- Email: [email protected]
Rational development of serum-free medium for Chinese hamster ovary cells
Process Biochemistry 41 (2006) 2314–2319 www.elsevier.com/locate/procbio
Rational development of serum-free medium for Chinese hamster ovary cells Chi-Hsien Liu *, Ting-Yan Chang Graduate Institute of Biochemical and Biomedical Engineering, Chang Gung University, Taoyuan, Taiwan Received 13 April 2006; received in revised form 29 May 2006; accepted 5 June 2006
Abstract Recombinant Chinese hamster ovary (CHO) cells which produce macrophage colony-stimulating factor (M-CSF) were used as a model to systematically develop the serum-free medium. Pre-weaning strategy combined with experimental design was adopted to develop the serum-free medium for CHO cells in this study. First, CHO cells were adapted to grow in a commercial serum-free medium. Then, these weaned cells were adopted to develop our own serum-free medium using experimental design. Insulin and SerEx had significantly stimulatory influence on M-CSF production (P < 0.05) among the seven screened supplements by use of fractional factorial design. Response surface methodology was consequently applied to find optimal concentrations of insulin and SerEx for M-CSF production. By virtue of the pre-weaning strategy and experimental designs, the development of serum-free medium could be simplified and accelerated. # 2006 Elsevier Ltd. All rights reserved. Keywords: CHO; M-CSF; Serum-free medium; Experimental design; Pre-weaning strategy
1. Introduction Macrophage colony-stimulating factor (M-CSF) is a multiple functional cytokine not only for hemopoietic system, but also for non-hemopoietic systems, such as breast function, pregnancy, and osteoclastogenesis. M-CSF also acts as the principal regulator of the survival, proliferation, and differentiation of macrophages and their precursors. In addition, MCSF plays important roles in infections as an inflammatory cytokine [1]. M-CSF secreted Chinese hamster ovary (CHO) cells were used as a model to rationally develop the serum-free medium in this study. Serum is the most important supplement in the traditional medium for the culture of animal cells. However, serum has many shortages such as batch variability, limited supply, high cost, interference with purification of recombinant product, and possibility of viral contamination. The worsening situation worldwide in the supply of serum, the pressure from biotechnology for easier product purification, and the need for consistent and defined conditions for the culture of animal cell will force the adoption of serum-free media [2]. There are
* Corresponding author. Tel.: +886 3 2118800x3146; fax: +886 3 2118700. E-mail address: [email protected] (C.-H. Liu). 1359-5113/$ – see front matter # 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2006.06.008
several commercial serum-free media available for CHO cells. However, their compositions are proprietary, further improvement of these media is difficult, and their prices are expensive. New recombinant strains might have different nutrient requirement for their unique recombinant products. Sometimes biotechnologists would incorporate new ingredients into the serum-free formula. Therefore, we need to develop new serumfree media. Compositions in serum, including cell adhesion factors, peptides, essential nutrients, and hormones, account for the proliferation effects on cultured cells. The availability of purified serum composition contributes to the development of serum-free medium. It is a very time-consuming and laborious process using the traditional one-factor at a time approach to develop the serum-free medium. However, the introduction of statistical design significantly reduces the labors of composition screening and concentration optimization. Castro et al. [3], Kim et al. [4], and Lee et al. [5] chose Plackett–Burman designs to design medium compositions for CHO cells. Every ingredient is tested at high and low levels (or concentrations). Components with stimulatory influence (P < 0.1) are added at high concentrations to complete the optimal media. Nevertheless, optimal concentrations of stimulatory ingredients could not be determined by use of this approach. There are several authors using different statistical methods to find optimal
C.-H. Liu, T.-Y. Chang / Process Biochemistry 41 (2006) 2314–2319
