Comparative study on human and bovine AT-SC isolation methods

Mammalian adipose tissue derived stem cells (AT-SC) have a tremendous potential in regenerative medicine for tissue engineering and somatic nuclear transfer (SNT). The isolation methods of human and bovine adipose tissue derived stem cells are compared in this paper to determine the feasibility and optimum method of isolation. The optimum isolation method will reduce the processing time, efforts and money as isolation is the first crucial and important step in stem cells research. Human abdominal subcutaneous adipose tissue and bovine abdominal subcutaneous adipose tissue are digested in three collagenase type 1 concentration 0.075%, 0.3% and 0.6% agitated at 1 h and 2 h under 37 °C in 5% CO₂ incubator. The cultures are then morphologically characterised. Human adipose tissue stem cells are found to be best isolated using abdominal subcutaneous depot, using 0.075% collagenase type 1 agitated at 1 h under 37 °C in CO₂ incubator. While bovine adipose tissue derived stem cells are best isolated using abdominal subcutaneous depot, using 0.6% collagenase type 1 agitated at 2 h under 37 °C in CO₂ incubator.     

The bicarbonate ion is essential for efficient DNA synthesis by primary cultured rat hepatocytes

Summary Bicarbonate in the culture medium is essential for DNA synthesis of primary cultured rat hepatocytes stimulated by epidermal growth factor (EGF). When primary cultured hepatocytes in supplemented Leibovitz L15 medium were placed in a 100% air incubator, no increase in DNA synthesis was observed even after stimulation by EGF. However, when these cells were cultured with NaHCO₃ and EGF and placed in a 5% CO₂:95% air incubator, a stimulus of DNA synthesis more than 10-fold greater than in cultures in air only was seen, and many mitotic figures could be identified. Furthermore, NaHCO₃ added to supplemented DMEM/F12 medium enhanced the DNA synthesis of primary cultured rat hepatocytes in this medium. The ideal pH of the medium for DNA synthesis of cultured hepatocytes was in the range of 7.6 to 8.0. A dose response of NaHCO₃ in several media showed that DNA synthesis of the cells increased as the concentration of NaHCO₃ increased and that 25 to 30 mM NaHCO₃ in the medium was optimal for the replication of DNA by primary cultured rat hepatocytes. The investigations described in this study were supported in part by grants CA-07175, CA-22484, and CA-45700 from the National Cancer Institute, Bethesda, MD.     

Exposure to hyperbaric oxygen induces cell cycle perturbation in prostate cancer cells

Summary Cell cycle synchronization of tumor cells by exposure to hyperbaric oxygenation (HBO) may increase the efficacy of chemotherapy or radiation by placing cells into a chemosensitive portion of the cycle. The purpose of the current study was to examine oxygen pressure-dependent relationships with respect to the cell cycle in prostate tumor cells in vitro. LNCaP cells were grown in an incubator at 21% O₂ and then exposed to 100% oxygen at pressures up to 6 atmospheres (atm) for 1.5 h. Cells were then returned to the incubator and evaluated for DNA content by propidium iodide and new DNA synthesis with a pulse-chase experiment. Cell cycle effects were evaluated by flow cytometry. Exposure to HBO increased the percentage of cells synthesizing new DNA in a dose-dependent fashion: 0 atm, 44%; 6 atm, 65%. Cells that synthesize new DNA accumulate in G₂/M as a function of partial pressure of oxygen. These results suggest that HBO induces cells to enter the cell cycle and accumulate in G₂/M. Cell cycle synchronization and entry of senescent cells into the cell cycle suggest that HBO may be a useful adjuvant to chemotherapy or radiation in the treatment of prostate cancer. There are two potential mechanisms of action that may make HBO efficacious in the treatment of prostate cancer. HBO may potentiate cancer chemotherapeutic agents that cause damage to DNA during DNA synthesis or HBO may inhibit cell division causing accumulation in G₂/M.     

Automatic gas tank switching system for CO2 incubators

Summary The use and construction of an automatic gas tank switching system are described. This device monitors the gas pressure in a CO₂ incubator gas system and automatically switches to a reserve tank when the main supply tank is depleted. The unit contains an alarm system that signals either a loss of power or of gas pressure in the supply system. This research was supported by National Cancer Institute Contract No 1-CP-33226, grants CA 13058 and CA 14680 and an institutional grant to the Michigan Cancer Foundation by the United Foundation of Greater Detroit.     

