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Preparation and Cultivation of icroencapsulated Recombinant CHO Cells
CHINESE JOURNAL OF BIOTECHNOLOGY Volume 23, Issue 3, May 2007 Online English edition of the Chinese language journal Cite this article as: Chin J Biotech, 2007, 23(3), 502−507.
RESEARCH PAPER
Preparation and Cultivation of Microencapsulated Recombinant CHO Cells ZHANG Ying1,2, WANG Wei1, LÜ Guo-Jun1,2, YU Wei-Ting1, GUO Xin1, XIONG Ying1, MA Xiao-Jun1,* 1
Laboratory of Biomedical Material Engineering, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China
2
Graduate School of the Chinese Academy of Sciences, Chinese Academy of Sciences, Beijing 100039, China
Abstract: The transplantation of microencapsulated recombinant cells is a novel alternative approach for the treatment of tumors through gene therapy, whereas its clinical application was retarded because the technique for large-scale preparation and culture of microencapsulated cells is still immature. Optimization of the preparation and culture conditions is needed to acquire biological microcapsule with high cell viability and protein production. In this study, the effects of different preparation and culture conditions on microencapsulated recombinant CHO cell growth and endostatin production were studied. The results showed that the growth phase of the inoculum cells and the seeding density potently affected the growth and endostatin production of the recombinant CHO cells in the microcapsule. The exponential growth phase of the recombinant CHO cells with a seeding density of 1×106–2×106 cells/mL microcapsule favored cell growth and endostatin production. The preparation time was another important factor that affected cell viability; the preparation time should be controlled within 5 h to avoid more damage to cells. There would be some damage to cells in the microencapsulation process, and the in vitro culture of microencapsulated cells was a suitable method to recover the cell viability. The highest viable cell density and endostatin production were acquired when the microcapsule percentage was 5% in the culture of microencapsulated cells. Key Words:
cell transplantation; anti-angiogenic therapy; microcapsule preparation and culture; recombinant CHO cells; endostatin
The formation of new blood vessels is of great importance for tumor growth and metastasis because the tumor tissue needs new blood vessels to supply nutrients constantly and eliminate metabolic wastes when the tumor volume exceeds 1 mm3. Inhibiting neovascularization may lead to tumor cell dormancy and induce cell apoptosis; therefore, tumors can be treated by inhibiting tumor cell growth[1,2]. At present, anti-angiogenic therapy has become a novel and alternative strategy in tumor treatment all over the world and is thought to be an important method for the treatment of tumors. In recent years, more than 20 kinds of anti-angiogenic drugs have entered I–III phase clinical experiments[3–7]. One of these angiogenesis inhibitors is endostatin[8,9], a 20 kD C-terminal
globular domain of collagen XVIII, which strongly inhibits proliferation, migration, and angiogenesis of endothelial cells by inducing apoptosis process, and enters I-II phase clinical experiments[10–13]. However, there are many problems in clinical experiment, such as short serum half-life, large injection dosage, and poor tolerance of patients due to the repeated administration of recombinant endostatin. Microencapsulation of recombinant cell technology can overcome these problems. Read et al. and Joki et al. have reported a novel tumor therapeutic strategy using microencapsulated recombinant implantation and acquired a good efficacy in 2001. It has been proved that microencapsulation could favor the long-term secretion of
Received: August 23, 2006; Accepted: November 8, 2006. * Corresponding author. Tel/Fax: +86-411-84379139; E-mail: [email protected] This work was supported by the grants from the National Basic Research Program of China (Nos. 2002CB713804 and 2005CB522702) and the National Natural Science Foundation of China (Nos. 20236040 and 30472102). Copyright © 2007, Institute of Microbiology, Chinese Academy of Sciences and Chinese Society for Microbiology. Published by Elsevier BV. All rights reserved.
ZHANG Ying et al. / Chinese Journal of Biotechnology, 2007, 23(3): 502–507
endostatin from genetically engineered cells and allow the treatment of malignant tumor[14,15]. The semipermeable membrane of the microcapsule allows the bidirectional diffusion of small molecules, such as nutrients, oxygen, and wastes, and prevents antibodies and immunocytes from entering the microcapsule. It may protect cells from its host’s immune rejection, increase the efficiency of exogenous gene expression, and reduce the need for frequent injection. However, preparation and culture of microencapsulated cells was still at the stage of small-scale research in the laboratory and, therefore, could not be used in clinical therapy. If this strategy was applied to clinical therapy, it is necessary to optimize the large-scale preparation and culture conditions to acquire a great deal of microcapsules with high cell viability and protein production[16]. The cells would be damaged due to lack of medium during the preparation of the microencapsulated cells, and the degree of injury may be higher in large-scale preparation. Therefore, it is very important to maintain the viability of the recombinant cells and recombinant protein expression. In this study, the effects of different preparations and culture conditions on microencapsulated recombinant CHO cell growth and endostatin production were studied, which is expected to provide the foundation for large-scale preparation and culture of microencapsulated recombinant cells.
