The cultivation of animal cells at controlled dissolved oxygen partial pressure. Reprinted from Biotechnology and Bioengineering Vol. X, Issue 6, Pages 801-814 (1968)

A 3-liter culture vessel has been developed for the growth of animal cells in suspension at controlled pH and dissolved oxygen partial pressure (pO(2)). The culture technique allows metabolically produced CO(2) to be measured; provision can be made to control the dissolved CO(2) partial pressure. In cultures containing a low serum concentration, gas sparging to control pO(2) was found to cause cell damage. This could be prevented by increasing the serum concentration to 10%, or by adding 0.02% of the surface-active polymer Pluronic F68. The growth of mouse LS cells in batch culture without pO(2) control was found to be limited by the availability of oxygen. Maximum viable cell populations were obtained when dissolved pO(2) was controlled at values within the range 40-100 mm Hg.     

Process control during the suspension culture of a human melanoma cell line in a mechanically stirred loop bioreactor

A human melanoma cell line was cultivated for more than 5 months in a serum-free medium without macromolecular growth factors. A mechanically stirred loop bioreactor was used for the culture of the melanoma cells. The tip speed of the marine impellers was 1.5 m s⁻¹. This cell line was able to endure tip speeds of up to 3.5 m s⁻¹ for a few hours without significant cell damage. By using process control it was possible to obtain growth rates and cell numbers close to those found in medium with serum. The pO₂ was controlled at 125 mbar and the pH at 7.15. The signal of an on-line fluorometer, although not caused by the cells, correlates with cell number. The partial pressure of CO₂ in the culture medium and the redox potential of the medium were monitored by on-line sensors.     

The influence of dissolved oxygen partial pressure on the level of various enzymes in mouse LS cells

The intracellular levels of seven enzymes in mouse LS cells growing in suspension culture at controlled dissolved oxygen partial pressures (pO₂) have been measured. During the growth of each culture large fluctuations were observed in the levels of some enzymes, particularly aldolase and cytochrome oxidase. Mean values for the concentration of each enzyme during the growth phase have been calculated. These results are discussed in relation to previous observations made on the growth of mouse LS cells.     

The scale-up of plant cell culture: Engineering considerations

The enormous versatility of plants has continued to provide the impetus for the development of plant tissue culture as a commercial production strategy for secondary metabolites. Unfortunately problems with slow growth rates and low products yields, which are generally non-growth associated and intracellular, have made plant cell culture-based processes, with a few exceptions, economically unrealistic. Recent developments in reactor design and control, elicitor technology, molecular biology, and consumer demand for natural products, are fuelling a renaissance in plant cell culture as a production strategy. In this review we address the engineering consequences of the unique characteristics of plant cells on the scale-up of plant cell culture.Abbreviations a gas-liquid interfacial area per volume - C dissolved oxygen concentration - C^* liquid phase oxygen concentration in equilibrium with the partial pressure of oxygen in the bulk gas phase - K_L overall mass transfer coefficient - k_L liquid film mass transfer coefficient - m_O2 cell maintenance coefficient for oxygen - OTR oxygen transfer rate - OUR oxygen uptake rate - pO₂ partial pressure of oxygen - STR stirred-tank reactor - v.v.m. volume of gas fed per unit operating volume of reactor per minute - X biomass concentration - Y_x/O2 biomass yield coefficient for oxygen - specific growth rate     

Accurate control of oxygen level in cells during culture on silicone rubber membranes with application to stem cell differentiation

Oxygen level in mammalian cell culture is often controlled by placing culture vessels in humidified incubators with a defined gas phase partial pressure of oxygen (pO_2gas). Because the cells are consuming oxygen supplied by diffusion, a difference between pO_2gas and that experienced by the cells (pO_2cell) arises, which is maximal when cells are cultured in vessels with little or no oxygen permeability. Here, we demonstrate theoretically that highly oxygen‐permeable silicone rubber membranes can be used to control pO_2cell during culture of cells in monolayers and aggregates much more accurately and can achieve more rapid transient response following a disturbance than on polystyrene and fluorinated ethylene‐propylene copolymer membranes. Cell attachment on silicone rubber was achieved by physical adsorption of fibronectin or Matrigel. We use these membranes for the differentiation of mouse embryonic stem cells to cardiomyocytes and compare the results with culture on polystyrene or on silicone rubber on top of polystyrene. The fraction of cells that are cardiomyocyte‐like increases with decreasing pO₂ only when using oxygen‐permeable silicone membrane‐based dishs, which contract on silicone rubber but not polystyrene. The high permeability of silicone rubber results in pO_2cell being equal to pO_2gas at the tissue‐membrane interface. This, together with geometric information from histological sections, facilitates development of a model from which the pO₂ distribution within the resulting aggregates is computed. Silicone rubber membranes have significant advantages over polystyrene in controlling pO_2cell, and these results suggest they are a valuable tool for investigating pO₂ effects in many applications, such as stem cell differentiation. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

