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Processes 2013, 1(1), 3-11; doi:10.3390/pr1010003
Published: 26 March 2013
Abstract: We investigated the cultivation of bacterial, mammalian and insect cells in an orbitally-shaken 250-mL disposable tube that incorporates a helical track (HT) on its inside wall. The mass transfer coefﬁcient of oxygen (kLa) was 200%–400% higher in the HT tube than in a shake flask. Bacterial growth and plasmid production were 30% higher in the HT tube than in a 1-L Erlenmeyer flask. Mammalian cell cultures achieved a 25% higher cell density in the HT tube as compared to a 250-mL square-shaped bottle while insect cells grew as well in HT tubes as in 250-mL shake flasks. Because of their performance, disposability, and compact size, we conclude that 250-mL HT tubes are a useful alternative to other shaken containers for the cultivation of bacterial, mammalian and insect cells.
|c*G||interfacial oxygen concentration in the gas phase [M]|
|cG||oxygen concentration in the atmospheric air [M]|
|cL||dissolved concentration of oxygen [M]|
|c*L||dissolved oxygen concentration at saturation [M]|
|kGa||oxygen mass transfer coefﬁcient in the gas phase [h−1]|
|kLa||oxygen mass transfer coefﬁcient in the liquid phase [h−1]|
|OTR||oxygen transfer rate through the gas-liquid interface [M·h−1]|
|OTRplug||oxygen transfer rate through the sterile closure [M·h−1]|
The suspension cultivation of microbes and animal cells is often performed in shaken flasks and bottles because of their ease of use and cost effectiveness. In most cases, however, shaken containers are neither monitored nor controlled for the dissolved oxygen concentration (cL) or pH. Consequently, the culture may become oxygen or pH limited, leading to reduced biomass accumulation [1,2]. In shaken flasks, mass transfer limitations can occur across the sterile closure or at the gas-liquid interface. Mass transfer resistance studies have shown that the closure can, in fact, be the limiting factor in passively aerated flasks [3,4]. In this study we tested a new orbitally shaken container for the cultivation of microbes and animal cells. The cylindrical 250-mL helical-track (HT) tube was developed to improve gas transfer by increasing the gas–liquid interface area. In addition, the tube has a ventilated cap with a membrane filter to improve gas transfer across the sterile closure. Cylindrical HT bioreactors ranging in working volume from 50 mL to 55 L, but not including the 250-mL HT tube described here, have been used for the cultivation of mammalian cells in suspension . Here we determined the volumetric mass transfer coefﬁcient of oxygen (kLa) in a disposable 250-mL HT tube. We also compared the growth of bacterial, mammalian and insect cells in the HT tube to that in conventional shaken flasks and bottles.
2. Experimental Section
2.1. The 250-mL HT Tube and Other Standard Vessels
The 250-mL polypropylene HT tube (kindly provided by Sartorius AG, Göttingen, Germany) was equipped with vented caps having a 0.22-µm polytetrafluoroethylene ﬁlter as a sterile barrier (Thermo Fisher Scientiﬁc, Waltham, MA, USA). The 0.8 cm-wide track started at the bottom of the tube and reached a height of 10.6 cm (Figure 1a). The outside diameter of the tube was 5 cm. The other standard vessels used here included 1-L Erlenmeyer flasks (Bellco Glass, Vineland, NJ, USA) with a stainless steel closure for bacterial cultures (Figure 1b); 250-mL square-shaped (Figure 1c) and cylindrical glass bottles with a screw cap (Figure 1d) (SciLabware, Stone, UK) for mammalian cell cultures; and 250-mL flat-base Erlenmeyer flasks equipped with vented caps (BD Falcon, Franklin Lakes, NJ, USA) for insect cells cultures (Figure 1e).
2.2. Gas Transfer
The volumetric mass transfer coefficient (kLa) was measured at 37 °C using a dynamic method as described . Non-invasive O2 sensors (PreSens, Regensburg, Germany) positioned below the liquid level measured the dissolved oxygen concentration (cL) in a 250-mL HT tube containing 100 mL of deionized water. The tube, without a closure, was agitated at various speeds on an orbital shaker. Nitrogen was flushed into the water until the cL reached about 0% air saturation (air sat). Air was then flushed into the gas phase of the vessel to replace nitrogen, and the cL was recorded over time as the vessel was agitated. The kLa was calculated from Equation (1) based on the change of cL with time. As the vessels were not equipped with closures, the c*L was assumed to be constant and equal to 0.2 mM .
