Effects of incubation in an atmosphere of 20% CO2 in air on the syrian hamster embryo clonal transformation assay

Summary An atmosphere containing 10% CO₂ has been generally accepted as optimal for the growth of Syrian hamster embryo cells in a clonal transformation assay. Data presented in this paper show that 10% CO₂ may not be the optimum environment for this assay. Using 10 or 20% (analytically measured) CO₂ in air (1 atm pressure), hamster embryo cell pools were examined for clonal growth characteristics and transformability using five known carcinogens and a single noncarcinogenic compound. At 10% CO₂, only 2 of 11 pools weee transformed by the five carcinogens but not by the noncarcinogen. At 20% CO₂, six of seven pools were transformed by the five carcinogens and not by the noncarcinogen. Further, the transformation frequencies were found to be greater in cultures incubated in an atmosphere consisting of 20% CO₂ in air. The data also show that 20% CO₂ increased the cloning efficiency of these cells. A comparison of the 10 and 20% CO₂ data to results reported from other conflicting interlaboratory results with this assay system may be due, in part, to variations of CO₂ concentrations. In some instances, the CO₂ levels indicated by incubator flow meters vary considerably from analytically determined CO₂ values. To prevent these CO₂ discrepancies and their resultant effects on transformation and cloning efficiency, methods for monitoring the CO₂ environment other than flow meters are recommended. The observation of increased cloning efficiencies and transformation rates strongly suggests that culture incubation at 20% CO₂ is a preferred environment for the conduct of this assay.     

Bcl‐xL overexpression does not enhance specific erythropoietin productivity of recombinant CHO cells grown at 33°C and 37°C

Overexpression of bcl‐xL in recombinant Chinese hamster ovary (rCHO) cells has been known to suppress apoptotic cell death and thereby extend culture longevity during batch culture. However, its effect on specific productivity (q) of rCHO cells is controversial. This study attempts to investigate the effect of bcl‐xL overexpression on q of rCHO cells producing erythropoietin (EPO). To regulate the bcl‐xL expression level, the Tet‐off system was introduced in rCHO cells producing EPO (EPO‐off‐bcl‐xL). The bcl‐xL expression level was tightly controlled by doxycycline concentration. To evaluate the effect of bcl‐xL overexpression on specific EPO productivity (q_EPO) at different levels, EPO‐off‐bcl‐xL cells were cultivated at the two different culture temperatures, 33°C and 37°C. The q_EPO at 33°C and 37°C in the presence of 100 ng/mL doxycycline (without bcl‐xL overexpression) were 4.89 ± 0.21 and 3.18 ± 0.06 μg/10⁶cells/day, respectively. In the absence of doxycycline, bcl‐xL overexpression did not affect q_EPO significantly, regardless of the culture temperature, though it extended the culture longevity. Taken together, bcl‐xL overexpression showed no significant effect on the q_EPO of rCHO cells grown at 33°C and 37°C. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

The effect of CO2 ventilation on kinetics and yields of cell-mass and ethanol in batch cultures ofZymomonas mobilis

Summary The effect of CO₂ removal by continuous sparging of N₂ in batch cultures ofZymomonas mobilis (ATCC10988) was examined. N₂ sparging considerably reduces lag times in batch cultures, possibly because of continuous removal of CO₂ from the culture media. Ventilation of CO₂ from culture media results in an increase of about 15% in the average specific growth rate and about 12% in the cell-mass yield with no noticeable trend in the average specific glucose uptake and ethanol production rates. The overall ethanol yield on glucose, however, decreases slightly by 5%. Analysis of ventilated experiments show that the CO₂ production is directly coupled with the ethanol formation but not necessarily with the cell-mass production, indicating a decoupling of growth from ethanol production. Further, comparison of ventilated and non-ventilated experiments rules out the possibility of CO₂ accumulation in the culture media as a factor responsible for increasing growth inhibition and decoupling of growth from ethanol fermentation at increasing initial glucose concentrations in batch cultures.     