1
Materials and methods
1.1 Materials 1.1.1 Cells and chemicals: The recombinant CHO cells transfected with the endostatin gene were kindly donated by Prof. Jian Fei (Shanghai Institute for Biological Sciences, Chinese Academy of Sciences). The cells were routinely cultivated in 250 mL T-flasks and incubated at 37°C in a humidified 5% CO2 atmosphere. The medium was DMEM/ F12 (1:1) medium (Sigma, USA.) supplemented with 10% fetal bovine serum (FBS, Sanli Biology Beijing, China), 100 u/mL penicillin, 100 µg/mL streptomycin, and 5 µg/mL puromycin (Sigma, USA). Alginate, Poly-L-lysine, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazoliumbromide (MTT) were purchased from Sigma, USA, and all the other chemicals were of analytical grade and purchased from the local market. 1.1.2 Instruments: MS-353 plate reader (Labsystems Co. Ltd, Finland), Heraeus BB16UV CO2 incubator (Heal Force Development Co. Ltd, China), and Electrostatic droplet generator (Dalian Institute of Chemical Physics, Chinese Academy of Sciences, China). 1.2 Methods 1.2.1 Preparation of APA microencapsulated recombinant CHO cells: Alginate-poly-L-lysine-alginate (APA) microcapsules containing recombinant CHO cells were prepared as described previously with some modifications. In brief,
recombinant CHO cells in exponential growth phase were harvested and resuspended in 1.5% (W/V) filter-sterilized sodium alginate solution (Sigma, USA). The cell suspension was then extruded through a 0.4-mm needle into a 100 mmol/L CaCl2 solution using an electrostatic droplet generator (Dalian Institute of Chemical Physics, Chinese Academy of Sciences, China) to obtain calcium alginate gel beads. After being hardened for 20 min, the gel beads were incubated with 0.05% (W/V) poly-L-lysine (Mw 20 700; Sigma, USA) for 10 min to form alginate-poly-L-lysine membrane around the surface. A total of 0.15% (W/V) alginate was then added to counteract the excess charge on the membrane for 5 min. Finally, membrane-enclosed gel beads were liquidized with 55 mmol/L of sodium citrate to obtain APA microcapsule with liquid core[17]. 1.2.2 Culture of microencapsulated recombinant CHO cells: The microcapsules containing recombinant CHO cells were suspended in DMEM/F12 (1:1) medium, inoculated into a 24-well plate with 0.1 mL of microcapsules, and 2 mL of medium in each well, and incubated at 37°C in a humidified 5% CO2 atmosphere. The culture was fed with fresh medium every 4 days, and the media were collected and kept frozen at −20°C for later analysis to determine the concentration of endostatin. 1.2.3 Measurement of viable cells in microcapsules: MTT assay was performed as described previously with some modifications[18]. In brief, MTT solution (5 mg/mL; Sigma, USA) was added into a 24-well plate with 100 μL in each well and incubated at 37°C for additional 24 h. The medium containing MTT was removed and microcapsules were washed twice with 0.9% saline, and then 1 mL of DMSO was added to solubilize the MTT tetrazolium crystal. The absorbance (A) was determined at 570 nm using a plate reader (Wellscan MK3, Labsystems, Finland) and 630 nm was used as reference. The viable cell number was calculated from the value of OD570 according to a standard curve of viable cell number versus value of OD570. 1.2.4 Determination of endostatin concentration: Concentration of endostatin in conditioned medium was determined by ELISA (Accucyte Human endostatin kit, R&D system, USA) according to the manufacturer’s instructions. The OD value was determined at 450 nm using a plate reader (Wellscan MK3, Labsystems, Finland) and 630 nm was used as reference. Endostatin concentration was proportional to the OD450 value; therefore, it could be calculated from the standard curve of endostatin concentration versus OD450 value. 1.2.5 Experimental setup: (1) To study the effect of the seeding density on microencapsulated recombinant CHO cell growth and endostatin production, the seed cells were in the lag phase, the exponential growth phase, and the stable phase of the cell growth. The other conditions were defined as: the seeding density was 2×106 cells/mL microcapsule, microcapsule
ZHANG Ying et al. / Chinese Journal of Biotechnology, 2007, 23(3): 502–507
percentage was 5%, and the preparation time was 3 h. (2) The seeding density was set up to be 1×107 cells/mL microcapsule, 5×106 cells/mL microcapsule, 2×106 cells/mL microcapsule, 1×106 cells/mL microcapsule, and 5×105 cells/mL microcapsule, respectively, in the experiment to study the effect of the seeding density on microencapsulated recombinant CHO cell growth and endostatin production. The other conditions were defined as: recombinant CHO cells were in the exponential growth phase, microcapsule percentage was 5%, and the preparation time was 3 h. (3) The preparation time was set up to be 3 h, 5 h, and 7 h, respectively, in the experiment to study the effect of the preparation time on microencapsulated recombinant CHO cell growth and endostatin production. The other conditions were: recombinant CHO cells were in the exponential growth phase, the seeding density was 2×106 cells/mL microcapsule, and microcapsule percentage was 5%. (4) The microcapsule percentage was set up to be 5%, 10%, 15%, and 20%, respectively, in experiment to study the effect of the microcapsule percentage on microencapsulated recombinant CHO cell growth and endostatin production. The other conditions were: recombinant CHO cells were in the exponential growth phase, the seeding density was 2×106 cells/mL microcapsule, and the preparation time was 3h. 1.2.6 Calculations and level of significance: The specific growth rate (μ) was calculated by the general formula[19]: µ=(lnX2−lnX1)/(t2−t1) (1) where X1 and X2 are the viable cell densities at time t1 and t2, respectively.
