inactivated trypsin media mixture is removed, and cells resuspended in pre-warmed HuES media. An appropriate split ratio is calculated (generally 1:10 to 1:20), and cells re-plated onto a 1-2 day old plate containing a monolayer of irradiated mouse embryonic fibroblast feeder cells. The newly seeded HuES culture plate is left undisturbed for 48 hrs, then media is changed every day thereafter. It is important not to trpsinize down to a single cell suspension, as this increases the risk of introducing karyotypic abnormalities.     

Frozen storage (—20° and —80°C) of soybean Bradyrhizobium cell suspensions

Liquid cultures of Bradyrhizobium japonicum were added in a 1:1 ratio to 20% aqueous skim milk, or centrifuged and the cells resuspended in 10% skim milk. The suspensions were stored at —20° or —80°C for 7 months and cell survival assessed. At —20°C, there was a decrease in the viable count of about two logs in liquid culture whilst for cells resuspended in 10% skim milk the decrease was limited to one log. The temperature of —80°C was found to be in itself protective and the surviving rhizobial cells maintained their infectivity and effectiveness. Thus appropriate freezing conditions provide a suitable method to store soybean rhizobia cells prior to preparing the legume inoculant.     

Injury and Death of Streptococcus lactis due to Freezing and Frozen Storage

Cells of Streptococcus lactis were harvested in the early stationary phase, washed, and resuspended in either skim milk (10% nonfat milk) or buffered distilled water (0.0003 m dipotassium phosphate, pH 7.2). Samples of each suspension were frozen and stored at -20 C for intervals up to 28 days. Colony counts of the frozen culture were made using lactic agar and a “restricted” lactic agar medium (Tryptone reduced to 0.5%) to determine injury and death. Death was determined by the difference in plate counts on lactic agar before and after freezing. Injured cells were determined by the difference in plate counts on the two plating media. Greatest injury of the cells occurred during early stages of frozen storage and decreased with time, and death continuously increased. Injury and death were more pronounced when cells were frozen in water than when frozen in 10% nonfat milk solids. Certain cultures survived better when frozen rapidly, whereas with others survival was greater when freezing was slow. Successive freezing, thawing, and propagation of the culture gradually eliminated cells which showed injury by freezing.     

Microimmunofluorescence using Terasaki plates and direct plate freezing method--rapid and reliable screening system of hybridomas

Microimmunofluorescence using Terasaki plates and a direct plate freezing method were combined for effective screening of hybridoma supernatants. The microplates, in which the fused cells (myeloma and spleen cells) were cultured and hybridoma colonies were growing, were frozen after harvest of supernatants and saved at -80 C for several weeks without affecting antibody production ability of hybridomas. Microimmunofluorescence was performed in Terasaki plates on which target cells were attached by poly-L-lysine and glutaraldehyde or by short time culture of the cells in Terasaki plates. The direct plate freezing method prevented initial hybridoma cells from changes or disappearance of antibody productions during screening of hybridoma supernatants; the microimmunofluorescence staining method permits fast and detailed estimation of specificity of antibodies of hybridomas by saving time and minimal consumption of supernatant for checking. The combination of these two methods is a powerful tool for obtaining desired monoclonal antibodies.     

Cryopreservation by slow cooling with DMSO diminished production of Oct-4 pluripotency marker in human embryonic stem cells

We tested a "standard" cryopreservation protocol (slow cooling with 10% DMSO) on the human embryonic stem cell (hESC) line H9 containing an Oct-4 (POU5F1) promoter-driven, enhanced green fluorescent protein (EGFP) reporter to monitor maintenance of pluripotency. Cells were cooled to -80 degrees C in cryovials and then transferred to a -80 degrees C freezer. Cells were held at -80 degrees C for 3 days ("short-term storage") or 3 months ("long-term storage"). Vials were thawed in a +36 degrees C water bath and cells were cultured for 3, 7, or 14 days. Propidium iodide (PI) was used to assess cell viability by flow cytometry. Control cells were passaged on the same day that the frozen cells were thawed. The majority of cells in control hESC cultures were Oct-4 positive and almost 99% of EGFP+ cells were alive as determined by exclusion of PI. In contrast, the frozen cells, even after 3 days of culture, contained only 50% live cells, and only 10% were EGFP-positive. After 7 days in culture, the proportion of dead cells decreased and there was an increase in the Oct-4-positive population but microscopic examination revealed large patches of EGFP-negative cells within clusters of colonies even after 14 days of culturing. After 3 months of storage at -80 degrees C the deleterious effect of freezing was even more pronounced: the samples regained a quantifiable number of EGFP-positive cells only after 7 days of culturing following thawing. It is concluded that new protocols and media are required for freezing hESC and safe storage at -80 degrees C as well as studies of the mechanisms of stress-related events associated with cell cryopreservation.     

An appraisal of the efficacy of pre-enrichment for the isolation of Campylobacter jejuni from water and food

Cells of Campylobacter jejuni exposed to heating or freezing were progressively less able to grow at 43°C, particularly on selective media. This influenced the recovery of damaged cells from naturally and artificially contaminated samples. With broth culture the isolation rate could be increased by pre-enrichment in basal or selective media at 37°C for 4 h. With membrane filtration or surface plating techniques the inclusion of agents that quench toxic derivatives of oxygen was more important.     