Characterization of site-specific recombination mediated by Cre recombinase during the development of erythropoietin producing CHO cell lines

The establishment of erythropoietin (EPO) producing Chinese hamster ovary (CHO) cell lines was conducted using Cre-mediated cassette exchange. The characterization of site-specific recombination mediated by Cre-recombinase during the cell line development was also performed. A total of six parental clones, which had various green fluorescence levels ranging from high to low and containing a single copy of insertion vector (pEGFP-m2), were screened. The EPO targeting vector (pIC-m2-EPO) was targeted into the 6 parental clones by Cre-mediated cassette exchange. Correctly targeted clones were obtained from 4 out of 6 parental clones with 0∼15% of targeting efficiencies. Moreover, there was a positive relationship (R² = 0.87) between fluorescence levels of the parental clones before Cre-mediated cassette exchange and specific EPO productivities (q _EPO ) of the correctly targeted clones after Cre-mediated cassette exchange. Therefore, it was verified that the chromosomal loci’s characteristic gene expression level was not modified even after cassette exchange mediated by Cre recombinase during the development of EPO producing CHO cell lines. This finding implies that the reproducible development of CHO cell lines largely producing a desired protein is expected to be achieved by Cre-mediated cassette exchange.     

Bcl-x(L) overexpression delays the onset of autophagy and apoptosis in hyperosmotic recombinant Chinese hamster ovary cell cultures

Hyperosmolality in recombinant Chinese hamster ovary (rCHO) cell cultures induces autophagy and apoptosis. To investigate the effect of Bcl-x_L overexpression on autophagy and apoptosis in hyperosmotic rCHO cell cultures, an erythropoietin (EPO)-producing rCHO cell line with regulated Bcl-x_L overexpression was subjected to hyperosmolality resulting from NaCl addition in a batch culture and nutrient supplementation in a fed-batch culture. In the batch culture, Bcl-x_L overexpression suppressed apoptosis, as evidenced by a decreased amount of cleaved caspase-7 and PARP. Concurrently, Bcl-x_L overexpression also delayed autophagy, as indicated by reduced LC3 conversion, from LC3-I to LC3-II. As a result, the cell viability and EPO production were improved by Bcl-x_L overexpression. In the fed-batch culture, the simultaneous application of Bcl-x_L overexpression and nutrient feeding increased the culture longevity and maximum EPO concentration. Taken together, Bcl-x_L overexpression delayed autophagy and apoptosis in hyperosmotic rCHO cell cultures, resulting in increased EPO production.     

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.     

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.     

Innovative development of membrane sparger for carbon dioxide supply in microalgae cultures

The present study was aimed to develop a membrane sparger (MS) integrated into a tubular photobioreactor to promote the increase of the carbon dioxide (CO₂) fixation by Spirulina sp. LEB 18 cultures. The use of MS for the CO₂ supply in Spirulina cultures resulted not only in the increase of DIC concentrations but also in the highest accumulated DIC concentration in the liquid medium (127.4 mg L⁻¹ d⁻¹). The highest values of biomass concentration (1.98 g L⁻¹), biomass productivity (131.8 mg L⁻¹ d⁻¹), carbon in biomass (47.9% w w⁻¹), CO₂ fixation rate (231.6 mg L⁻¹ d⁻¹), and CO₂ use efficiency (80.5% w w⁻¹) by Spirulina were verified with MS, compared to the culture with conventional sparger for CO₂ supply. Spirulina biomass in both culture conditions had high protein contents varying from 64.9 to 69% (w w⁻¹). MS can be considered an innovative system for the supply of carbon for the microalgae cultivation and biomass production. Moreover, the use of membrane system might contribute to increased process efficiency with a reduced cost of biomass production.     

Productivity enhancement of recombinant protein in CHO cells via specific promoter activation by oncogenes

To construct a recombinant protein highly producing cell lines, we have previously developed the Oncogene Activated Production (OAP) system by using BHK-21 cells. Here we verified the availability of the OAP system in CHO cells. We firstly generated ‘primed’ ras amplified CHO cells, ras clone I, by introducing human c-Ha-ras oncogene into CHO cells. This ras clone I enables quick and easy establishment of recombinant protein hyper producing cell lines by introduction reporter gene of interest. Then we generated I13 by introducing human interleukin 6 (hIL-6) gene as a reporter gene, which showed enhanced productivity rate as compared to A7 established by conventional method. Furthermore, we found that hIL-6 production level of I13 was slightly improved by raising the CO₂ concentration from 5 to 8% possibly because of the enhanced growth rate. We further introduced the E1A oncogene, which has been shown to have a synergistic effect on the recombinant protein production of the ras-amplified BHK-21 cells, then evaluated the productivity. When culture in 5% CO₂ condition, only the slight effect can be seen. However when cultured in 8% CO₂ condition, not only cell number, but also productivity increased significantly, resulted in great augmentation of hIL-6 production, maximum production being 88.6 μg/ml/3 days. This study demonstrates that recombinant protein production level reached commercially desirable level by utilizing our OAP system in CHO cells and optimizing the culture condition. This revised version was published online in August 2006 with corrections to the Cover Date.     