The supply of oxygen to submerged cultures of BHK 21 cells

Oxygen solution rates were measured in 4, 30, and 100 liter culture vessels, and the oxygen demand of growing BHK 21 cells estimated. This data was used to calculate the minimal sparged air rates necessary to satisfy oxygen demand throughout the cell growth cycle, and in this way adequate oxygen was supplied without the damaging effects of excessive sparging. Comparable results were obtained when oxygen was supplied by this method and when pO₂ was controlled at 80 mmHg, but both cell growth rate and maximum cell density were reduced when pO₂ was controlled at other values.     

Large-scale production of Erythroid Differentiation Factor (EDF) by gene-engineered Chinese hamster ovary (CHO) cells in suspension culture

From Chinese hamster ovary (CHO) cells which were producing Erythroid Differentiation Factor (EDF) in a culture medium, anchorage-independent cells, named as CHO-SPN, were produced by repeated cultivating in a suspension system. The growth time and maximum cell density of the CHO-SPN cells were 48 h and 7.8×10⁵ viable cells/ml. CHO-SPN cells accumulated 8,000 units/ml (corresponding to 4 mg/ml) of EDF in 4d. After 20 cycles of culture, CHO-SPN cells still possessed the same EDF productivity and the same growth kinetics. Furthermore, in an appropriate dissolved oxygen concentration and pH controlled culture system, the growth time and cell density became 24 h and 1×10⁶ viable cells/ml. The critical level of dissolved oxygen for cell growth was 0.015 atm. The maximum oxygen demand was 3.3×10⁻⁹ mole of O₂/ml/min.Fetal bovine serum (FBS) was indispensable for cell growth. However, a FBS-free medium (ASF201) was available for maintenance of the CHO-SPN cells, and EDF production occurred in the same medium.     

The effect of oxygen partial pressure in bioreactors on cell proliferation and subsequent differentiation of somatic embryos of Cyclamen persicum

The effect of the relative oxygen partial pressure (pO₂) in bioreactors on cell proliferation and subsequent differentiation of somatic embryos from suspension cultures of Cyclamen persicum Mill. was investigated. The growth rate of cell line 3738-VIII in growth-regulator containing medium in bioreactors at 5% pO₂ was slightly reduced in comparison to 10% and 20% pO₂. Cultures growing at 40% pO₂ had a lower growth rate, a markedly reduced cell viability and showed a decrease of the medium pH to 3.5. Because a pH-control with a setpoint of 3.3 caused cell death within 4 days, it was assumed, that the reason for the poor cell proliferation and viability in the cultures at 40% pO₂ was an effect of medium acidification rather than of the high O₂ partial pressure. A significantly higher number of germinating embryos was obtained from the cultures grown at 40% pO₂ than from those grown in flasks or in bioreactors at 5%, 10% and 20% pO₂. These results were specific for cell line 3738-VIII. Another cell line, 3736-12, did not show marked differences in cell proliferation, viability, pH or subsequent regeneration of somatic embryos when grown at different O₂ partial pressures. This revised version was published online in June 2006 with corrections to the Cover Date.     