The kGa represents the rate at which oxygen is transferred from the surroundings into the gas phase of the vessel . Thus, the kGa is related to the mass transfer resistance of the closure. The HT tube with 100 mL deionized water was agitated at 200 rpm at 37 °C. It was flushed with nitrogen until the cL reached 0% air sat as measured by non-invasive O2 sensors positioned in the gas phase. The HT tube was then fitted with its closure, and the cG was recorded over time as the container was agitated. Assuming that the gas phase was well mixed, Equation (2) was used to calculate the kGa. The maximal OTR through the sterile closure (OTRplug) was calculated from Equation (2) [6,8].
2.3. Bacterial Cultivation and Plasmid Production
The dual expression vector pXLGCHO-A2 carrying the human anti-Rhesus D IgG1 heavy and light chain cDNAs was constructed as described for pXLGCHO-A3 except that the expression cassettes for the light and heavy chain genes were in a head-to-tail rather than a head-to-head orientation . E. coli DH5α were transformed with pXLGCHO-A2 and grown overnight at 37 °C in a 1-L Erlenmeyer flask in 100 mL of Terrific Broth (TB) (Difco Laboratories, Franklin Lakes, NJ, USA) with 100 mg ampicillin/L (AppliChem, Darmstadt, Germany) with agitation at 200 rpm on an ES-X shaker (Kühner, Birsfelden, Switzerland). The culture was diluted 10 times in TB containing 100 mg ampicillin/L, and aliquots of 100 mL were transferred into 250-mL HT tubes and 1-L Erlenmeyer flasks with a metal closure (Figure 1a,b). The cultures were grown at 37 °C for 7 h. Turbidity was assessed by optical density at 600 nm (OD600) using a spectrophotometer (Eppendorf, Hamburg, Germany). The pH was measured off-line, and the dissolved oxygen concentration cL was measured on-line with non-invasive O2 sensors (PreSens) positioned below the liquid level.
For plasmid production, duplicate cultures of 100 mL were grown overnight in 250-mL HT tubes and 1-L Erlenmeyer flasks. Plasmid was recovered from each culture using a Nucleobond PC 500 maxiprep kit (Macherey-Nagel, Düren, Germany) according to the manufacturer’s protocol. The DNA was resuspended in 500 µL of TE (10 mM Tris-HCl, 1 mM EDTA, pH 7.4), and its concentration was determined with a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientiﬁc).
2.4. Mammalian Cell Cultivation
The DG44 strain of Chinese hamster ovary (CHO-DG44) cells was routinely cultivated in ProCHO5 medium (Lonza, Verviers, Belgium) supplemented with 13.6 mg hypoxanthine/L, 3.9 mg thymidine/L, and 4 mM glutamine. The cells were incubated at 37 °C with 5% CO2 in cylindrical glass bottles of various sizes with agitation at 110 rpm in an ISF4-W incubator (Kühner) . For cell growth studies, 100 mL of culture was transferred at a density of 0.3 × 106 cells/mL into 250-mL cylindrical and square-shaped glass bottles and 250-mL HT tubes. The bottles were agitated at 110 rpm as previously described  and the HT tubes at 160 rpm. The cultures were incubated at 37 °C in the presence of 5% CO2 in an ISF4-W incubator with a shaking diameter of 5 cm. Cell density and viability were determined by the Trypan blue exclusion method. The biomass was determined by the packed cell volume (PCV) method using PCV tubes (TPP, Trasadingen, Switzerland) as described .
2.5. Insect Cell Cultivation
Spodoptera frugiperda Sf-9 insect cells were routinely cultivated in 5 mL of Sf900 II SFM medium (Invitrogen, Carlsbad, CA, USA) at 28 °C in TubeSpin® bioreactor 50 tubes (TubeSpins) (TPP) with agitation at 180 rpm in an ISF4-W incubator as described . For cell growth studies, the cells were inoculated at a density of 0.6 × 106 cells/mL in 250-mL flat-base Erlenmeyer flasks equipped with vented caps and 250-mL HT tubes at working volumes of 50 and 100 mL, respectively. The Erlenmeyer flasks were agitated at 130 rpm with a shaking diameter of 2.5 cm, and the HT tubes at 140 rpm with a shaking diameter of 5 cm. The cultures were incubated at 28 °C in an ISF4-W incubator. The cell density, viability and PCV were assessed as described for mammalian cells.
3. Results and Discussion
3.1. kLa in 250-mL HT Tubes
The kLa was measured in 100 mL of deionized water in 250-mL HT tubes without a cap at agitation rates from 90 to 200 rpm. Over this range, the kLa increased with the agitation rate (Figure 2). At agitation rates used for insect (140 rpm) and mammalian cells (160 rpm), the kLa reached 25 and 50 h−1, respectively (Figure 2). At the agitation rate used for E. coli cultures (200 rpm), the kLa was 90 h−1 (Figure 2).