An appraisal of the efficacy of pre-enrichment for the isolation of Campylobacter jejuni from water and food

Cells of Campylobacter jejuni exposed to heating or freezing were progressively less able to grow at 43 degrees C, particularly on selective media. This influenced the recovery of damaged cells from naturally and artificially contaminated samples. With broth culture the isolation rate could be increased by pre-enrichment in basal or selective media at 37 degrees C for 4 h. With membrane filtration or surface plating techniques the inclusion of agents that quench toxic derivatives of oxygen was more important.     

Effects of Hyperoxia upon Microorganisms: I. Membrane Culture Technique for Exposing Cells Directly to Test Atmospheres

A membrane culture technique was developed for directly exposing microorganisms to test atmospheres. Inhibition and killing were calculated from comparisons with air-grown cultures. Direct colony counts were used with low inocula. With mass inocula, plate colony counts and optical-density measurements were made on resuspended filter populations. Bacteria, including Escherichia coli, were more sensitive to oxygen than previously reported. With inocula of a few hundred cells per membrane, five of seven species failed to produce colonies while exposed to oxygen at one atmosphere. Upon reincubation in air, the survival of five species ranged from near 0 to 12% of the cells. Aerobacter aerogenes was neither inhibited nor killed. With this technique, bacteria are in direct contact with the test atmosphere and cells which survive are detected but do not obscure the response of other cells in the population.     

A Replica Plating Method for Plant Cells

A replica plating method is described for plant cells growing in Petri dishes. The method involved a uniform application of plant cells (Morinda citrifolia L.) by spraying cells evenly on agar plates containing 60% conditioned medium. Subsequently the cells were allowed to grow through a nylon net. The net was removed from the master plate and placed upside down on replica plates. Cells from colonies adhering to the threads of the net were thus transferred to the replica plate and yielded colonies that, after a growth period of about 10–20 days, corresponded in position to the colonies on the master plate. An 80% transfer of colonies from the master plate to the copy plate was possible.     

Caspase Inhibitor Z-VAD-FMK Enhances the Freeze-Thaw Survival Rate of Human Embryonic Stem Cells

Previous study demonstrated that the low survival of human embryonic stem cells (hESC) under conventional slow-cooling cryopreservation protocols is predominantly due to apoptosis rather than cellular necrosis. Hence, this study investigated whether a synthetic broad-spectrum irreversible inhibitor of caspase enzymes, Z-VAD-FMK can be used to enhance the post-thaw survival rate of hESC. About 100 mM Z-VAD-FMK was supplemented into either the freezing solution, the post-thaw culture media or both. Intact and adherent hESC colonies were cryopreserved so as to enable subsequent quantitation of the post-thaw cell survival rate through the MTT assay, which can only be performed with adherent cells. Exposure to 100 mM Z-VAD-FMK in the freezing solution alone did not significantly enhance the post-thaw survival rate (10.2% vs. 9.9%, p > 0.05). However, when 100 mM Z-VAD-FMK was added to the post-thaw culture media, there was a significant enhancement in the survival rate from 9.9% to 14.4% (p < 0.05), which was further increased to 18.7% when Z-VAD-FMK was also added to the freezing solution as well (p < 0.01). Spontaneous differentiation of hESC after cryopreservation was assessed by morphological observations under bright-field microscopy, and by immunocytochemical staining for the pluripotency markers SSEA-3 and TRA-1-81. The results demonstrated that exposure to Z-VAD-FMK did not significantly enhance the spontaneous differentiation of hESC within post-thaw culture.     

An Air-Drying Procedure for Mammalian Male Meiotic Chromosomes, Following Softening in Gluconic Acid and Cell Separation by An Ethanol-Acetic Mixture

A I cm³ sample of tubules from testes is placed in 5 ml of 0.7% Na-citrate for 20-30 min, then 5 ml of glacial acetic acid is added, mixed well, and allowed to stand for 30 min. The mixture is centrifuged, the supernatant removed, and 3 ml of 3 M gluconic acid is mixed with the tissue and allowed to act for 3 hr. The gluconic acid is removed with a pipette and the tissue is suspended in 5 ml of a freshly made 1:1 absolute ethanol-glacial acetic acid mixture. The tissue is drawn into and discharged from a syringe several times through an 18, a 20, and finally a 22 gauge needle to separate and suspend the cells. The cells are centrifuged and resuspended several times in fresh fixative to remove the gluconic acid. Finally, the cells are suspended in sufficient fixative to give a smear of suitable density, and air-dried preparations are made, or the suspension may be stored at 0-5 C for several days. The cells can be stained by any of the usual stains for chromosomes. This technique results in the improved spreading produced by the air-drying technique and permits recovery of all stages of meiosis and mitosis present.     