Response to oxygen of diazotrophic Azospirillum brasilense — Arthrobacter giacomelloi mixed batch culture

Azospirillum brasilense and Arthrobacter giacomelloi were grown together in batch culture under different oxygen pressures. The response to oxygen of growth, nitrogenase activity and respiration rate was determined. The two microorganisms were found to be able to coexist all over the range of partial oxygen pressures examined, that is from 0.004–0.20 bar. Nitrogenase activity by mixed culture of A. brasilense and A. giacomelloi always appeared higher than that of A. brasilense pure culture. Low respiratory activity at partial oxygen pressures higher than 0.02 bar by both pure and mixed cultures seemed not to account for the high nitrogenase activity and improved oxygen tolerance of the mixed culture.Abbreviations pO₂ partial oxygen pressure     

Model‐based optimization and pO2 control of fed‐batch Escherichia coli and Saccharomyces cerevisiae cultivation processes

A model‐based approach for optimization and cascade control of dissolved oxygen partial pressure (pO₂) and maximization of biomass in fed‐batch cultivations is presented. The procedure is based on the off‐line model‐based optimization of the optimal feeding rate profiles and the subsequent automatic pO₂ control using a proposed cascade control technique. During the model‐based optimization of the process, feeding rate profiles are optimized with respect to the imposed technological constraints (initial and maximal cultivation volume, cultivation time, feeding rate range, maximal oxygen transfer rate and pO₂ level). The cascade pO₂ control is implemented using activation of cascades for agitation, oxygen enrichment, and correction of the preoptimized feeding rate profiles. The proposed approach is investigated in two typical fed‐batch processes with Escherichia coli and Saccharomyces cerevisiae. The obtained results show that it was possible to achieve sufficiently high biomass levels with respect to the given technological constraints and to improve controllability of the investigated processes.     

Expansion of mesenchymal stem cells under atmospheric carbon dioxide

Stem cells are needed for an increasing number of scientific applications, including both fundamental research and clinical disease treatment. To meet this rising demand, improved expansion methods to generate high quantities of high quality stem cells must be developed. Unfortunately, the bicarbonate buffering system – which relies upon an elevated CO₂ environment – typically used to maintain pH in stem cell cultures introduces several unnecessary limitations in bioreactor systems. In addition to artificially high dissolved CO₂ levels negatively affecting cell growth, but more importantly, the need to sparge CO₂ into the system complicates the ability to control culture parameters. This control is especially important for stem cells, whose behavior and phenotype is highly sensitive to changes in culture conditions such as dissolved oxygen and pH. As a first step, this study developed a buffer to support expansion of mesenchymal stem cells (MSC) under an atmospheric CO₂ environment in static cultures. MSC expanded under atmospheric CO₂ with this buffer achieved equivalent growth rates without adaptation compared to those grown in standard conditions and also maintained a stem cell phenotype, self‐renewal properties, and the ability to differentiate into multiple lineages after expansion. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1298–1306, 2013     

Metabolic-flux analysis of hybridoma cells under oxidative and reductive stress using mass balances

Hybridoma cells were grown at steady state under both reductiveand oxidative stress and the intracellular fluxes weredetermined by mass-balancing techniques. By decreasing the dissolved oxygen pressure (pO₂) in the bioreactor, the reduced formof nicotinamide adenine nucleotide (NADH) was enhanced relativeto the oxidized form (NAD⁺). Oxidative stress, as a resultof which the NAP(P)⁺/NAD(P)H-ratio increases, was generatedby both the enhancement of the pO₂ to 100% air saturationand by the addition of the artificial electron acceptorphenazine methosulphate (PMS) to the culture medium. It wasfound that fluxes of dehydrogenase reactions by which NAD(P)H isproduced decreased under hypoxic conditions. For example, thedegradation rates of arginine, isoleucine, lysine and theglutamate dehydrogenase flux were significantly lower at oxygenlimitation, and increased at higher pO₂ levels and when PMSwas added to the culture medium. In contrast, the prolinesynthesis reaction, which requires NADPH, decreased under PMSstress. The flux of the NADH-requiring lactate dehydrogenase reaction also strongly decreased from 19 to 3,4 pmol/cell/day,under oxygen limitation and under PMS stress, respectively. Thedata show that metabolic-flux balancing can be used to determinehow mammalian respond to oxidative and reduction stress.     

