High density and scale-up cultivation of recombinant Chinese Hamster Ovary cells (CHO) using microcarrier-based stirred bioreactor.
Mammalian cells are the preferred expression system for making recombinant proteins for human use such as vaccines, hormones, and antibodies. The majority of animal cells are anchorage-dependent and require attachment to a surface for their survival and replication (1). Microcarriers technology is replacing conventional monolayer cell culture methods as the extremely supporting all anchorage dependent animals cells to grow in suspension cultures, and is used in both batch and perfusion culture systems. Microcarriers materials may be composed by natural polymers and their derivative such as glutin, collagen, fibrin, chitin, dextran or cellulose (2-3). Microcarrier techniques are extensively applied in cell culture are used for the production of viruses and other biologicals products (4-5). Microcarriers have also been investigated to culture different cell-lines such CHO, and Vero cells used for production of recombinant therapeutics (6-7). The purpose of the present study was to evaluate the performance of CHO cells culture in a Cytodex1 microcarrier using spinner flasks bioreactors.
Materials and methods
The mammalian cell-line CHO (Chinese Hamster Ovary) cells form biotechnology center-EIPICO-Egypt was used in this study.
Culture medium and chemicals
Dulbecco's Modified Eagle's medium (DMEM), RPMI 1640, Minimum Essential Medium (MEM), Ffetal bovine serum, glutamine, streptomycin, penicillin, gentamycin and tyrosine were from (Sigma-Aldrich). Cyrodoex1 microcarrier was from (GE-Healthcare)
Microcarriers were prepared according to the manufacturers instructions. Dry microcarriers were hydrated in phosphate-buffered saline. After three washings in [Ca+.sub.2] and [Mg+.sub.2] free PBS, the microcarriers were autoclaved at 121[degrees]C for 30 min and stored at 4[degrees]C. Sterile, hydrated microcarriers were then dispensed into spinner flask containing growth medium to give a final concentration of 5 mg/ml.
The viability and concentration of cells in suspension were measured by using the Trypan blue dye exclusion method and the adhered cells were quantified by counting the stained nuclei with crystal violet. Daily counts was taken until culture reached stationary phase.
Two to three drops of the microcarriers culture were transferred into a multiwell plate, and the cells were stained with 5 drops of the crystal violet reagent for 3 min. After washing three times with 2-ml portions of PBS, the stained cells on the microcarriers were examined with a microscope.
Thaw from cryopreservation
Froze vials of CHO cells were thawed and placed into pre-warmed media and 2mM L-glutamine. Viable cell concentration was determined via trypan blue exclusion and dilutions made to seed T-Flask and spinner flask.
Preparation of seed cells
CHO cells were inoculated in 25cm T-Flask then the cells from this flak were seeded into 175cm T-flask. During cell passage, confluent monolayer cells were washed with PBS containing 0.02% EDTA and were then digested to a suspension of single cell using a 0.02% trypsin solution.
Cell cultivation in spinner flasks bioreactor
Sterile spinner flask (Bellco-Biotechnology) containing medium was placed in an incubator for 2 hours prior to cultivation so that the temperature and pH of the medium were equilibrated at 37C and 7.0 respectively, to eliminate the possibility that cell attachedment rate would be influenced sharply by the increase and decrease in pH and temperature.
After attaining a sufficient quantity of cells in culture T flasks (Greiner bio one), 70 mL of culture medium, equilibrated with a predefined amount of microcarrier in a 500 mL spinner flask were inoculated with 105 cel/mL. For the first six hours, in order to achieve a uniform and efficient cell attachment, the culture was performed with only 1/3 of the final volume and with intermittent agitation (5 minutes of stirring at 25-30 rpm every 30 minutes). After cell attachment, the volume of culture was made up to 200 mL and the agitation was kept constant at 60 rpm until the end of the experiment. Medium replacement was carried out according to the requirements of each individual experiment; as soon as the pH of the medium decreased to approximately 7.1, 50% of its working volume was replaced by fresh medium. For cell adhesion measurements, samples were taken from the spinner flask every 1 h approximately over the first 6 h of culture. Samples for quantification of cell density and viability were taken daily.
Cell culture techniques have become vital to the study of animal cell structure and function and for the production of many important biological materials such as vaccines, hormones, antibodies, enzymes, and nucleic acids. Different methods of mass cultivation of mammalian cells have been devised to meet the growing demand for these cells. Microcarrier techniques becomes the natural choice where cells are used for the production of biologicals products.
Effect of culture media
CHO cells were cultured on microcarriers in either DMEM, RPMI or MEM. As shown in figure, culture of CHO in DMEM has generated the maximum cell concentration which was [1.7 x 10.sup.6] cell/ml. This is followed by culture of CHO in RPMI media which has yield [1.3 x 10.sup.6] cell/ml, and MEM 1 x [10.sup.6]. Growth of CHO cells in DMEM was in lag phase from 0h until 40h, the culture entered exponential phase and reached the maximum cell concentration within 6 days. Once reached the maximum, cell concentration dropped continuously until the end of the culture. Again culture of CHO cells in DMEM had achieved the best result. Based on these results, DMEM was selected to be used in further stages of the study because it has the best performance in terms of maximum cell concentration yielded.