球形棕囊藻游离单细胞的密度与囊体形成的关系研究

球形棕囊藻(Phaeocystis globosa Scherffel)主要以囊体形态形成赤潮,由单细胞向囊体形态的转变是赤潮爆发的关键。本研究推测囊体形成的前提是游离单细胞达到一定密度阈值,当密度低于该阈值时,囊体无法形成。基于此,本文探究了不同条件(温度、营养充气搅动、摄食压力、初始密度)下囊体形成时游离单细胞的密度。结果显示:不同培养条件下,囊体形成所需的游离单细胞密度不一致,但都达到了104cells/mL的数量级;稀释试验表明,利用f/2培养基稀释使游离单细胞的密度小于104cells/mL时,囊体不能形成,而密度大于104cells/mL的游离单细胞对照组,在24 h内便有囊体形成。总的来说,游离单细胞在高密度情况下更容易形成囊体。     

Selective enumeration of spores of Clostridium species in dried foods

The suitability of a variety of media and procedures for the enumeration of sulphite-reducing clostridia in food was investigated. The most suitable procedure was pasteurization of the 1/10 macerate for at least 1 min at 80 degrees C; followed by culture at 30 degrees C for up to 3 d in a sulphite-based, differential reinforced clostridium medium, without bicarbonate or lactate but with an increased iron concentration, and sulphite and iron added after sterilization. Black sulphite-reducing colonies were finally tested for sensitivity to metronidazole and confirmation of their failure to grow on agar slopes under aerobic conditions.     

Selective enumeration of spores of Clostridium species in dried foods

The suitability of a variety of media and procedures for the enumeration of sulphite-reducing clostridia in food was investigated. The most suitable procedure was pasteurization of the 1/10 macerate for at least 1 min at 80°C; followed by culture at 30°C for up to 3 d in a sulphite-based, differential reinforced clostridium medium, without bicarbonate or lactate but with an increased iron concentration, and sulphite and iron added after sterilization. Black sulphite-reducing colonies were finally tested for sensitivity to metronidazole and confirmation of their failure to grow on agar slopes under aerobic conditions.     

PVP contrasted with dextran and a multifactor theory of cryoprotection

Washed human erythrocytes were suspended in 0, 5, 10, 15, and 20% PVP in phosphate-buffered saline (PBS). Fifty lambda samples were frozen in alcohol baths at temperatures ranging from ?10 ° to ?80 °C. The specimens were frozen either for 1 or 16 min, rapidly thawed, and resuspended in PBS or PBS plus PVP. Percent hemolysis was determined colorimetrically. Results indicate that there is a high degree of latent damage when red cells are frozen in the presence of PVP. This damage is evident from the large increase in hemolysis when freeze-thawed, intact red cells are resuspended in the PBS. Under some circumstances 16 min freezing is significantly less damaging than 1 min freezing. This indicates a partial recovery from the freezing stress during subzero storage of the red cells.The general cryoprotective properties of PVP were described in terms of: (1) latent damage; (2) storage damage; (3) optimal cooling and rewarming rates (as a function of freezing bath temperature); (4) optimum PVP concentration; and (5) post-thaw cryoprotection. The data were compared with that from a similar study using dextran-40. This comparison indicated six similarities and ten differences in the cryoprotective properties of dextran and PVP. The remarkable differences between dextran and PVP was counted as an important common characteristics of macromolecular cryoprotective agents. That is, their cryoproteetive properties cannot be reduced to one or a few physical characteristics held in common. Nine other common characteristics were listed. Several of these, which include latent damage and recovery from latent damage, cannot be explained by current theories of cryoprotection. A multifactor theory was proposed to account for these ten common features of macromolecular cryoprotective agents.     

The role of bacterial growth autoregulators (alkyl hydroxybenzenes) in the response of staphylococci to stresses

The investigation of the response of a batch culture of Staphylococcus aureus to exogenous alkyl-substituted hydroxybenzenes (AHBs), chemical analogues of anabiosis autoinducers, showed that C1-AHB at concentrations from 5 microM to 1.5 mM did not influence the culture growth, whereas the more hydrophobic C6-AHB inhibited it at concentrations of 0.5 mM and higher. Either of the AHBs drastically enhanced the phenotypic dissociation of staphylococcal cultures, which manifested itself in an increase in the fraction of cells producing small nonhemolyzing colonies of G type when plated on solid media with erythrocytes. In a submerged staphylococcal culture, the relative number of cells producing G-type colonies varied from 10 to 90%, depending on the concentration of the AHB added. The growth of S. aureus in the presence of AHBs also enhanced cell tolerance to heat shock (heating at 45 or 60 degrees C for 10 min). The role of AHBs, which are structural modifiers of membranes and possess chaperone activity, in the mechanisms responsible for cell tolerance and phenotypic dissociation of microbial populations is discussed.     

A method to maintain mammalian cells for days alive at 4 °C

This paper describes a method for the temporary storage of cultured cells. Cells from recently completed cell monolayers were trypsinized and then centrifuged. After centrifugation, the supernatant and pellet were kept at 4 °C for one week. After storage, the supernatant was discarded, the cells were resuspended and used for seeding new flasks and for titration of virus. The cells not only remained viable, but also rapidly formed new monolayers and allowed immediate infection and growth of viruses. We conclude that this method can be a helpful asset to cell culture experiments.     

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.