Arterial Levels of Oxygen Stimulate Intimal Hyperplasia in Human Saphenous Veins via a ROS-Dependent Mechanism

Saphenous veins used as arterial grafts are exposed to arterial levels of oxygen partial pressure (pO₂), which are much greater than what they experience in their native environment. The object of this study is to determine the impact of exposing human saphenous veins to arterial pO₂. Saphenous veins and left internal mammary arteries from consenting patients undergoing coronary artery bypass grafting were cultured ex vivo for 2 weeks in the presence of arterial or venous pO₂ using an established organ culture model. Saphenous veins cultured with arterial pO₂ developed intimal hyperplasia as evidenced by 2.8-fold greater intimal area and 5.8-fold increase in cell proliferation compared to those freshly isolated. Saphenous veins cultured at venous pO₂ or internal mammary arteries cultured at arterial pO₂ did not develop intimal hyperplasia. Intimal hyperplasia was accompanied by two markers of elevated reactive oxygen species (ROS): increased dihydroethidium associated fluorescence (4-fold, p<0.05) and increased levels of the lipid peroxidation product, 4-hydroxynonenal (10-fold, p<0.05). A functional role of the increased ROS saphenous veins exposed to arterial pO₂ is suggested by the observation that chronic exposure to tiron, a ROS scavenger, during the two-week culture period, blocked intimal hyperplasia. Electron paramagnetic resonance based oximetry revealed that the pO₂ in the wall of the vessel tracked that of the atmosphere with a ~30 mmHg offset, thus the cells in the vessel wall were directly exposed to variations in pO₂. Monolayer cultures of smooth muscle cells isolated from saphenous veins exhibited increased proliferation when exposed to arterial pO₂ relative to those cultured at venous pO₂. This increased proliferation was blocked by tiron. Taken together, these data suggest that exposure of human SV to arterial pO₂ stimulates IH via a ROS-dependent pathway.     

Desorption of carbon dioxide from fermentation broth

Rates of CO₂ desorption from fermentation broths under actual operating conditions were determined by measuring the CO₂ partial pressure in the exit gas. The concentrations of CO₂ physically dissolved in the broths were measured by the so-called tubing method. Values of k_La for CO₂ desorption calculated from these values agreed well with the k_La values for oxygen absorption corrected for the difference in gas diffusivities. The dissolved CO₂ concentration in the broth, which seems to bean important operating parameter, can easily be estimated from the CO₂ partial pressure in the exit gas, a more easily measurable quantity, if the k_La value is known. For a given value of k_La, assumption of perfect mixing or plug flow in the gas phase made little difference in the calculated values of the dissolved CO₂ concentration, indicating that the gas phase was probably in between perfect mixing and plug flow. In industrial fermentors, the CO₂ partial pressure in the exit gas can practically be assumed to be in equilibrium with the dissolved CO₂ concentration.     

Comparative growth characteristics of vero cells on gas-permeable and conventional supports

We have evaluated the gas-permeable membrane, etched FEP-teflon, and compared it with other cell supports for tissue culture, in particular Falcon plastic and glass. VERO cells grown on FEP-teflon in 5% CO₂, 95% air reach 1.7 times the density/unit surface area as on other cell surfaces. Cells propagated on FEP-teflon tend to form multiple layers and maintain maximum cell density for periods unusually long for VERO. pH regulation of atmospheric pCO₂, to maintain stable medium pH, increases the maximum cell density on Falcon plastic by 20%, and allows growth on FEP-teflon at serum levels 1.0%. Cells on FEP-teflon eventually form multilayers at these low serum levels. Comparison of the cell morphology in confluent sheets on glass and the top layer of multilayers on FEP-teflon show no significant differences in overall morphology. However, the submerged cells in piled-up foci appear denuded of the small microvilli typical of this cell line. The growth characteristics of FEP-teflon appear related to the permeability of this material to oxygen and/or carbon dioxide. We discuss our findings in terms of the influence of pericellular pO₂, pCO₂ and pH on serum requirements and contact inhibition.     

Changes associated with tyrosine phosphorylation during short-term hypoxia in retinal microvascular endothelial cells in vitro