2
Results and discussion
2.1 Effect of the inoculum cells growth phase on the microencapsulated recombinant CHO cell growth and endostatin production The effect of the growth phase of inoculum cells on microencapsulated recombinant CHO cell growth and endostatin production is shown in Fig. 1. The viability of microencapsulated cells in the exponential growth phase was the best; the lag phase of those cells was the shortest, and the
cell growth entered the exponential growth phase on day 2. The specific growth rate (μ) was the largest on day 2, reaching 0.863/d, and then decreased during the culture. The cell growth entered the stable phase after 14 days of culture, and the viable cell density was 4.17×107 cells/mL microcapsule. The growth of microencapsulated cells in the lag phase was slower; the lag phase of those cells prolonged and the cell growth entered the exponential growth phase on day 4. The specific growth rate was the highest on day 4, reaching 0.611/d, and the highest viable cell density of 3.64×107 cells/mL microcapsule was reached on day 16 of culture. The viability of microencapsulated cells in the stable phase was poor and the cell growth was the slowest. The maximal specific growth rate (μmax) was only 0.453/d, and the maximal viable cell density reached only 3.64×107 cells/mL microcapsule on day 16 of culture. The endostatin production corresponded to the cell growth. Endostatin production was the highest and reached 753.5 ng/mL on day 16 of culture when seed cells were in the exponential growth phase. Endostatin production decreased when the seed cells were in the lag phase; it was the lowest and only reached 112 ng/mL on day 16 of culture when the seed cells were in the stable phase. The cell growth included the lag phase, the exponential growth phase, the stable phase, and the dead phase during the in vitro culture of cells. The cells differed in their adaptabilities to unfavorable conditions because the viabilities of the cells differed with growth phases. Most of the recombinant cells died after microencapsulation because of the death of the cells in the stable phase and because of poor viabilities; therefore, microencapsulated cell growth was the slowest and the endostatin production was the lowest. Whereas for the seed cells in the exponential growth phase and the lag phase, the cell growth was rapid and the endostatin production was high because the cells possessed good viabilities and high adaptabilities to unfavorable conditions. Therefore, the seed cells in the exponential growth phase were the best in the course of the microcapsule preparation, and this phase was favorable to microencapsulated cell growth and endostatin production.
Fig. 1 Effect of the growth phase of inoculum cells on microencapsulated recombinant CHO cell growth and endostatin production ■ Lag phase; ● Exponential growth phase; ▲ Stable phase.
ZHANG Ying et al. / Chinese Journal of Biotechnology, 2007, 23(3): 502–507
2.2 Effect of the seeding density on microencapsulated recombinant CHO cell growth and endostatin production The growth of microencapsulated recombinant CHO cells at different seeding densities is shown in Fig. 2. With an initial seeding density of 2×106 cells/mL microcapsule and 1×106 cells/mL microcapsule, the cell growth was the best and the maximal viable cell density was 4.17×107 and 4.03×107 cells/mL microcapsule, respectively. A decrease in the seeding density to 5×105 cells/mL microcapsule slowed down the growth of cells, and the maximal cell density reached only 1.27×107 cells/mL microcapsule on day 18 of the culture. The cell growth was faster during the first 4 days when the seeding density increased to 1×107 cells/mL microcapsule and 5×106 cells/mL microcapsule, then the cell growth ceased and the cell density remained stable, and the maximal cell density reached only 2.02×107 and 2.47×107 cells/mL microcapsule, respectively. The μmax was 0.863/d and 0.966/d when the seeding density was 2×106 and 1×106cells/mL microcapsule and the μmax decreased to 0.312/d and 0.625/d when the seeding density increased to 1×107 and 5×106 cells/mL microcapsule, respectively. The μmax was only 0.904/d when the seeding density was 5×105 cells/mL microcapsule, similar to the μmax of 2×106 and 1×106 cells/mL microcapsule. The result of the endostatin production showed that the endostatin expression was the highest when the seeding density was 2×106 and 1×106 cells/mL microcapsule, reaching 753.5 ng/mL and 722.9 ng/mL, respectively, on day 16. Both the increase and the decrease in the seeding density could lead to a decrease in the endostatin production. One of the major parameters in immobilized cell culture was the initial cell density. It was necessary to inoculate with an adequate cell density for the quick growth of the cells and insufficient inoculum could result in the retardation of cell growth and reduction in cell proliferation. Hu reported that the cell growth was the best when the seeding density was 4×105 cells/mL,
and the cell proliferation could be inhibited with the decrease in the seeding density. The cell growth ceased when the seeding density was less than 7 cells per microcarrier[20,21]. Arús showed that the cell seeding density also affected microencapsulated hybridoma cell growth and antibody production; the cell growth was the best and the antibody production was the highest when the initial cell density was 1×107 cells/mL microcapsule. The cell growth was inhibited and the antibody production decreased when the cell density decreased to 1×106 and 5×106 cells/mL microcapsule[22]. However, the initial cell density of 1×107 cells/mL microcapsule was very high, which was even higher than the final cell density in the suspension culture, thereby leading to difficulty in carrying out seed cell culture; therefore, it was necessary to optimize the seeding density and decrease it to an appropriate extent. The result of this study showed that an initial cell density of 1×106–2×106 cells/mL microcapsule was the best among the conditions being tested according to cell proliferation and endostatin production. The μmax and the maximal viable cell density decreased with increasing of the seeding density. Although the μmax when the seeding density decreased to 5×105 cells/mL microcapsule was similar to the μmax when the seeding density was 1×106–2×106 cells/mL microcapsule, the cells did not grow in many microcapsules because the seed cell numbers in them were less than 7 cells per microcapsule. (The mean size in terms of the diameter of the microcapsule was 300 μm. There were about 70 000 microcapsules per mL, so the average cell number per microcapsule was 7. As the seed cells randomly distribute in the microcapsules during the preparation of the microcapsule, there were more than 7 cells in some of the microcapsules, whereas less than 7 cells in others.) The cells showed good growth only in a few microcapsules, with the seeding density beyond 7 cells per microcapsule; therefore, the maximal cell density only reached 1.27×107 cells/mL microcapsule.