ingredient concentrations for serum-free media. For example, Liu et al. used the steepest ascent method to find optimal concentrations of stimulatory ingredients for CHO cells [6]. Central composite design was applied to optimize concentrations of glucose, glutamine, and inorganic salts for cultivation of a CHO cell line [7]. For the development of serum-free medium, biotechnologists usually weaned animal cells from the serum supplemented to the serum-reduced condition. Sequentially, they have to simultaneously screen components and find their optimal concentrations under the serum-free condition. This approach has to carefully maintain the cell viability and density, if not, cells will die completely. This study used pre-weaning strategy to increase the probability of successful growth in development process for serum-free medium. Here CHO cells were firstly adapted to grow in a commercial serum-free medium. Then, these weaned cells were used as seed to statistically develop our own serum-free medium. 2. Materials and methods 2.1. Cell line and cell culture Genetically-engineered CHO cells, obtained from ATCC (CRL-10154), maintained the production of M-CSF under the pressure of 1 mM methotrexate. The cells were initially maintained in DMEM/F12 (a mixture of DMEM and F12; Sigma–Aldrich, St. Louis, MO, USA) supplemented with 5% fetal bovine serum (FBS; Hyclone, Logan, UT, USA), and sequentially adapted to grow in a commercial serum-free medium (HyQ SFM4; Hyclone) according to product’s protocol. In brief, CHO cells in a conventional medium were seeded into a 1:1 ratio of the serum-free medium and serum-supplemented medium with the cell density of 5 105 viable cells/ml. The culture was cultivated until viable cells continued one to two population doublings. Then the culture was seeded into a 2:1 ratio of the serum-free medium and serum-supplemented medium. Weaned cells were subcultured in this manner until the serum concentration was reduced below 0.1 % and the viable cell concentration is greater than 2 106 cells/ml. These serum-free medium adapted CHO cells were used for further experiment. Cell culture was performed in triplicate in 12-well tissue culture plates (BD Biosciences, Rockville, MD, USA), containing 1 ml of DMEM/F12 medium and tested supplements. The basal medium used here was DMEM/F12 according to the suggestion of Kitano [8]. CHO cells were inoculated at a concentration of 105 cells/ml and harvested after 96 h of cultivation. The viability was determined by the trypan blue exclusion method and the cell concentration was determined by Multisizer 3 (Beckman Counter, Beds, England, UK). Aliquots of spent media were taken and kept frozen at 20 8C for further ELISA analysis.
2.2. M-CSF assay The concentration of M-CSF in media samples after appropriate dilution was detected by use of ELISA kit (DuoSet DY216; R&D systems, Minneapolis, MN, USA). A solution of 3,30 ,5,50 -tetramethyl-benzidine and H2O2 (Kirkeguard & Perry Laboratories Inc., Galthersburg, MD, USA) was added as the peroxidase substrate. After 20-min incubation, 2N H2SO4 solution was added to stop the reaction and the plates were measured at a wavelength of 450 nm. Each sample was tested in duplicate to reduce the uncertainty of ELISA measurement.
2.3. Experimental design and statistical analysis The rules and design of factorial experiments are provided in the statistical textbook of Montgomery [9]. Fractional factorial design and central composite design data were regressed and analyzed by running the glm and rsreg procedures in SAS software (Cary, NC, USA). The linear-regression coefficients of fractional factorial design can be applied in selection of important
2315
medium components. In the screening tests, the magnitude and sign of the regression constants can be used to identify the significance of the variables on responses such as cell density and M-CSF production. If the coefficient is relatively large, it has more significant effects on the response as compared to the small one does. Furthermore, the variable with positive fitted constant is helpful to the response and that one with negative coefficient has inhibitory effects on the response. The three-dimension and contour plots were generated by Sigmaplot package (Systat Co., Point Richmond, USA).
2.4. Chemicals Insulin, meat peptone, Pluronic F68, bovine serum albumin (BSA), linoleic acid–albumin, methotrexate, and dextran sulfate (Average MW 5000 by low angle laser light scattering method) were purchased from Sigma–Aldrich. Yeast extract and SerEx were obtained from Difco (Sparks, MD, USA) and PAA Laboratories (Linz, Austria), respectively.