The occlusion of capillary vessels results in low oxygen tension in adjacent tissues which triggers a signaling cascade that culminates in neovascularization. Using bovine retinal capillary endothelial cells (BRCEC), we investigated the effects of short-term hypoxia on DNA synthesis, phosphotyrosine induction, changes in the expression of basic fibroblast growth factor receptor (bFGFR), protein kinase C (PKCα), heat shock protein 70 (HSP70), and SH2-containing protein (SHC). The effect of protein tyrosine kinase (PTK) and phosphatase inhibitors on hypoxia-induced phosphotyrosine was also studied. Capillary endothelial cells cultured in standard normoxic (pO₂ = 20%) conditions were quiesced in low serum containing medium and then exposed to low oxygen tension or hypoxia (pO₂ = 3%) in humidified, 5% CO₂, 37°C, tissue culture chambers, on a time-course of up to 24 h. DNA synthesis was potentiated by hypoxia in a time-dependent manner. This response positively correlated with the cumulative induction of phosphotyrosine and the downregulation of bFGFR (M_r ～ 85 kDa). Protein tyrosine kinase inhibitors, herbimycin-A, and methyl 2,5-dihydroxycinnamate, unlike genistein, markedly blocked hypoxia-induced phosphotyrosine. Prolonged exposure of cells to phosphatase inhibitor, sodium orthovanadate, also blocked hypoxia-induced phosphotyrosine. The expression of HSP70, PKCα, and SHC were not markedly altered by hypoxia. Taken together, these data suggest that short-term hypoxia activates endothelial cell proliferation in part via tyrosine phosphorylation of cellular proteins and changes in the expression of the FGF receptor. Thus, endothelial cell mitogenesis and neovascularization associated with low oxygen tension may be controlled by abrogating signaling pathways mediated by protein tyrosine kinase and phosphatases. © 1995 Wiley-Liss, Inc.     

CO2 in large-scale and high-density CHO cell perfusion culture

Productivity in a CHO perfusion culture reactor was maximized when pCO₂ was maintained in the range of 30–76 mm Hg. Higher levels of pCO₂ (> 150 mm Hg) resulted in CHO cell growth inhibition and dramatic reduction in productivity. We measured the oxygen utilization and CO₂ production rates for CHO cells in perfusion culture at 5.55×10^-17 mol cell^-1 sec^-1 and 5.36×10^-17 mol cell^-1 sec^-1 respectively. A simple method to directly measure the mass transfer coefficients for oxygen and carbon dioxide was also developed. For a 500 L bioreactor using pure oxygen sparge at 0.002 VVM from a microporous frit sparger, the overall apparent transfer rates (k_La+k_AA) for oxygen and carbon dioxide were 0.07264 min^-1 and 0.002962 min^-1 respectively. Thus, while a very low flow rate of pure oxygen microbubbles would be adequate to meet oxygen supply requirements for up to 2.1×10⁷ cells/mL, the low CO₂ removal efficiency would limit culture density to only 2.4×10⁶ cells/mL. An additional model was developed to predict the effect of bubble size on oxygen and CO₂ transfer rates. If pure oxygen is used in both the headspace and sparge, then the sparging rate can be minimized by the use of bubbles in the size range of 2–3 mm. For bubbles in this size range, the ratio of oxygen supply to carbon dioxide removal rates is matched to the ratio of metabolic oxygen utilization and carbon dioxide generation rates. Using this strategy in the 500 L reactor, we predict that dissolved oxygen and CO₂ levels can be maintained in the range to support maximum productivity (40% DO, 76 mm Hg pCO₂) for a culture at 10⁷ cells/mL, and with a minimum sparge rate of 0.006 vessel volumes per minute.A = volumetric agitated gas-liquid interfacial area at the top of the liquid, 1/mB = cell broth bleeding rate from the vessel, L/minCER = carbon dioxide evolution rate in the bioreactor, mol/min[CO₂] = dissolved CO₂ concentration in liquid, M[CO₂]^* = CO₂ concentration in equilibrium with sparger gas, M[CO₂]^** = CO₂ concentration in equilibrium with headspace gas, MCO₂(1) = dissolved carbon dioxide molecule in water[C_T] = total carbonic species concentration in bioreactor medium, M[C_T]_F = total carbonic species concentration in feed medium, MD = bioreactor diameter, mD_I = impeller diameter, mD_b = the initial delivered bubble diameter, mF = fresh medium feeding rate, L/minH_L = liquid height in the vessel, mk_A = carbon dioxide transfer coefficient at liquid surface, m/mink _infA ^supO = oxygen transfer coefficient at liquid surface, m/minNomenclature     

Carbon dioxide inhibition of yeast growth in biomass production

Saccharomyces cerevisiae was grown under aerobic and substrate-limiting conditions for efficient biomass production. Under these conditions, where the sugar substrate was fed incrementally, the growth pattern of the yeast cells was found to be uniform, as indicated by a constant respiratory quotient during the entire growing period. The effect of carbon dioxide was investigated by replacing portions of the nitrogen in the air stream with carbon dioxide, while maintaining the oxygen content at the normal 20% level, so that identical oxygen transfer rate and atmospheric pressure were maintained for all experiments with different partial pressures of carbon dioxide. Inhibition of yeast growth was negligible below 20% CO₂ in the aeration mixture. Slight inhibition was noted at the 40% CO₂ level and significant inhibition was noted above the 50% CO₂, level, corresponding to 1.6 × 10^?2M of dissolved CO₂ in the fermentor broth. High carbon dioxide content in the gas phase also inhibited the fermentation activity of baker's yeast.     