Fig. 2 Effect of the seeding density on microencapsulated recombinant CHO cell growth and endostatin production ■ 1×107 cells/mL microcapsule; ● 5×106 cells/mL microcapsule; ▲ 2×106 cells/mL microcapsule; ▼ 1×106 cells/mL microcapsule; ◄ 5×105 cells/mL microcapsule.
ZHANG Ying et al. / Chinese Journal of Biotechnology, 2007, 23(3): 502–507
2.3 Effect of the preparation time on microencapsulated recombinant CHO cell growth and endostatin production The cells would be damaged due to lack of medium during the preparation of microencapsulated cells, and the degree of injury may be larger with the prolongation of the preparation time in large-scale preparation. Fig. 3 shows the effect of preparation time on microencapsulated recombinant CHO cell growth and endostatin production. The lag phase of the cell growth was short when the preparation time was 3 h. The cell growth entered the exponential growth phase on day 2, and the μmax was 0.863/d. The viable density reached the highest on day 14, which was 4.17×107 cells/mL microcapsule. The cell growth entered the exponential growth phase on day 4 after a transitory delay when the preparation time was 5 h and the μmax was 0.728/d. The maximal viable cell density reached the
highest of 3.22×107 cells/mL microcapsule on day 14 and then remained stable. The cell growth was slow when the preparation time was 7 h. The maximal viable cell density and the μmax reached only 6.59×106 cells/mL microcapsule and 0.241/d, respectively. The endostatin production was the highest when the preparation time was 3 h, which was 753.5 ng/mL. The endostatin production was 514.1 ng/mL with a preparation time of 5 h and only 88.7 ng/mL with a preparation time of 7 h. It was necessary to limit the preparation time strictly to maintain the cell viability and endostatin production because the cells would be damaged during the preparation of microencapsulated cells. The result of this experiment showed that the preparation time, as well as the duration for which the cells remained without medium should be less than 5 h.
Fig. 3 Effect of the preparation time on microencapsulated recombinant CHO cell growth and endostatin production ■ 3 h; ● 5 h; ▲ 7 h.
2.4 Effect of the microcapsule percentage on microencapsulated recombinant CHO cell growth and endostatin production Fig. 4 shows the effect of the microcapsule percentage on microencapsulated recombinant CHO cell growth and endostatin production. The cell growth was the best when the microcapsule percentage was 5% and the maximal viable cell density was 4.86×107 cells/mL microcapsule. The cell growth was inhibited with the increase in the microcapsule percentage, and the maximal viable cell density was only 3.52×107 cells/mL microcapsule, with a microcapsule percentage of 10%. The maximal viable cell density decreased to 2.74×107 cells/mL microcapsule and 2.31×107 cells/mL microcapsule when the microcapsule percentage increased to 15% and 20%, respectively. The μmax was 0.728/d when the microcapsule percentage was 5%, which then decreased with the increase in the microcapsule percentage. When the microcapsule percentage increased to 20%, the μmax was only 0.278/d. The result of the endostatin production showed that although the microcapsule percentage considerably affected the recombinant cell growth, it has no significant effect on endostatin production. The endostatin production was
approximately 840 ng/mL on day 16 of culture. The result of this experiment showed that the microcapsule percentage of 5% favored cell growth and endostatin production. The cell proliferation decreased with the increase in the microcapsule percentage due to lack of nutrients, especially oxygen. The increase in the microcapsule percentage increased the cell density in medium and led to enhanced demand of oxygen. The lower solubility and low transfer coefficient of oxygen resulted in poor oxygen supply and thus affected the cell growth. In this study, the effect of different preparation and culture conditions on microencapsulated recombinant CHO cell growth and endostatin production were studied. The results showed that microencapsulated recombinant cells could proliferate and express endostatin in the long-term; preparation and culture conditions considerably affected cell growth and endostatin production. The recombinant CHO cells in the exponential growth phase with a seeding density of 1×106–2×106 cells/mL microcapsule favored cell growth and endostatin production. The preparation time also had a considerable effect on cell viability and endostatin production; longer preparation time means more damage to cells, so the
ZHANG Ying et al. / Chinese Journal of Biotechnology, 2007, 23(3): 502–507
preparation time should be controlled within 5 h to maintain the cell viability and endostatin production. The highest viable cell density and endostatin production were acquired when the microcapsule percentage was 5% in the in vitro culture of
microencapsulated cells. The biological microcapsule with high cell viability and endostatin expression could be acquired by optimization of the preparation conditions to meet the demand of microencapsulated cell transplantation.