3. Results and discussion 3.1. Additives screening Based on the results of preliminary experiments and literature review, seven ingredients including insulin, meat peptone, yeast extract, SerEx, BSA, linoleic acid–BSA, and dextran sulfate were selected to evaluate their stimulatory effects on CHO cells under serum-free conditions. Bovine serum albumin (BSA) has many physical functions including acting as a carrier for fatty acids, trace mineral, and functioning as a detoxifier for H2O2. Exogenous lipids or their precursors such as linoleic acid were required for cell proliferation. Insulin is an important supplement in a serum-free medium. It can stimulate the proliferation of animal cells [10]. SerEx, containing growth factors and hormones, is completely defined and free of animal derived components. Dextran sulfate with MW 12,500 was reported to be an effective agent for inducing single cell cultures and enhancing recombinant protein production of insect cells under serum-free conditions [11]. Therefore, insulin, BSA, linoleic acid–BSA, and SerEx were included in this screening test. Other additives such as meat peptone, yeast extract, and dextran sulfate were also considered. Meat peptone was adopted owing to its potential role in promoting cell growth and specific antibody production [6,12]. Yeast extract can partially replace serum in culture of BHK-21 cells [13] and can be a low-cost additive to serum-free medium of CHO cells [14]. The fractional factorial design was applied to test the stimulatory effects of seven ingredients on cell growth and MCSF production in this study. From the principle of the sparsity of effects [9], a system or process is likely to be driven primarily by some main and low-order interaction effects. Effects of interaction were neglected in our first-order linear model to simplify and accelerate the screening process. However, the interaction between the selected key components will be discussed in the following optimization Section 3.2. The 273 fractional factorial design could efficiently test 7 compounds with 16 trials and preserve enough degrees of freedom to evaluate performance of these supplements. The concentration of each component and results of cell growth, M-CSF production and specific M-CSF productivity are demonstrated in Table 1. Regression results of fractional factorial design (Table 2) indicated that addition of insulin and
2316
C.-H. Liu, T.-Y. Chang / Process Biochemistry 41 (2006) 2314–2319
Table 1 Matrix and results of 273 fractional factorial design
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
A
B
C
D
E
F
G
M-CSF production (ng/ml)
Cell growth (105 cells/ml)
Specific M-CSF production rate (ng/105 cells/day)
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
204 475 138 556 213 792 231 667 688 776 600 729 870 806 563 656
0.80 3.71 1.11 6.33 1.21 7.76 1.43 5.42 5.82 7.40 5.61 7.64 5.01 5.93 8.55 7.11
64 32 31 22 44 26 40 31 30 26 27 24 43 34 16 23
A: insulin, 5 mg/L; B: meat peptone, 2.5 mg/ml; C: yeast extract, 2.5 mg/ml; D: SerEx, 1%; E: BSA, 1 mg/ml; F: linoleic acid–albumin, 1 mg/ml; G: dextran sulfate, 500 mg/ml. 1: addition, 1: no addition. Cell was cultured in DMEM/F12 supplemented with indicated ingredients for 4 days.
SerEx greatly facilitated cell growth of CHO cells at significant level of 0.01. Additionally, meat peptone, yeast extract, linoleic acid–BSA had stimulatory effects on cell growth. Whereas, BSA and dextran sulfate had inhibitory effects on cell growth in the tested concentration range. Regression results of M-CSF production (Table 2) indicated that addition of insulin and SerEx enhanced M-CSF production by CHO cells at significant level of 0.05. Besides, yeast extract had stimulatory effects on M-CSF production. Meat peptone, BSA, linoleic acid–BSA, and dextran sulfate had inhibitory effects on M-CSF production in the tested concentration range. Furthermore, product of the specific cellular productivity and the integrated viable cell density can determine the final volumetric production. Besides effects of tested components on cell growth, effects of tested components on specific M-CSF productivity were also evaluated. As shown in Table 1, poor cell growth often accompanied good specific M-CSF productivity. Different culture conditions such as hyperosmotic pressure, hypothermia and chemical were reported to inhibit specific growth rate in hybridoma and CHO cell cultures [15–17]. The enhanced recombinant protein productivity also accompanied the
decreased cell growth in these mammalian cells. The real mechanisms of decreased cell growth and enhanced productivity merit more investigation. In Table 2, insulin, meat peptone and linoleic acid were found to significantly (P < 0.05) inhibit specific M-CSF productivity by regression analysis. A marginal improvement of specific M-CSF productivity was achieved when yeast extract and BSA were supplemented in the basal medium (Table 2). However, it should be noted that the aim of our optimization was to maximize volumetric productivity of M-CSF. Therefore, the most important ingredients on M-CSF volumetric production, i.e. SerEx and insulin, were optimized to find their formulation by the following two central composite design. 3.2. Optimization experiments Based on the results of screening experiments, insulin and SerEx were finally selected as supplements for serum-free medium. Central composite design and response surface methodology were adopted as an efficient way to find an optimal culture medium for M-CSF production by CHO cells.
Table 2 Regression coefficients and significance analysis of a fractional factorial design for M-CSF production, specific M-CSF production and cell growth M-CSF production (ng/ml)
Constant Insulin Meat peptone Yeast extract SerEx BSA Linoleic acid–BSA Dextran sulfate *
P < 0.05.