Effect of elevated carbon dioxide on chicken eggs during the early and late incubation periods

Carbon dioxide (CO₂) is always maintained at ambient levels by ventilation in commercial egg incubators. However, elevated CO₂ levels during the early and late periods have been reported to improve the quality of chicks and shorten the hatch window. This study investigated the effect of precise CO₂ supplementation during the early and late periods of incubation on embryo growth and incubation performance by developing and using a CO₂ supplementation system to increase the CO₂ level in an experimental egg incubator. The CO₂ level was maintained at 1% in the early period (from the beginning to the 10th day of incubation, E0–E10) and in the late period (from internal pipping (IP) to the 21st day of incubation (E21), IP–E21) in an incubator for the treatment group, whereas the CO₂ level was maintained at the ambient level in the other incubators for the control group. A comparative assessment of embryonic development, hatching characteristics, and hormone and nutrient levels was conducted for each trial. The experiment comprised three trials, with 300 Jing Hong No. 1 breeding eggs in each incubator. The elevated CO₂ treatment significantly shortened the chick hatching time (H0) by 4 h (P < 0.05) and the hatch window by 3 h (P < 0.05) without affecting hatchability, chick weight at 1 d of age, brooding period, or quality score. At external pipping (EP), the heart weight, intestinal weight, relative intestinal weight, and relative heart weight in the treatment group were significantly higher than those in the control group (P < 0.05). In addition, the embryonic intestine, relative intestine, and relative heart weights of the newly hatched chicks in the treatment group were significantly higher than those in the control group (P < 0.05) at H0. The treatment significantly increased the concentration of corticosterone in the embryonic plasma during the period from IP to EP (P < 0.05), promoted the secretion of triiodothyronine and tetraiodothyronine (P < 0.05), and increased the glycogen content of the embryonic liver on E21 (P < 0.05). This result indicates that elevated CO₂ (1%) during the early and late periods of incubation accelerated embryonic organ development and shortened the chick hatching time and hatch window without affecting hatchability or hatchling quality, which can be explained by the synergistic functions of the secretion of plasma corticosterone and thyroid hormones and the accumulation of liver glycogen between the early and late periods of incubation.     

四种昆虫饲养设备在昆虫学实验室的应用比较

本文比较了智能人工气候箱、智能型光照培养箱、全自动控制养虫室和简易型养虫室等4种常用昆虫饲养设备的优点和不足，分析了不同设备的适用情况。以西北农林科技大学应用昆虫学重点实验室正在应用的昆虫饲养设备为例，详细介绍了几种设备使用中的注意事项及维护方法。智能型人工气候箱和全自动控制养虫室可用于饲养对温度、湿度及光照要求较高的寄主植物及昆虫；智能型光照培养箱适用于对温度和光照有一定要求而对湿度要求不严格的试验；简易养虫室造价低，较容易建立，适用于饲养普通试验观察的昆虫种群。本文对4种设备的比较为农业科学研究中昆虫饲养设备的选择及管理维护提供了参考。     

Neuromodulation and Mitochondrial Transport: Live Imaging in Hippocampal Neurons over Long Durations

To understand the relationship between mitochondrial transport and neuronal function, it is critical to observe mitochondrial behavior in live cultured neurons for extended durations^1-3. This is now possible through the use of vital dyes and fluorescent proteins with which cytoskeletal components, organelles, and other structures in living cells can be labeled and then visualized via dynamic fluorescence microscopy. For example, in embryonic chicken sympathetic neurons, mitochondrial movement was characterized using the vital dye rhodamine 123⁴. In another study, mitochondria were visualized in rat forebrain neurons by transfection of mitochondrially targeted eYFP⁵. However, imaging of primary neurons over minutes, hours, or even days presents a number of issues. Foremost among these are: 1) maintenance of culture conditions such as temperature, humidity, and pH during long imaging sessions; 2) a strong, stable fluorescent signal to assure both the quality of acquired images and accurate measurement of signal intensity during image analysis; and 3) limiting exposure times during image acquisition to minimize photobleaching and avoid phototoxicity.Here, we describe a protocol that permits the observation, visualization, and analysis of mitochondrial movement in cultured hippocampal neurons with high temporal resolution and under optimal life support conditions. We have constructed an affordable stage-top incubator that provides good temperature regulation and atmospheric gas flow, and also limits the degree of media evaporation, assuring stable pH and osmolarity. This incubator is connected, via inlet and outlet hoses, to a standard tissue culture incubator, which provides constant humidity levels and an atmosphere of 5-10% CO₂/air. This design offers a cost-effective alternative to significantly more expensive microscope incubators that don''t necessarily assure the viability of cells over many hours or even days. To visualize mitochondria, we infect cells with a lentivirus encoding a red fluorescent protein that is targeted to the mitochondrion. This assures a strong and persistent signal, which, in conjunction with the use of a stable xenon light source, allows us to limit exposure times during image acquisition and all but precludes photobleaching and phototoxicity. Two injection ports on the top of the stage-top incubator allow the acute administration of neurotransmitters and other reagents intended to modulate mitochondrial movement. In sum, lentivirus-mediated expression of an organelle-targeted red fluorescent protein and the combination of our stage-top incubator, a conventional inverted fluorescence microscope, CCD camera, and xenon light source allow us to acquire time-lapse images of mitochondrial transport in living neurons over longer durations than those possible in studies deploying conventional vital dyes and off-the-shelf life support systems.     