[FIGURE 1 OMITTED]
Cell growth on spinner flask
CHO-cell line was cultured for 10days in a spinner flask culture system with DMEM/FBS.
Figure 2 shows atypical growth curves of CHO cells in the microcarrier system, demonstrating that there was no drop in cell density below the starting cell number during Lag phase (first 24 hours in culture) which often occurs when culture initiation conditions are not optimal. Lag time before logarithmic phase growth about 24h followed by an exponential phase leading to maximal cell number at day 7. The rate of growth and doubling time was 17-18h until day 8, when the growth rates was considerably diminished, remaining at the low level during. In addition the final cell density was [2.5x 10.sup.6] cell/ml by day 8.
[FIGURE 2 OMITTED]
Effect on cell viability
Viable cell densities obtained are represented in figure 3. It is possible to conclude that microcarrier was able to support CHO-cell line expansion. Throughout the experiments, the cell viabilities were always above 90%, and the fraction of the cells found in suspension was always less than 5%. Visual examination of microcarrier culture showed healthy cells that remained firmly attached on the bead surface. Cell attached occurred within a few hours, and cells were uniformly distributed over the microcarrier surface and complete monolayer was formed on at least 95% of the microcarrier. Some cell detachment was observed in cultures by the 8th day.
[FIGURE 3 OMITTED]
Effect of the buffer used with DMEM
CHO cells were cultured on microcarriers in DEME containing either sodium bicarbonate or HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffers. After 10 days the culture containing sodium bicarbonate buffer had twice the cell density of that containing HEPES (Fig 4).
[FIGURE 4 OMITTED]
Effect of serum concentration on cell growth
To examine the effect of serum concentration(during the growth phase) on growth rate (and final cell density), CHO cells were grown on microcarners at various serum concentrations. The growth rates and maximum cell densities observed at 2.5, 5.0,7.5 and 10% were essentially the same (Fig 5). A lag time of 2 days was seen, after which the cells entered log phase and grew to a density bout 106 cells per ml. At a 20% serum concentration, a 3-day lag period was seen, and the cells grew to slightly under [10.sup.6] cells per ml. Morphologically, the cells appeared much healthier and less cell detachment was observed at the lower senun concentrations (2.5 and 5.0%).
[FIGURE 5 OMITTED]
Microcarrier culture introduces new possibilities allows practical high-yield culture of anchorage-dependent cell. The advantages of using microcarrier for mammalian cells culture production, include increased productivity, reduced cost, labor-saving technique allowing the elimination of hundreds of flasks and reduced risk of contamination compared to other conventional methods. Microcarrier culture of CHO cell line was studied by using Cytodex1 and slow stirring spinner flask bioreactor. The growth rate of the CHO cells in DMEM was more rapid than those of the RPMI and MEM this due to the higher concentration of glucose, glutamine and essential amino in DEME which are essential source for carbon and nitrogen.
These finding are going parallel with (8). This study reveals that, there is no observation of immediate cell growth on microcarrier but the lag phase was about 24 and cells reached confluence six days after inoculation. This adaptation period was may be due to replacement of glycocalyx elements which losting during trypsinization and to attached to substrate and spread out. These finding coupled with those of (9). It is possible that, with microcarrier or environmental optimization, the lag times could be reduced. The results of this investigation demonstrate that cytodex 1 microcarrier is suitable substrates for the growth of CHO cell line. It is obvious from the growth curves shown in figure 2 that both cell growth rates and maximum attainable cell density occur with this beads. In addition, in all cases, more than 90% of cells were remained viable for more than 8 days. Also the cells appeared healthy throughout the growth period, and formed complete monolayers on the microcarrier surfaces. Similar results founded by (10). The reasons for the increase of cells numbers due to improvement of surface area in culture system for the cell growth using cytodex 1 which increase the surface area to [6000 cm.sup.2] compared to [28 cm.sup.2] of the common using culture flask. Also because the cytodox is composed of cross-linked detran matrix with positively charge DEAE (N,N-diethylaminoethyl) groups so that it can attached strongly cell membrane (11).
In this study, DMEM was found to be the most suitable culture medium for growing CHO cells when compared to, RPMI 1640, and MEM. The cell viability in Cytodex cultures was maintained for significantly longer periods, leading to higher integrated viable cell densities. The cytodex, which not only provide possibilities for anchorage-dependent cells but also for cells growing suspension, can be used in homogeneous spinner bioreactors. This study achieved the maximum cell numbers to 2.6 x [10.sup.6] with improvement the growth rate by10 fold and demonstrated the efficacy of using the microcarrier system for propagation of CHO cells on spinner bioreactor.
I gratefully acknowledge Prof Omar El-Ahmady for helpful suggestions and supporting this research. I would also like to thank all members of the Biotechnology department in EIPICO for the valuable helping and technical assistance in this work.
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Abdel Nasser, A., El-Moghazy
Department of Microbiology and Immunology, Faculty of Pharmacy, Al-Azhar University and Biotechnology Center-EIPICO-Egypt
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|Author:||Nasser, Abdel; El-Moghazy, A.|
|Publication:||International Journal of Biotechnology & Biochemistry|
|Date:||Dec 1, 2010|
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