Specific M-CSF production rate (ng/105 cells/day)
Cell growth (105 cells/ml)
Coefficients
Significance
Coefficient
Significance
Coefficient
Significance
560 122 43 40 151 11 12 32
0.000 0.013* 0.298 0.334 0.004* 0.784 0.753 0.427
5.05 1.36 0.35 0.25 1.58 0.48 0.61 0.41
0.000 0.007* 0.381 0.523 0.003* 0.236 0.143 0.308
32.06 4.81 5.31 0.06 4.19 0.06 4.81 0.56
0.000 0.038* 0.026* 0.975 0.063 0.975 0.038* 0.779
C.-H. Liu, T.-Y. Chang / Process Biochemistry 41 (2006) 2314–2319
2317
Table 3 Experimental design and results of the central composite designa
1 2 3 4 5 6 7 8 9(C) 10(C) 11(C) 12(C) 13(C) a
Insulin (mg/l)
SerEx (%)
Cell number S.D. (105 cells/ml)
M-CSF S.D. (ng/ml)
0.064 0.128 0.064 0.128 0.051 0.141 0.096 0.096 0.096 0.096 0.096 0.096 0.096
0.14 0.14 0.28 0.28 0.21 0.21 0.11 0.31 0.21 0.21 0.21 0.21 0.21
3.62 0.43 2.99 0.04 2.85 0.29 4.12 0.07 3.13 0.02 3.05 0.16 5.13 0.64 5.48 0.58 5.33 0.90 5.46 0.06 5.11 0.28 4.97 0.41 2.95 0.18
552 47 479 24 373 35 657 64 386 43 420 36 888 131 741 65 746 106 948 43 806 134 754 49 389 143
Standard deviation (S.D.) was determined in duplicate experiments.
From preliminary experiment, the concentration interval studied in the final optimization was 0.051–0.141 mg/l insulin and 0.113–0.315% SerEx. The concentration for each supplement, central composite design matrix, and experimental results are shown in Table 3. Five repeats are included at the center of the design, and the total number of test runs for this design is 13. The full second-order polynomial model for MCSF production obtained by the rsreg procedure of SAS software are shown in the following equation: M-CSF ¼ 808:82 þ 32:64 insulin 26:11 SerEx 227:40 insulin2 þ 89:25 insulin SerEx 20:67 SerEx2 (1)
Fig. 2. Contour plot of M-CSF production by addition of insulin and SerEx from the response surface model (Eq. (1)).
respectively. The F-value indicated that the second-order response surface model was significant at 0.5% level. Accordingly, M-CSF production by CHO cells is well represented by the second order model. Besides, the fact that F-value of the lack of fit test was insignificant (F-value = 1.26, probability > 0.4008) indicated that the model was not influenced by the error of lack of fit. Canonical analysis provided by SAS software is a mathematical procedure applied to simplify a second-order polynomial model and find the extreme value of the response surface model. The results of canonical analysis demonstrated that the response surface had a maximum M-CSF production at the composition of 0.093 mg/l of insulin and 0.15% of SerEx. The maximum M-CSF
2
The F-value and determination coefficient (R ) for the full quadratic equation for M-CSF production are 10.83 and 0.8856,
Fig. 1. Response surface of M-CSF production by addition of insulin and SerEx from the response surface model (Eq. (1)).
Fig. 3. Effect of insulin and SerEx concentration on M-CSF production. The short dash line shows the theoretical prediction calculated from the response surface model (Eq. (1)). (~) Experimental data and (*) medium composition.
2318
C.-H. Liu, T.-Y. Chang / Process Biochemistry 41 (2006) 2314–2319
Fig. 4. Trends of M-CSF production in different media, including DMEM/F12IS, HyQ, and DMEM/F12 + 5% FBS. Results were expressed as means of duplicate samples and coefficient of variation was less than 10%.