CO2 Dynamics and Growth in Photoautotrophic and Photomixotrophic Apple Cultures

The daily dynamics of CO₂ concentration in the culture vessels and the photoautotrophic or photomixotrophic growth capacity of apple (Malus pumila hybrid MM 106 paradisiaca× Northern Spy) cultures were studied. The photoautotrophic cultures were grown on a sugar-free growth medium and submitted (0S+CO₂) or not (0S-CO₂) to periodic injections of exogenous CO₂. The photomixotrophic cultures were grown in the presence of 30 g dm⁻³ sucrose, with (30S+CO₂) or without (30S-CO₂) CO₂ enrichment. The photosynthetic photon flux density applied was of 210 ± 5 μmol m⁻²s⁻¹. In the 0S-CO₂ treatment, CO₂ showed rather uniform and narrow light-dark fluctuations throughout the culturing cycle. In the 30S-CO₂ treatment, the daily ratio between CO₂ produced during the dark period and that uptaken during the following light period, was almost always above 1 with the only exception of a few days (from the 5^th to the 9^th day) when the amount of photosynthesised CO₂ was equal to or higher than that produced during dark respiration. The 0S+CO₂ cultures needed to be enriched all days with exogenous CO₂ to avoid periods of gas deficiency while in 30S+CO₂ the CO₂ injected the first culturing day was uptaken over 5 d; thereafter, daily injections were necessary. Culture fresh and dry mass, number of newly formed shoots and number of nodes per shoot in 0S+CO₂ treatment did not statistically differ from the values obtained with 30S−CO₂. The highest growth was observed in 30S+CO₂ treatment. The increase in culture fresh mass due to 1 μmol of CO₂ added to the culture vessels was 1.54 and 1.36 mg for 30S and 0S respectively, while in terms of dry mass the increase was about 2.5 times higher in the sugar-enriched treatment. CO₂ enrichment accounted for 77.3 % and 21.2 % of the final fresh mass in 0S+CO₂ and 30S+CO₂, respectively. This revised version was published online in July 2006 with corrections to the Cover Date.     

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.     

Effect of carbon dioxide on cell growth and saponin production in suspension cultures of Panax ginseng

The effects of carbon dioxide supply within the range of 1–5 % (along with purified air), on cell culture of Panax ginseng were investigated in a balloon type bubble bioreactor containing 4 dm³ of Murashige and Skoog (MS) medium supplemented with 7.0 mg dm⁻³ indolebutyric acid, 0.5 mg dm⁻³ kinetin and 30 g dm⁻³ sucrose. A 1 % CO₂ supply was found beneficial for the production of cell mass; however, increasing CO₂ concentration to 2.5 and 5 % decreased the biomass accumulation. CO₂ enrichment was not beneficial for saponin production and 1, 2.5, and 5 % CO₂ supply resulted in decrease in saponin accumulation up to 11.6, 19.5, and 50.6 %, respectively.     

Carbon monoxide-dependent evolution of hydrogen by the homoacetate-fermenting bacteriumClostridium thermoaceticum

Clostridium thermoaceticum was found to form H₂ when cultivated heterotrophically on dextrose under a carbon monoxide (CO) gas phase. In contrast, when cultivated under CO₂, only minimal levels of hydrogen were detected. Resting cells from the CO-grown cultures also formed H₂ when incubated under CO with dextrose, while a comparative study with resting cells from CO₂-grown cultures demonstrated that the CO₂-grown cells were not competent in H₂ formation when incubated under CO. When dextrose was deleted, CO-cultivated resting cells did not form H₂ when incubated under CO.     

Activities of Ribulose Bisphosphate Carboxylase and Phosphoenolpyruvate Carboxylase and C-Bicarbonate Fixation during in Vitro Culture of Pinus radiata Cotyledons