Fig. 4 Effect of the microcapsule percentage on microencapsulated recombinant CHO cell growth and endostatin production ■ 20%; ● 15%; ▲ 10%; ▼ 5%.
3772–3784.
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RESEARCH PAPER
Preparation and Cultivation of Microencapsulated Recombinant CHO Cells ZHANG Ying1,2, WANG Wei1, LÜ Guo-Jun1,2, YU Wei-Ting1, GUO Xin1, XIONG Ying1, MA Xiao-Jun1,* 1
Laboratory of Biomedical Material Engineering, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China
2
Graduate School of the Chinese Academy of Sciences, Chinese Academy of Sciences, Beijing 100039, China
Abstract: The transplantation of microencapsulated recombinant cells is a novel alternative approach for the treatment of tumors through gene therapy, whereas its clinical application was retarded because the technique for large-scale preparation and culture of microencapsulated cells is still immature. Optimization of the preparation and culture conditions is needed to acquire biological microcapsule with high cell viability and protein production. In this study, the effects of different preparation and culture conditions on microencapsulated recombinant CHO cell growth and endostatin production were studied. The results showed that the growth phase of the inoculum cells and the seeding density potently affected the growth and endostatin production of the recombinant CHO cells in the microcapsule. The exponential growth phase of the recombinant CHO cells with a seeding density of 1×106–2×106 cells/mL microcapsule favored cell growth and endostatin production. The preparation time was another important factor that affected cell viability; the preparation time should be controlled within 5 h to avoid more damage to cells. There would be some damage to cells in the microencapsulation process, and the in vitro culture of microencapsulated cells was a suitable method to recover the cell viability. The highest viable cell density and endostatin production were acquired when the microcapsule percentage was 5% in the culture of microencapsulated cells. Key Words:
cell transplantation; anti-angiogenic therapy; microcapsule preparation and culture; recombinant CHO cells; endostatin
The formation of new blood vessels is of great importance for tumor growth and metastasis because the tumor tissue needs new blood vessels to supply nutrients constantly and eliminate metabolic wastes when the tumor volume exceeds 1 mm3. Inhibiting neovascularization may lead to tumor cell dormancy and induce cell apoptosis; therefore, tumors can be treated by inhibiting tumor cell growth[1,2]. At present, anti-angiogenic therapy has become a novel and alternative strategy in tumor treatment all over the world and is thought to be an important method for the treatment of tumors. In recent years, more than 20 kinds of anti-angiogenic drugs have entered I–III phase clinical experiments[3–7]. One of these angiogenesis inhibitors is endostatin[8,9], a 20 kD C-terminal
globular domain of collagen XVIII, which strongly inhibits proliferation, migration, and angiogenesis of endothelial cells by inducing apoptosis process, and enters I-II phase clinical experiments[10–13]. However, there are many problems in clinical experiment, such as short serum half-life, large injection dosage, and poor tolerance of patients due to the repeated administration of recombinant endostatin. Microencapsulation of recombinant cell technology can overcome these problems. Read et al. and Joki et al. have reported a novel tumor therapeutic strategy using microencapsulated recombinant implantation and acquired a good efficacy in 2001. It has been proved that microencapsulation could favor the long-term secretion of
Received: August 23, 2006; Accepted: November 8, 2006. * Corresponding author. Tel/Fax: +86-411-84379139; E-mail: [email protected] This work was supported by the grants from the National Basic Research Program of China (Nos. 2002CB713804 and 2005CB522702) and the National Natural Science Foundation of China (Nos. 20236040 and 30472102). Copyright © 2007, Institute of Microbiology, Chinese Academy of Sciences and Chinese Society for Microbiology. Published by Elsevier BV. All rights reserved.
ZHANG Ying et al. / Chinese Journal of Biotechnology, 2007, 23(3): 502–507
endostatin from genetically engineered cells and allow the treatment of malignant tumor[14,15]. The semipermeable membrane of the microcapsule allows the bidirectional diffusion of small molecules, such as nutrients, oxygen, and wastes, and prevents antibodies and immunocytes from entering the microcapsule. It may protect cells from its host’s immune rejection, increase the efficiency of exogenous gene expression, and reduce the need for frequent injection. However, preparation and culture of microencapsulated cells was still at the stage of small-scale research in the laboratory and, therefore, could not be used in clinical therapy. If this strategy was applied to clinical therapy, it is necessary to optimize the large-scale preparation and culture conditions to acquire a great deal of microcapsules with high cell viability and protein production[16]. The cells would be damaged due to lack of medium during the preparation of the microencapsulated cells, and the degree of injury may be higher in large-scale preparation. Therefore, it is very important to maintain the viability of the recombinant cells and recombinant protein expression. In this study, the effects of different preparations and culture conditions on microencapsulated recombinant CHO cell growth and endostatin production were studied, which is expected to provide the foundation for large-scale preparation and culture of microencapsulated recombinant cells.