production predicted by the model is 817 ng/ml of M-CSF. The developed serum-free medium, including DMEM/F12, 0.093 mg/l of insulin, and 0.15% of SerEx, was termed as DMEM/F12-IS. The influence of insulin and SerEx on M-CSF production was visualized by virtue of three-dimension plot (Fig. 1) and contour plot (Fig. 2). A set of experiments with different concentrations of insulin and SerEx were performed to check how experimental data fit the data predicted by the model. As shown in Fig. 3, an experimental value of 1165 ng/ ml M-CSF was obtained at the optimal medium composition. Good agreement was shown to exist between the experimental points and the values predicted by the response model. In order to make a comparison between the DMEM/F12-IS, HyQ, and DMEM/F12 + 5% FBS in regard to M-CSF production, CHO cells were cultivated in three media for 6 days (Fig. 4). Interestingly, M-CSF production did not increase after day 4 in DMEM/F12-IS medium, which was different from the trend in HyQ and DMEM/F12 + 5% FBS cultures. The cease of M-CSF production was related to the viable cell growth. CHO cells
stopped growing after 4-day culture in DMEM/F12-IS medium as shown in Fig. 5. Whereas the formulation of DMEM/F12-IS medium was designed according to 4-day culture results, the formulated supplements were only enough for CHO cells to grow in a 4-day period. Accordingly, cease of growth and MCSF production might be due to the limitation of key ingredients (insulin and SerEx) after the elongated cultivation. The average specific M-CSF production rates for HyQ, DMEM/F12-IS and DMEM/F12 + 5% FBS were 46, 55 and 56 ng M-CSF/105cells/day, respectively. Although CHO cells in DMEM/F12-IS had high specific M-CSF production rates, the cell density was lowest among three tested media. Consequently, product of specific M-CSF production rates and viable cell concentration, i.e. M-CSF volumetric production in this optimized DMEM/F12-IS, was 91% of that in DMEM/F12 + 5% FBS and 83% of commercial HyQ medium after 4-day culture. The better M-CSF production in HyQ medium may be due to the CHO cells were already adapted to grow in HyQ medium. Further adaptation of the CHO cells in the new developed medium was needed for the better growth and M-CSF production. Taken together, pre-weaning strategy combined with experimental designs could simplify and accelerate the procedures for serum-free medium optimization. 4. Conclusion We combined pre-weaning strategy and central composite design to successfully develop the serum-free medium for MCSF by Chinese hamster ovary (CHO) cells. The optimized serum-free medium (DMEM/F12-IS) included the following compositions: DMEM/F12, 0.093 mg/l of insulin, and 0.15% of SerEx. An M-CSF titer in this DMEM/F12-IS medium was 91% of that in DMEM/F12 + 5% FBS. The protocol presented here could be applied to develop serum-free medium for other mammalian and insect cells with reduced effort. Acknowledgements This work was supported by grants NSC 94-2214-E-182-008 and CMRPD 32051. References
Fig. 5. Trends of cell growth in different media, including DMEM/F12-IS, HyQ, and DMEM/F12 + 5% FBS. Results were expressed as means of duplicate samples and coefficient of variation was less than 15%.
[1] Li G, Song YH, Wu KF, Lin YM, Cao ZY, Zheng GG. Clone and expression of mutant M-CSF and its receptor from human leukemic cell line J6-1. Leukemia Res 2002;26:377–82. [2] Freshney RI, Serum-free media. In culture of animal cells: a manual of basic technique, 4th ed., New York: Wiley; 2000. p. 105–20. [3] Castro PML, Hayter PM, Ison AP, Bull AP. Application of a statistical design to the optimization of culture medium for recombinant interferongamma production by Chinese hamster ovary cells. Appl Microbiol Biotechnol 1992;38:84–90. [4] Kim EJ, Kim NS, Lee GM. Development of a serum-free medium for the production of humanized antibody from Chinese hamster ovary cells using statistical design. In Vitro Cell Dev Biol Anim 1998;34:757–61. [5] Lee GM, Kim EJ, Kim NS, Yoon SK, Ahn YH, Song JY. Development of a serum-free medium for the production of erythropoietin by suspension culture of recombinant Chinese hamster ovary cells using a statistical design. J Biotechnol 1999;69:85–93.