An in vitro system to screen for diarrheagenic chemicals

We examined an in vitro system to screen for diarrheagenic chemicals using an established intestinal cell line (T₈₄ human colonic carcinoma). The cells were grown on Millicell-PCF (polycarbonate membrane) wells. The cells were seeded at approximately 5 × 10₆ cells/30mm well and incubated for 9–11 days in a 5% C0₂ incubator saturated with water at 37°C. The culture medium was a 1:1 mixture of Ham's F12 and Dulbecco's MEM with 5% fetal bovine serum and 25 pglml gentamicin sulfate. The well containing cells was removed from the incubator and mounted in a modified Ussing chamber for measurement of shortcircuit current (I_sc). Chemical-induced increases in Psc are usually indicative of electrogenic epithelial Cl^– secretion, which is associated with diarrheagenic effects in animals and humans. T₈₄ cells grown on Millicell-PCF membrane responded with an increase in Isc after basolateral addition of the cholinergic (muscarznic) agonist carbachol, prostaglandin E₂, 16,16-dimethylprostaglandin E₂, and forskolin, while non-diarrheagenic prostaglandin D₂ did not affect I_sc. Based on our results, this in vitro system has the potential to be adapted as a rapid screen for detecting diarrheagenic chemicals.Abbreviations dmPGE₂ 16,16-dimethylPGE₂ - EC₅₀ 50% of maximum effective concentration - EDTA ethylenediaminetetraacetate - I_SC short-circuit current - PGD₂ prostaglandin D₂ - PGE₂ prostaglandin E₂ - PD potential difference - R_T transepithelial resistance     

Design and validation of a pulsatile perfusion bioreactor for 3D high cell density cultures

This study presents the design and validation of a pulsatile flow perfusion bioreactor able to provide a suitable environment for 3D high cell density cultures for tissue engineering applications. Our bioreactor system is mobile, does not require the use of traditional cell culture incubators and is easy to sterilize. It provides real‐time monitoring and stable control of pH, dissolved oxygen concentration, temperature, pressure, pulsation frequency, and flow rate. In this bioreactor system, cells are cultured in a gel within a chamber perfused by a culture medium fed by hollow fibers. Human umbilical vein endothelial cells (HUVEC) suspended in fibrin were found to be living, making connections and proliferating up to five to six times their initial seeding number after a 48‐h culture period. Cells were uniformly dispersed within the 14.40 mm × 17.46 mm × 6.35 mm chamber. Cells suspended in 6.35‐mm thick gels and cultured in a traditional CO₂ incubator were found to be round and dead. In control experiments carried out in a traditional cell culture incubator, the scarcely found living cells were mostly on top of the gels, while cells cultured under perfusion bioreactor conditions were found to be alive and uniformly distributed across the gel. Biotechnol. Bioeng. 2009; 104: 1215–1223. © 2009 Wiley Periodicals, Inc.     

The Super-Spinner: A low cost animal cell culture bioreactor for the CO2 incubator

The production of small quantities of monoclonal antibodies and recombinant proteins was carried out using a new low cost production system, the Super Spinner. Into a 1 1 standard Duran^® flask a membrane stirrer equipped with a polypropylene hollow fiber membrane was installed to improve the oxygen supply by bubble-free aeration. The aeration was facilitated by using the CO₂ conditioned incubator gas, which was pumped through the membrane stirrer via a small membrane pump. The maximal oxygen transfer rate (OTR_max) of the Super Spinner was detected. For this purpose one spinner flask was equipped with an oxygen electrode. The OTR_max was measured by the dynamic method. The ratio of membrane length to culture volume was adapted corresponding to the oxygen uptake rate of the cells according to the desired cell density. A balanced nutrient supply resulted in an optimal formation and yield of products.     