1
Materials and methods
1.1 Materials 1.1.1 Cells and chemicals: The recombinant CHO cells transfected with the endostatin gene were kindly donated by Prof. Jian Fei (Shanghai Institute for Biological Sciences, Chinese Academy of Sciences). The cells were routinely cultivated in 250 mL T-flasks and incubated at 37°C in a humidified 5% CO2 atmosphere. The medium was DMEM/ F12 (1:1) medium (Sigma, USA.) supplemented with 10% fetal bovine serum (FBS, Sanli Biology Beijing, China), 100 u/mL penicillin, 100 µg/mL streptomycin, and 5 µg/mL puromycin (Sigma, USA). Alginate, Poly-L-lysine, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazoliumbromide (MTT) were purchased from Sigma, USA, and all the other chemicals were of analytical grade and purchased from the local market. 1.1.2 Instruments: MS-353 plate reader (Labsystems Co. Ltd, Finland), Heraeus BB16UV CO2 incubator (Heal Force Development Co. Ltd, China), and Electrostatic droplet generator (Dalian Institute of Chemical Physics, Chinese Academy of Sciences, China). 1.2 Methods 1.2.1 Preparation of APA microencapsulated recombinant CHO cells: Alginate-poly-L-lysine-alginate (APA) microcapsules containing recombinant CHO cells were prepared as described previously with some modifications. In brief,
recombinant CHO cells in exponential growth phase were harvested and resuspended in 1.5% (W/V) filter-sterilized sodium alginate solution (Sigma, USA). The cell suspension was then extruded through a 0.4-mm needle into a 100 mmol/L CaCl2 solution using an electrostatic droplet generator (Dalian Institute of Chemical Physics, Chinese Academy of Sciences, China) to obtain calcium alginate gel beads. After being hardened for 20 min, the gel beads were incubated with 0.05% (W/V) poly-L-lysine (Mw 20 700; Sigma, USA) for 10 min to form alginate-poly-L-lysine membrane around the surface. A total of 0.15% (W/V) alginate was then added to counteract the excess charge on the membrane for 5 min. Finally, membrane-enclosed gel beads were liquidized with 55 mmol/L of sodium citrate to obtain APA microcapsule with liquid core[17]. 1.2.2 Culture of microencapsulated recombinant CHO cells: The microcapsules containing recombinant CHO cells were suspended in DMEM/F12 (1:1) medium, inoculated into a 24-well plate with 0.1 mL of microcapsules, and 2 mL of medium in each well, and incubated at 37°C in a humidified 5% CO2 atmosphere. The culture was fed with fresh medium every 4 days, and the media were collected and kept frozen at −20°C for later analysis to determine the concentration of endostatin. 1.2.3 Measurement of viable cells in microcapsules: MTT assay was performed as described previously with some modifications[18]. In brief, MTT solution (5 mg/mL; Sigma, USA) was added into a 24-well plate with 100 μL in each well and incubated at 37°C for additional 24 h. The medium containing MTT was removed and microcapsules were washed twice with 0.9% saline, and then 1 mL of DMSO was added to solubilize the MTT tetrazolium crystal. The absorbance (A) was determined at 570 nm using a plate reader (Wellscan MK3, Labsystems, Finland) and 630 nm was used as reference. The viable cell number was calculated from the value of OD570 according to a standard curve of viable cell number versus value of OD570. 1.2.4 Determination of endostatin concentration: Concentration of endostatin in conditioned medium was determined by ELISA (Accucyte Human endostatin kit, R&D system, USA) according to the manufacturer’s instructions. The OD value was determined at 450 nm using a plate reader (Wellscan MK3, Labsystems, Finland) and 630 nm was used as reference. Endostatin concentration was proportional to the OD450 value; therefore, it could be calculated from the standard curve of endostatin concentration versus OD450 value. 1.2.5 Experimental setup: (1) To study the effect of the seeding density on microencapsulated recombinant CHO cell growth and endostatin production, the seed cells were in the lag phase, the exponential growth phase, and the stable phase of the cell growth. The other conditions were defined as: the seeding density was 2×106 cells/mL microcapsule, microcapsule
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percentage was 5%, and the preparation time was 3 h. (2) The seeding density was set up to be 1×107 cells/mL microcapsule, 5×106 cells/mL microcapsule, 2×106 cells/mL microcapsule, 1×106 cells/mL microcapsule, and 5×105 cells/mL microcapsule, respectively, in the experiment to study the effect of the seeding density on microencapsulated recombinant CHO cell growth and endostatin production. The other conditions were defined as: recombinant CHO cells were in the exponential growth phase, microcapsule percentage was 5%, and the preparation time was 3 h. (3) The preparation time was set up to be 3 h, 5 h, and 7 h, respectively, in the experiment to study the effect of the preparation time on microencapsulated recombinant CHO cell growth and endostatin production. The other conditions were: recombinant CHO cells were in the exponential growth phase, the seeding density was 2×106 cells/mL microcapsule, and microcapsule percentage was 5%. (4) The microcapsule percentage was set up to be 5%, 10%, 15%, and 20%, respectively, in experiment to study the effect of the microcapsule percentage on microencapsulated recombinant CHO cell growth and endostatin production. The other conditions were: recombinant CHO cells were in the exponential growth phase, the seeding density was 2×106 cells/mL microcapsule, and the preparation time was 3h. 1.2.6 Calculations and level of significance: The specific growth rate (μ) was calculated by the general formula[19]: µ=(lnX2−lnX1)/(t2−t1) (1) where X1 and X2 are the viable cell densities at time t1 and t2, respectively.