Dissolved carbon dioxide accumulation in a large scale and high density production of TGFβ receptor with baculovirus infected Sf-9 cells

Production of a TGF receptor with high density baculovirus infected Sf-9 cells (7×10⁶cells ml^-1) served as a test run for a retrofitted 150 L microbial fermentor. The entire 110 L batch run was performed in serum free medium, with an addition of a concentrated amino acid and yeastolate mixture at the time of infection. This addition strategy has been proven effective at a small scale by enabling cultures to maintain maximum product yield. In the bioreactor however, while cellular growth was comparable to that of the smaller scale control, TGF receptor production was three fold below the control. To minimize the mechanical stress, low flow rate of pure oxygen was used to control the dissolved oxygen at 40%. As a consequence, it seems that this aeration strategy involved an accumulation of dissolved carbon dioxide that in turn inhibited the protein production. A model has been developed that estimated the CO₂ partial pressure in the culture to be in the vicinity of 0.15 atm. The effect of dissolved CO₂ at this concentration has been assessed at smaller scale for TGF receptor and -gal expression, in controlled atmosphere incubators.Abbreviations CST cumulative sparging time (s) - dpi days post-infection (d) - CO₂/O₂ diffusivities ratio (=0.784 at 27 °C) - DO % saturation of dissolved oxygen (%) - hpi hours post-infection (h) - He Henry coefficient (He_{CO 2}=32.1×10^-3M atm^-1, He_{O 2}=1.23×10^-3 M atm^-1 at 27 °C) - k_La volumetric liquid-side mass transfer coefficient (h^-1) - LDH Lactate dehydrogenase - MOI multiplicity of infection (pfu cell^-1) - N agitation speed (rpm) - OTR oxygen transfer rate (mole O₂ ml^-1 h^-1) - OUR oxygen uptake rate (mole O₂ ml^-1 h^-1) - p partial pressure (atm) - P total pressure (atm) - pfu plaque forming units - q specific consumption or production rate (mole cell^-1 h^-1) - Q_{H, OUT} headspace outlet gas flow rate - Q_S,NOM nominal volumetric sparged gas flow rate (92 ml s^-1 at bioreactor conditions) - R ideal gas constant (82.05 ml atm mole^-1 K^-1) - SR_{V L} molar sparging rate per unit liquid volume (mole ml^-1 h^-1) - SSR specific sparging rate (mole cell^-1 h^-1) - T temperature (C or K) - V_L culture volume (ml) - VVD volume of feed per volume of culture per day (d^-1) - X cell concentration (cell ml^-1) - Y yield coefficient Indexes CO₂, O₂ related to CO₂ or O₂, respectively - glc glucose - H headspace gas phase or gas/liquid interface - L liquid phase - lact lactate - S sparged phase or gas/liquid interface - V viable     

Effects of various partial pressures of oxygen and carbon dioxide on different stages of somatic embryogenesis in Picea abies

Effects of various partial pressures of oxygen (5, 20 and 45 kPa) and carbon dioxide (0.03 and 6 kPa) on initiation, proliferation and maturation of somatic embryos in Picea abies were studied. The pO₂ had a significant effect on the initiation of embryogenic tissue from mature zygotic embryos. However, the effect of pO₂ was dependent on the strength of the basal medium. Low pO₂ stimulated the formation of embryogenic tissue when the zygotic embryos were incubated on full strength medium, but was inhibitory when half-strength medium was used. Proliferation of embryogenic tissue was stimulated by higher partial pressures of both CO₂ and O₂. The effect of the gas phase on maturation of somatic embryos varied between different cell lines. However, there was a general tendency for 5 kPa O₂ and 6 kPa CO₂ to stimulate maturation.Abbreviations ABA abscisic acid - BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - ET embryogenic tissue