Optimization of the production of the lantibiotic mutacin 1140 in minimal media

Mutacin 1140 is produced by Streptococcus mutans and belongs to the type A lantibiotic family. Experiments were done to optimize production of mutacin 1140 in minimal media enabling a more cost efficient downstream purification method. The development of a small volume fermentation method enabled a rapid screen of several variables in a standard shaking incubator. This method provided a fast approach for determining components that promote mutacin 1140 production in minimal media broth. Lactose was determined to be the optimal carbon source for mutacin 1140 production. High concentrations of CaCl₂ (0.3%, w/v) and MgSO₄ (0.77%, w/v) promoted an increase in mutacin 1140 production, while ZnCl₂ and FeCl₃ appeared to impair production. Optimization of mutacin 1140 production in minimal media resulted in more than a 100-fold increase in production compared to the base medium used to begin our optimizations. The yield has been estimated by RP-HPLC to be ∼10 mg/L.     

TEMPERATURE VARIABILITY WITHIN BACTERIOLOGICAL INCUBATORS

SUMMARY: Temperature variability within four bacteriological incubators regulated for 30° has been studied using readings taken from the normal thermometers and from others placed on the incubator shelves.
During 5-day incubation periods, differences between readings from individual thermometers and between thermometers on different shelves in the same incubator rarely exceeded 0·5° and 1° respectively. The mean temperature recorded on the top shelf was significantly higher ( P <0·05) than that on the bottom shelf for all incubators, and than that indicated by the normal thermometers for two incubators.
Colony counts for pasteurized milk were not materially affected by within incubator temperature differences in this experiment nor, at three of the four centres, by removal from the incubators for brief periods for counting after intermediate incubation times.     

An efficient method of generating transgenic mice by pronuclear microinjection

Genetic transformation of mice using pronuclear microinjection was demonstrated by a number of groups in rapid succession in the early 1980’s. Since that time, studies using transgenic animals have produced major advances in biomedical sciences and molecular genetics. More important, it is possible to study the molecular basis for tissue and stage-specific expression of genes. We recently used this method to generate transgenic mice. DNA fragment (transgene) was injected into the pronucleus of one-cell embryos. We describe this simplified protocol, which is reliable. With the use of buffered medium M2 for the whole process, it is not mandatory to have a CO₂ incubator.     

Culture of fetal mouse liver in plastic chambers: A new technique for organ culture

Summary A new technique for organ culture which uses plastic culture chambers and the advantages of the cellophane-sheet technique is described with the results of a study of cultivations of fetal mouse liver. Two chambers, each containing cells, were placed in gas permeable roller tubes and rotated at 0.1 rpm in a CO₂-air gassed incubator. The fetal mouse liver cells developed electron microscopic features similar to those of the in vivo adult liver by 9 days of cultivation. The albumin content and tyrosine aminotransferase (TAT) activity were detected in the cultivated liver. TAT activity was further induced by prednisolone. These results indicate the potential of this culture method for the study of physiological and pathological processes. This work was supported in part by a Grant-in-Aid for Cancer Research from the Ministry of Education, Science and Culture, Japan and Science Technology Agency, Japan.     

Extremely low frequency (ELF) stray magnetic fields of laboratory equipment: a possible co-exposure conducting experiments on cell cultures

Abstract

Measurements of extremely low frequency (ELF) magnetic fields were conducted in the environment of commercial laboratory equipment in order to evaluate the possible co-exposure during the experimental processes on cell cultures. Three types of device were evaluated: a cell culture CO₂ incubator, a thermostatic water bath and a laboratory shaker table. These devices usually have electric motors, heating wires and electronic control systems, therefore may expose the cell cultures to undesirable ELF stray magnetic fields. Spatial distributions of magnetic field time domain signal waveform and frequency spectral analysis (FFT) were processed. Long- and short-term variation of stray magnetic field was also evaluated under normal use of investigated laboratory devices. The results show that the equipment under test may add a considerable ELF magnetic field to the ambient environmental magnetic field or to the intentional exposure to ELF, RF or other physical/chemical agents. The maximum stray magnetic fields were higher than 3?µT, 20?µT and 75?µT in the CO₂ incubator, in water bath and on the laboratory shaker table, respectively, with high variation of spatial distribution and time domain. Our investigation emphasizes possible confounding factors conducting cell culture studies related to low-level ELF-EMF exposure due to the existing stray magnetic fields in the ambient environment of laboratory equipment.     