2
Results and discussion
2.1 Effect of the inoculum cells growth phase on the microencapsulated recombinant CHO cell growth and endostatin production The effect of the growth phase of inoculum cells on microencapsulated recombinant CHO cell growth and endostatin production is shown in Fig. 1. The viability of microencapsulated cells in the exponential growth phase was the best; the lag phase of those cells was the shortest, and the
cell growth entered the exponential growth phase on day 2. The specific growth rate (μ) was the largest on day 2, reaching 0.863/d, and then decreased during the culture. The cell growth entered the stable phase after 14 days of culture, and the viable cell density was 4.17×107 cells/mL microcapsule. The growth of microencapsulated cells in the lag phase was slower; the lag phase of those cells prolonged and the cell growth entered the exponential growth phase on day 4. The specific growth rate was the highest on day 4, reaching 0.611/d, and the highest viable cell density of 3.64×107 cells/mL microcapsule was reached on day 16 of culture. The viability of microencapsulated cells in the stable phase was poor and the cell growth was the slowest. The maximal specific growth rate (μmax) was only 0.453/d, and the maximal viable cell density reached only 3.64×107 cells/mL microcapsule on day 16 of culture. The endostatin production corresponded to the cell growth. Endostatin production was the highest and reached 753.5 ng/mL on day 16 of culture when seed cells were in the exponential growth phase. Endostatin production decreased when the seed cells were in the lag phase; it was the lowest and only reached 112 ng/mL on day 16 of culture when the seed cells were in the stable phase. The cell growth included the lag phase, the exponential growth phase, the stable phase, and the dead phase during the in vitro culture of cells. The cells differed in their adaptabilities to unfavorable conditions because the viabilities of the cells differed with growth phases. Most of the recombinant cells died after microencapsulation because of the death of the cells in the stable phase and because of poor viabilities; therefore, microencapsulated cell growth was the slowest and the endostatin production was the lowest. Whereas for the seed cells in the exponential growth phase and the lag phase, the cell growth was rapid and the endostatin production was high because the cells possessed good viabilities and high adaptabilities to unfavorable conditions. Therefore, the seed cells in the exponential growth phase were the best in the course of the microcapsule preparation, and this phase was favorable to microencapsulated cell growth and endostatin production.
Fig. 1 Effect of the growth phase of inoculum cells on microencapsulated recombinant CHO cell growth and endostatin production ■ Lag phase; ● Exponential growth phase; ▲ Stable phase.
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2.2 Effect of the seeding density on microencapsulated recombinant CHO cell growth and endostatin production The growth of microencapsulated recombinant CHO cells at different seeding densities is shown in Fig. 2. With an initial seeding density of 2×106 cells/mL microcapsule and 1×106 cells/mL microcapsule, the cell growth was the best and the maximal viable cell density was 4.17×107 and 4.03×107 cells/mL microcapsule, respectively. A decrease in the seeding density to 5×105 cells/mL microcapsule slowed down the growth of cells, and the maximal cell density reached only 1.27×107 cells/mL microcapsule on day 18 of the culture. The cell growth was faster during the first 4 days when the seeding density increased to 1×107 cells/mL microcapsule and 5×106 cells/mL microcapsule, then the cell growth ceased and the cell density remained stable, and the maximal cell density reached only 2.02×107 and 2.47×107 cells/mL microcapsule, respectively. The μmax was 0.863/d and 0.966/d when the seeding density was 2×106 and 1×106cells/mL microcapsule and the μmax decreased to 0.312/d and 0.625/d when the seeding density increased to 1×107 and 5×106 cells/mL microcapsule, respectively. The μmax was only 0.904/d when the seeding density was 5×105 cells/mL microcapsule, similar to the μmax of 2×106 and 1×106 cells/mL microcapsule. The result of the endostatin production showed that the endostatin expression was the highest when the seeding density was 2×106 and 1×106 cells/mL microcapsule, reaching 753.5 ng/mL and 722.9 ng/mL, respectively, on day 16. Both the increase and the decrease in the seeding density could lead to a decrease in the endostatin production. One of the major parameters in immobilized cell culture was the initial cell density. It was necessary to inoculate with an adequate cell density for the quick growth of the cells and insufficient inoculum could result in the retardation of cell growth and reduction in cell proliferation. Hu reported that the cell growth was the best when the seeding density was 4×105 cells/mL,
and the cell proliferation could be inhibited with the decrease in the seeding density. The cell growth ceased when the seeding density was less than 7 cells per microcarrier[20,21]. Arús showed that the cell seeding density also affected microencapsulated hybridoma cell growth and antibody production; the cell growth was the best and the antibody production was the highest when the initial cell density was 1×107 cells/mL microcapsule. The cell growth was inhibited and the antibody production decreased when the cell density decreased to 1×106 and 5×106 cells/mL microcapsule[22]. However, the initial cell density of 1×107 cells/mL microcapsule was very high, which was even higher than the final cell density in the suspension culture, thereby leading to difficulty in carrying out seed cell culture; therefore, it was necessary to optimize the seeding density and decrease it to an appropriate extent. The result of this study showed that an initial cell density of 1×106–2×106 cells/mL microcapsule was the best among the conditions being tested according to cell proliferation and endostatin production. The μmax and the maximal viable cell density decreased with increasing of the seeding density. Although the μmax when the seeding density decreased to 5×105 cells/mL microcapsule was similar to the μmax when the seeding density was 1×106–2×106 cells/mL microcapsule, the cells did not grow in many microcapsules because the seed cell numbers in them were less than 7 cells per microcapsule. (The mean size in terms of the diameter of the microcapsule was 300 μm. There were about 70 000 microcapsules per mL, so the average cell number per microcapsule was 7. As the seed cells randomly distribute in the microcapsules during the preparation of the microcapsule, there were more than 7 cells in some of the microcapsules, whereas less than 7 cells in others.) The cells showed good growth only in a few microcapsules, with the seeding density beyond 7 cells per microcapsule; therefore, the maximal cell density only reached 1.27×107 cells/mL microcapsule.