3.2. Bacterial Cultivation and Plasmid Production
E. coli were cultivated in 1-L Erlenmeyer flasks and 250-mL HT tubes at 200 rpm. During the ﬁrst 4 h of cultivation, the OD600 values were nearly identical in the two containers (Figure 3a). From 5–7 h of cultivation, slightly higher OD600 values were observed in the HT tubes than in the flasks (Figure 3a). Over time, the pH decreased from 7.2 to 6.4 (Figure 3b) and the cL reached about 0% air sat in both cultivation systems (Figure 3c). Within 7 h of cultivation, the cG decreased to 60% air sat in the flask and 90% air sat in the HT tube (Figure 3c). After 4 h of cultivation, the OTR reached a plateau in both vessels (Figure 3d). Plasmid DNA was puriﬁed from overnight cultures. The amount of plasmid DNA and the OD600 were 40% higher for the cultures grown in HT tubes as compared to those grown in flasks (Table 1).
|Table 1. Plasmid DNA production in E. coli.|
|Yield||1-L Erlenmeyer flasks||250-mL HT tubes|
|a Total amount of plasmid||1.9 ± 0.1 mg||2.7 ± 0.3 mg|
|b OD600||21 ± 0.5||29 ± 0.7|
a Recovered from a 100-mL overnight culture using Nucleobond PC 500 maxiprep kits; b Measured in overnight cultures before cell harvest.
It is known that bacterial growth is affected by the gas transfer in the culture vessel, and in the case of aerobic growth of E. coli in shaken cultures, the OTR may become limiting . Here, there was not a difference between cultures in the HT tubes and 1-L Erlenmeyer flasks in terms of the dissolved oxygen concentration at saturation (cL) (Figure 3c). However, the cG and OTR values were higher in the HT tubes than in the flasks (Figure 3c,d). At the agitation rates used for the cell growth, the kLa values were 33 and 90 h−1 in the 1-L Erlenmeyer and in the HT tubes, respectively. These differences translated into greater biomass and higher plasmid production in HT tubes (Figure 3e).
3.3. Cultivation of Mammalian Cells
CHO-DG44 cells were cultivated in 250-mL cylindrical and square-shaped bottles and in 250-mL HT tubes. At the agitation rates used for cell growth, the kLa values were 7, 8, and 50 h−1 in the three vessels, respectively. The cells cultivated in the 250-mL HT tubes achieved the highest density (Figure 4a), but there were not any noticeable differences in cell viability among the three cultures (Figure 4b). The biomass was also the highest in the HT tube (Figure 4c). The pH decreased from 7.1 to 6.5 (cylindrical bottle), 6.6 (square-shaped bottle), and 6.7 (HT tube). While the cL reached 0% air sat in 2 days in the cylindrical bottles, by the end of the other cultures it had only decreased to 10% air sat in the square-shaped bottles and 50% in the HT tubes.
The maximal mammalian cell density was 25% higher in the HT tubes than in the cylindrical and square-shaped bottles (Figure 4a). The OTR was limiting in cylindrical and square-shaped bottles as judged by the cL values mentioned above. Moreover, higher dissolved CO2 concentrations (data not shown) and lower pH values were observed in the bottles as compared to the HT tubes. Altogether, these results showed that the better gas transfer provided in the HT tubes was beneﬁcial for CHO-DG44 cell growth in comparison with orbitally shaken glass bottles.
3.4. Cultivation of Insect Cells
Sf9 cells were cultivated in 250-mL Erlenmeyer flasks and HT tubes at 130 rpm and 140 rpm, respectively. At these shaking speeds the kLa values were 10 and 20 h−1 for the flasks and HT tubes, respectively. Over the course of the cultures, the cell density (Figure 5a) and viability (Figure 5b) were about the same, but the biomass was higher in the Erlenmeyer flasks than in the HT tubes (Figure 5c). In both containers the cL decreased after 2 days of culture from 100% air sat to about 80% air sat, and the pH varied between 6.0 and 6.3 over the course of the culture (data not shown). The dissolved CO2 remained about 1% in both vessels (data not shown). Since the gas transfer did not seem limiting in either the Erlenmeyer flasks or HT tubes, no difference in cell growth in these two containers was expected. However, in the HT tube the working volume was double that in the flask. Thus, for the HT tube relative to the flasks, more and larger cultures can be maintained in an incubator.
The versatility of 250-mL HT tubes may be of interest for the cultivation of bacteria and suspension-adapted animal cells for recombinant protein production . The HT tube showed a higher gas transfer efﬁciency than did shaken flasks and bottles, confirming previous results using other cylindrical orbitally shaken containers with an internal helical track [15,16]. This property was clearly beneﬁcial for bacterial and mammalian cell cultures but not necessarily for those of insect cells. The efficiency of gas transfer in the HT tube is probably due to the greater liquid free-surface area generated by the movement of liquid up the track. The 250-mL HT tube can be used with a working volume of at least 100 mL, it has a small footprint on the shaker, and is disposable. For these reasons, it is an attractive option for the cultivation of microbes and animal cells.