Influence of cysteamine supplementation and culture in portable dry-incubator on the in vitro maturation, fertilization and subsequent development of mouse oocytes

The need to transport oocytes and embryos between two laboratories have prompted us to evaluate the effects of in vitro maturation of immature mouse oocytes in a CO2-deficient dry heat portable incubator and subsequent in vitro development of these fertilized mouse oocytes in a standard CO2 incubator. In addition, the effects of cysteamine supplementation on maturation rate and embryonic development during in vitro maturation (IVM) and culture of embryos in the portable incubator were also investigated. Germinal vesicle stage mouse oocytes, recovered at 40-h post-FSH from 6- to 8-week-old C57BL/6xCBA F1 healthy female mice, were matured in vitro in a modified TCM-199 supplemented with or without 100 microM cysteamine in a standard incubator (5% CO2; 37 degrees C) or cultured in a CO2-deficient dry heat portable incubator for 5 h at 37 degrees C and thereafter transferred to a standard incubator for further culture. The addition of cysteamine in the IVM medium significantly improved maturation rates of the GV mouse oocytes to metaphase II stage. However, cysteamine supplementation in the culture medium did not significantly improve fertilization and blastocyst formation rates of IVM and ovulated oocytes, and in vivo-derived zygotes. Culture conditions in a CO2-deficient dry heat portable incubator did not adversely affect the developmental competence of in vivo-derived zygotes and in vitro matured mouse oocytes after IVF or parthenogenetic activation. Cysteamine supplement in the IVM medium could enhance nuclear maturation of these immature oocytes during shipment.     

Enhancement of recombinant erythropoietin production in CHO cells in an incubator without CO2 addition

The effect of low levels of carbon dioxide (CO₂) in the gas phase on the production of recombinant human erythropoietin (EPO)in CHO cells was explored. A T-flask culture in an incubator without CO₂ addition showed a slow cell growth initially followed by the cessation of growth, while other cultures incubated under 0.5–5% CO₂ concentrations grew normally at the same rate during the entire period of cultivation. Interestingly, the production of EPO in the culture incubated under no CO₂ supply was highest among the tested cultures. The cell specific secretion rate of EPO (q_EPO) of the culture under no CO₂ supply was about 3 times higher than that of the culture under 5% CO₂ supply. Western blot analysis and in vivo bioassay of EPO showed no apparent changes in EPO quality between the two cases of different CO₂ environments (air vs. 5% CO₂), suggesting robust glycosylation of EPO by CHO cells even under very reduced CO₂ environment. Various combinations of the two extreme cases, with 5% CO₂ supply (suitable for cell growth) and no CO₂ addition (better for EPO production), were made in order to maximize the volumetric productivity of EPO secretion (P_V) in CHO cells. The P_V of the cultures programmed with initial incubation under 5% CO₂ followed by no CO₂ supply was about 2 times superior to that of the culture incubated only under no CO₂ supply. The P_V of the culture under no CO₂ supply was slightly lower than that of culture grown under 5% CO₂. However, the q_EPO of the no CO₂ supply case was more than 5 times higher than that of the culture under 5% CO₂ supply. In conclusion, we have demonstrated that a simple programming of CO₂ supply to an incubator can enhance the production of EPO in CHO cells remarkably, without any apparent change of the EPO quality. This revised version was published online in August 2006 with corrections to the Cover Date.     

In vitro culture of macrobrachium eggs

Causes for the death of the eggs in the prawn Macrobrachium nobilii are: i) shedding of eggs by ovigerous female, and ii) infection by epibionts: a Saprolegnial fungus, bacteria (gram negative) and protozoans (Vorticellids and Paramecium). A cause for the death of freshly hatched larvae of some decapods is the reduction in reserve yolk energy in the larvae hatched in the last few batches. To circumvent these disadvantages, an artificial incubator was designed, in which 70% of the 3-day old eggs can successfully be incubated and hatched simultaneously. The isolted eggs are irrigated with filtered and aerated water over a diaphragm in the incubator; the water flushed from below through the diaphragm in the artificial incubator, sways and keeps the eggs continuously in a suspended motion, simulating the irrigation technique of the mother.Presented in the Second International Symposium on Invertebrate Reproduction held in Davis, California during August, 1979     

Modulation of glycolysis induction in primary cultures of rabbit kidney proximal tubule cells: the role of shaking,glucose and insulin.

We have assessed the impact of increasing oxygen availability on cellular phenotype expression of rabbit proximal tubule cells in primary culture developed with variable glucose and/or insulin contents. To mitigate hypoxia at the cell/medium interface, cells were shaken for the whole culture duration and their expressed phenotype was compared with those expressed by static cultures. O₂ and CO₂ tensions were kept constant in the incubator atmosphere. Glycolysis and gluconeogenesis pathways, detoxication system, and mitochondrial, apical and basolateral membrane marker enzyme activities were assessed. This study showed that the induction of glycolysis which appear in primary cultures of proximal tubule cells may be partially prevented by continuously shaking the cultures. This effect was more marked in the presence of glucose, suggesting better substrate oxidation in shaken cultures.