C.-H. Liu, T.-Y. Chang / Process Biochemistry 41 (2006) 2314–2319 [6] Liu CH, Chu IM, Hwang SM. Factorial designs combined with the steepest ascent method to optimize serumfree media for CHO cells. Enzyme Microb Technol 2001;28:314–21. [7] Deshpande RR, Wittmann C, Heinzle E. Microplates with integrated oxygen sensing for medium optimization in animal cell culture. Cytotechnol 2004;46:1–8. [8] Kitano M. Serum-free media. In: Ho CS, Wang DIC, editors. Animal cell bioreactors. Boston: Butterworth–Heinemann Press; 1991. p. 73–106. [9] Montgomery DC. Introduction to factorial designs. In: In design and analysis of experiments. New York: Wiley; 1991. p. 197–56. [10] Barnes D, Sato G. Methods for growth of culture cells in serum-free medium. Anal Biochem 1980;102:255–70. [11] Dee KU, Shuler ML, Wood HA. Inducing single-cell suspension of BTITN5B1-4 insect cells: the use of sulfated polyanions to prevent cell aggregation and enhance recombinant protein production. Biotechnol Bioeng 1997;54:191–205. [12] Jan DCH, Jones SJ, Emery AN, Al-Rubeai M, Peptone. a low-cost growthpromoting nutrient for intensive animal cell culture. Cytotechnol 1994;16:17–26.
2319
[13] Saha SN, Sen AK. Use of yeast extract of partial replacement of serum for growth of foot-to-mouth virus in BHK-21 cells. Indian J Anim Sci 1995;65:13058. [14] Sung YH, Chung JY, Lee GM, Lim SW. Yeast hydrolysate as a low-cost additive to serum-free medium for the production of human thrombopoietin in suspension cultures of Chinese hamster ovary cells. Appl Microbiol Biotechnol 2004;63:527–36. [15] Ryu JS, Kim TK, Chung JY, Lee GM. Osmoprotective effect of glycine betaine on foreign protein production in hyperosmotic recombinant chinese hamster ovary cell cultures differs among cell lines. Biotechnol Bioeng 2000;70:167–75. [16] Fox SR, Patel UA, Yap MG, Wang DIC. Maximizing interferon gamma production by Chinese hamster ovary cells through temperature shift optimization: experimental and modeling. Biotechnol Bioeng 2004;85:177–84. [17] Li J, Sun X, Zhang Y. Improvement of hepatitis B surface antigen expression by dimethyl sulfoxide in the culture of recombinant Chinese hamster ovary cells. Process Biochem 2006;41: 317–322.
Rational development of serum-free medium for Chinese hamster ovary cells Chi-Hsien Liu *, Ting-Yan Chang Graduate Institute of Biochemical and Biomedical Engineering, Chang Gung University, Taoyuan, Taiwan Received 13 April 2006; received in revised form 29 May 2006; accepted 5 June 2006
Abstract Recombinant Chinese hamster ovary (CHO) cells which produce macrophage colony-stimulating factor (M-CSF) were used as a model to systematically develop the serum-free medium. Pre-weaning strategy combined with experimental design was adopted to develop the serum-free medium for CHO cells in this study. First, CHO cells were adapted to grow in a commercial serum-free medium. Then, these weaned cells were adopted to develop our own serum-free medium using experimental design. Insulin and SerEx had significantly stimulatory influence on M-CSF production (P < 0.05) among the seven screened supplements by use of fractional factorial design. Response surface methodology was consequently applied to find optimal concentrations of insulin and SerEx for M-CSF production. By virtue of the pre-weaning strategy and experimental designs, the development of serum-free medium could be simplified and accelerated. # 2006 Elsevier Ltd. All rights reserved. Keywords: CHO; M-CSF; Serum-free medium; Experimental design; Pre-weaning strategy
1. Introduction Macrophage colony-stimulating factor (M-CSF) is a multiple functional cytokine not only for hemopoietic system, but also for non-hemopoietic systems, such as breast function, pregnancy, and osteoclastogenesis. M-CSF also acts as the principal regulator of the survival, proliferation, and differentiation of macrophages and their precursors. In addition, MCSF plays important roles in infections as an inflammatory cytokine [1]. M-CSF secreted Chinese hamster ovary (CHO) cells were used as a model to rationally develop the serum-free medium in this study. Serum is the most important supplement in the traditional medium for the culture of animal cells. However, serum has many shortages such as batch variability, limited supply, high cost, interference with purification of recombinant product, and possibility of viral contamination. The worsening situation worldwide in the supply of serum, the pressure from biotechnology for easier product purification, and the need for consistent and defined conditions for the culture of animal cell will force the adoption of serum-free media [2]. There are
* Corresponding author. Tel.: +886 3 2118800x3146; fax: +886 3 2118700. E-mail address: [email protected] (C.-H. Liu). 1359-5113/$ – see front matter # 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2006.06.008