Fig. 2 Effect of the seeding density on microencapsulated recombinant CHO cell growth and endostatin production ■ 1×107 cells/mL microcapsule; ● 5×106 cells/mL microcapsule; ▲ 2×106 cells/mL microcapsule; ▼ 1×106 cells/mL microcapsule; ◄ 5×105 cells/mL microcapsule.
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2.3 Effect of the preparation time on microencapsulated recombinant CHO cell growth and endostatin production The cells would be damaged due to lack of medium during the preparation of microencapsulated cells, and the degree of injury may be larger with the prolongation of the preparation time in large-scale preparation. Fig. 3 shows the effect of preparation time on microencapsulated recombinant CHO cell growth and endostatin production. The lag phase of the cell growth was short when the preparation time was 3 h. The cell growth entered the exponential growth phase on day 2, and the μmax was 0.863/d. The viable density reached the highest on day 14, which was 4.17×107 cells/mL microcapsule. The cell growth entered the exponential growth phase on day 4 after a transitory delay when the preparation time was 5 h and the μmax was 0.728/d. The maximal viable cell density reached the
highest of 3.22×107 cells/mL microcapsule on day 14 and then remained stable. The cell growth was slow when the preparation time was 7 h. The maximal viable cell density and the μmax reached only 6.59×106 cells/mL microcapsule and 0.241/d, respectively. The endostatin production was the highest when the preparation time was 3 h, which was 753.5 ng/mL. The endostatin production was 514.1 ng/mL with a preparation time of 5 h and only 88.7 ng/mL with a preparation time of 7 h. It was necessary to limit the preparation time strictly to maintain the cell viability and endostatin production because the cells would be damaged during the preparation of microencapsulated cells. The result of this experiment showed that the preparation time, as well as the duration for which the cells remained without medium should be less than 5 h.
Fig. 3 Effect of the preparation time on microencapsulated recombinant CHO cell growth and endostatin production ■ 3 h; ● 5 h; ▲ 7 h.
2.4 Effect of the microcapsule percentage on microencapsulated recombinant CHO cell growth and endostatin production Fig. 4 shows the effect of the microcapsule percentage on microencapsulated recombinant CHO cell growth and endostatin production. The cell growth was the best when the microcapsule percentage was 5% and the maximal viable cell density was 4.86×107 cells/mL microcapsule. The cell growth was inhibited with the increase in the microcapsule percentage, and the maximal viable cell density was only 3.52×107 cells/mL microcapsule, with a microcapsule percentage of 10%. The maximal viable cell density decreased to 2.74×107 cells/mL microcapsule and 2.31×107 cells/mL microcapsule when the microcapsule percentage increased to 15% and 20%, respectively. The μmax was 0.728/d when the microcapsule percentage was 5%, which then decreased with the increase in the microcapsule percentage. When the microcapsule percentage increased to 20%, the μmax was only 0.278/d. The result of the endostatin production showed that although the microcapsule percentage considerably affected the recombinant cell growth, it has no significant effect on endostatin production. The endostatin production was
approximately 840 ng/mL on day 16 of culture. The result of this experiment showed that the microcapsule percentage of 5% favored cell growth and endostatin production. The cell proliferation decreased with the increase in the microcapsule percentage due to lack of nutrients, especially oxygen. The increase in the microcapsule percentage increased the cell density in medium and led to enhanced demand of oxygen. The lower solubility and low transfer coefficient of oxygen resulted in poor oxygen supply and thus affected the cell growth. In this study, the effect of different preparation and culture conditions on microencapsulated recombinant CHO cell growth and endostatin production were studied. The results showed that microencapsulated recombinant cells could proliferate and express endostatin in the long-term; preparation and culture conditions considerably affected cell growth and endostatin production. The recombinant CHO cells in the exponential growth phase with a seeding density of 1×106–2×106 cells/mL microcapsule favored cell growth and endostatin production. The preparation time also had a considerable effect on cell viability and endostatin production; longer preparation time means more damage to cells, so the
ZHANG Ying et al. / Chinese Journal of Biotechnology, 2007, 23(3): 502–507
preparation time should be controlled within 5 h to maintain the cell viability and endostatin production. The highest viable cell density and endostatin production were acquired when the microcapsule percentage was 5% in the in vitro culture of
microencapsulated cells. The biological microcapsule with high cell viability and endostatin expression could be acquired by optimization of the preparation conditions to meet the demand of microencapsulated cell transplantation.
Fig. 4 Effect of the microcapsule percentage on microencapsulated recombinant CHO cell growth and endostatin production ■ 20%; ● 15%; ▲ 10%; ▼ 5%.
3772–3784.
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