The authors thank Divor Kiseljak and Yashas Rajendra for providing pXLGCHO–A2. We gratefully acknowledge the very considerable support for equipment and consumables by Adolf Kühner AG and Sartorius AG. The work presented here has been partially supported by the KTI Program of the Swiss Economic Ministry and by a grant from the Academy of Finland (decision no. 135820).
Conflict of Interest
The authors declare no conflict of interest.
- Büchs, J. Introduction to advantages and problems of shaken cultures. Biochem. Eng. J. 2001, 7, 91–98, doi:10.1016/S1369-703X(00)00106-6.
- Klöckner, W.; Büchs, J. Advances in shaking technologies. Trends Biotechnol. 2012, 30, 307–314, doi:10.1016/j.tibtech.2012.03.001.
- Schultz, J.S. Cotton closure as aeration barrier in shaken flask fermentations. Appl. Microbiol. 1964, 12, 305–310.
- Mrotzek, C.; Anderlei, T.; Henzler, H.J.; Büchs, J. Mass transfer resistance of sterile plugs in shaking bioreactors. Biochem. Eng. J. 2001, 7, 107–112.
- Zhang, X.; Stettler, M.; Reif, O.; Kocourek, A.; DeJesus, M.; Hacker, D.L.; Wurm, F.M. Shaken helical track bioreactors: Providing oxygen to high-density cultures of mammalian cells at volumes up to 1000 L by surface aeration with air. N. Biotechnol. 2008, 25, 68–75.
- Gupta, A.; Rao, G. A study of oxygen transfer in shake flasks using a non-invasive oxygen sensor. Biotechnol. Bioeng. 2003, 84, 351–358, doi:10.1002/bit.10740.
- Nikakhtari, H.; Hill, G.A. Closure effects on oxygen transfer and aerobic growth in shake flasks. Biotechnol. Bioeng. 2006, 95, 15–21, doi:10.1002/bit.20930.
- Amoabediny, G.; Buchs, J. Modelling and advanced understanding of unsteady-state gas transfer in shaking bioreactors. Biotechnol. Appl. Bioc. 2007, 46, 57–67.
- Rajendra, Y.; Kiseljak, D.; Baldi, L.; Hacker, D.L.; Wurm, F.M. Reduced glutamine concentration improves protein production in growth-arrested CHO-DG44 and HEK-293E cells. Biotechnol. Lett. 2011, 34, 619–626.
- Muller, N.; Girard, P.; Hacker, D.L.; Jordan, M.; Wurm, F.M. Orbital shaker technology for the cultivation of mammalian cells in suspension. Biotechnol. Bioeng. 2005, 89, 400–406.
- Stettler, M.; Zhang, X.; Hacker, D.L.; de Jesus, M.; Wurm, F.M. Novel orbital shake bioreactors for transient production of CHO derived IgGs. Biotechnol. Prog. 2007, 23, 1340–1346, doi:10.1021/bp070219i.
- Xie, Q.; Michel, P.O.; Baldi, L.; Hacker, D.L.; Zhang, X.; Wurm, F.M. TubeSpin bioreactor 50 for the high-density cultivation of Sf-9 insect cells in suspension. Biotechnol. Lett. 2011, 33, 897–902, doi:10.1007/s10529-011-0527-6.
- Krause, M.; Ukkonen, K.; Haataja, T.; Ruottinen, M.; Glumoff, T.; Neubauer, A.; Neubauer, P.; Vasala, A. A novel fed-batch based cultivation method provides high cell-density and improves yield of soluble recombinant proteins in shaken cultures. Microb. Cell Fact. 2010, 9, doi:10.1186/1475-2859-9-11.
- Nettleship, J.E.; Assenberg, R.; Diprose, J.M.; Rahman-Huq, N.; Owens, R.J. Recent advances in the production of proteins in insect and mammalian cells for structural biology. J. Struct. Biol. 2010, 172, 55–65, doi:10.1016/j.jsb.2010.02.006.
- Tissot, S.; Michel, P.O.; Hacker, D.L.; Baldi, L.; de Jesus, M.; Wurm, F.M. k(L)a as a predictor for successful probe-independent mammalian cell bioprocesses in orbitally shaken bioreactors. N. Biotechnol. 2012, 29, 387–394, doi:10.1016/j.nbt.2011.10.010.
- Zhang, H.; Williams-Dalson, W.; Keshavarz-Moore, E.; Shamlou, P.A. Computational-fluid-dynamics (CFD) analysis of mixing and gas-liquid mass transfer in shake flasks. Biotechnol. Appl. Bioc. 2005, 41, 1–8, doi:10.1042/BA20040082.
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