The activities of ribulose bisphosphate carboxylase (RuBPC) and phosphoenolpyruvate carboxylase (PEPC), as indicators of autotrophic and nonautotrophic CO₂ fixation, were measured in excised cotyledons of Pinus radiata D. Don cultured for 21 days under shoot-forming (SF) and nonshoot-forming (NSF) conditions. The activity of RuBPC was found to increase in both SF and NSF cultures during the initial 5 days of culture. However, it leveled off from day 5 to day 10 and subsequently began to decrease until the end of the culture period under the SF conditions. In contrast, in the NSF cultures, RuBPC activity increased until day 15, when it was twofold higher than the maximum activity found in the SF cultures. An increase in PEPC activity of about 2.5 times the level of activity in freshly excised cotyledons was observed during the initial 5 days of culture under the SF conditions. PEPC activity began to decline after day 5 until it reached the level of activity seen in NSF cotyledons by day 15. In contrast, the activity of PEPC did not show any significant increase during the initial 10 days of culture under the NSF conditions. The K_m (phosphoenolpyruvate) of PEPC from SF cotyledons was about 35% higher than that of NSF cotyledons. Cotyledons from two culture periods (days 5 and 15) were incubated for 15 seconds with NaH¹⁴CO₃. The label in the malate and asparatate fractions as a percentage of total ¹⁴C incorporation was 3 times higher in the SF cotyledons than in the NSF cotyledons. A higher incorporation of ¹⁴C into products of photosynthesis under the NSF conditions was also observed.     

Ribulose-1,5-bisphosphate carboxylase and phosphoenolpyruvate carboxylase activities of photoautotrophic callus of Platycerium coronarium (Koenig ex O.F. Muell.) Desv. under CO2 enrichment

The in vitro activities of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and phosphoenolpyruvate carboxylase (PEPC) were measured in cell-free extracts of Platycerium coronarium callus cultured for up to 42 days under photoautotrophic conditions with CO₂ enrichment. With an increase in CO₂ in the culture environment to 10% (v/v) at low light, the apparent photoautotrophic fixation of CO₂ by Rubisco declined, whereas the non-photoautotrophic CO₂ fixation by PEPC activity was enhanced. Hence, photosynthesis appears to play a lesser role in providing carbon skeletons and energy with prolonged culture in a CO₂-enriched environment. Instead, the anaplerotic supply of C-skeletons by PEPC may be important under such a situation. Short-term H¹⁴CO₃-fixation experiments indicated that photoautotrophic callus cultured for 3 weeks with 10% CO₂ enrichment assimilated less ¹⁴CO₂ than the control (0.03% CO₂). Analyses of ¹⁴C-metabolites indicated that about 50% of the total soluble ¹⁴CO₂ fixed was in the organic acid fraction and 35% in the amino acid fraction. Despite the changes in the in vitro Rubisco/PEPC activity-ratio, no significant change in the ¹⁴C distribution pattern was apparent in response to increasing sucrose or CO₂ concentrations. The suppression of Rubisco activity and total chlorophyll content in high sucrose or elevated CO₂ concentrations suggests an inhibition of the capacity for photoautotrophic callus growth under these conditions. This revised version was published online in June 2006 with corrections to the Cover Date.     

OBSERVATIONS ON CELL DEVELOPMENT IN CHLAMYDOMONAS SEGNIS (CHLOROPHYCEAE) AT LOW AND HIGH CARBON DIOXIDE TENSION1

Some cell cycle events were compared in Chlamydomonas segnis Ettl during its development in synchronous cultures (12:12 LD) supplied with air and air enriched with 5% CO₂. In cultures bubbled with air, growth resulted in production of 2 relatively small zoospores. In cultures provided with 5% CO₂, 4 large zoospores were formed but not released in darkness unless the cultures were bubbled with CO₂-free air and/or exposed to light. Respiration in zoospores was inhibited by high CO₂ tension. In cultures maintained under continuous illumination for one cell cycle, provision of 5% CO₂ led to enhanced growth, a relatively long S-phase and a 4 h delay of the second cell division. In such cultures, the DNA content of parental cells (12 h L) was insufficient to support two cell divisions. The RNA/DNA ratio of the resulting zoospores was 10 compared to 4 in air cultures. These results provided evidence that the delay of the second cell division was a consequence of the delay in DNA production.     

Production of biomass and recombinant human-like collagen in <Emphasis Type="Italic">Escherichia coli</Emphasis> processes with different CO<Subscript>2</Subscript> pulses

The evolution of CO₂ in a fed-batch culture of recombinant Escherichia coli containing human-like collagen (HLC) cDNA was determined with an O₂-enriched air supply (40%, v/v) in a 12.8 l fermentor; a maximum CO₂ concentration of 12.7% in the effluent gas was detected. The CO₂ pulse injection experiments showed that: (1) a 20% CO₂ pulse introduced in the batch cultivation phases inhibited cell growth but if introduced in the fed-batch cultivation phases slightly stimulated growth; and (2) CO₂ inhibited HLC expression only in the expression phase, where the final HLC concentration decreased by 34% under a 3 h 20% CO₂ pulse. The higher the CO₂ concentration and/or the longer the duration of the CO₂ pulse, the stronger the stimulatory or inhibitory effects. An erratum to this article can be found at