several commercial serum-free media available for CHO cells. However, their compositions are proprietary, further improvement of these media is difficult, and their prices are expensive. New recombinant strains might have different nutrient requirement for their unique recombinant products. Sometimes biotechnologists would incorporate new ingredients into the serum-free formula. Therefore, we need to develop new serumfree media. Compositions in serum, including cell adhesion factors, peptides, essential nutrients, and hormones, account for the proliferation effects on cultured cells. The availability of purified serum composition contributes to the development of serum-free medium. It is a very time-consuming and laborious process using the traditional one-factor at a time approach to develop the serum-free medium. However, the introduction of statistical design significantly reduces the labors of composition screening and concentration optimization. Castro et al. [3], Kim et al. [4], and Lee et al. [5] chose Plackett–Burman designs to design medium compositions for CHO cells. Every ingredient is tested at high and low levels (or concentrations). Components with stimulatory influence (P < 0.1) are added at high concentrations to complete the optimal media. Nevertheless, optimal concentrations of stimulatory ingredients could not be determined by use of this approach. There are several authors using different statistical methods to find optimal
C.-H. Liu, T.-Y. Chang / Process Biochemistry 41 (2006) 2314–2319
ingredient concentrations for serum-free media. For example, Liu et al. used the steepest ascent method to find optimal concentrations of stimulatory ingredients for CHO cells [6]. Central composite design was applied to optimize concentrations of glucose, glutamine, and inorganic salts for cultivation of a CHO cell line [7]. For the development of serum-free medium, biotechnologists usually weaned animal cells from the serum supplemented to the serum-reduced condition. Sequentially, they have to simultaneously screen components and find their optimal concentrations under the serum-free condition. This approach has to carefully maintain the cell viability and density, if not, cells will die completely. This study used pre-weaning strategy to increase the probability of successful growth in development process for serum-free medium. Here CHO cells were firstly adapted to grow in a commercial serum-free medium. Then, these weaned cells were used as seed to statistically develop our own serum-free medium. 2. Materials and methods 2.1. Cell line and cell culture Genetically-engineered CHO cells, obtained from ATCC (CRL-10154), maintained the production of M-CSF under the pressure of 1 mM methotrexate. The cells were initially maintained in DMEM/F12 (a mixture of DMEM and F12; Sigma–Aldrich, St. Louis, MO, USA) supplemented with 5% fetal bovine serum (FBS; Hyclone, Logan, UT, USA), and sequentially adapted to grow in a commercial serum-free medium (HyQ SFM4; Hyclone) according to product’s protocol. In brief, CHO cells in a conventional medium were seeded into a 1:1 ratio of the serum-free medium and serum-supplemented medium with the cell density of 5 105 viable cells/ml. The culture was cultivated until viable cells continued one to two population doublings. Then the culture was seeded into a 2:1 ratio of the serum-free medium and serum-supplemented medium. Weaned cells were subcultured in this manner until the serum concentration was reduced below 0.1 % and the viable cell concentration is greater than 2 106 cells/ml. These serum-free medium adapted CHO cells were used for further experiment. Cell culture was performed in triplicate in 12-well tissue culture plates (BD Biosciences, Rockville, MD, USA), containing 1 ml of DMEM/F12 medium and tested supplements. The basal medium used here was DMEM/F12 according to the suggestion of Kitano [8]. CHO cells were inoculated at a concentration of 105 cells/ml and harvested after 96 h of cultivation. The viability was determined by the trypan blue exclusion method and the cell concentration was determined by Multisizer 3 (Beckman Counter, Beds, England, UK). Aliquots of spent media were taken and kept frozen at 20 8C for further ELISA analysis.
2.2. M-CSF assay The concentration of M-CSF in media samples after appropriate dilution was detected by use of ELISA kit (DuoSet DY216; R&D systems, Minneapolis, MN, USA). A solution of 3,30 ,5,50 -tetramethyl-benzidine and H2O2 (Kirkeguard & Perry Laboratories Inc., Galthersburg, MD, USA) was added as the peroxidase substrate. After 20-min incubation, 2N H2SO4 solution was added to stop the reaction and the plates were measured at a wavelength of 450 nm. Each sample was tested in duplicate to reduce the uncertainty of ELISA measurement.
2.3. Experimental design and statistical analysis The rules and design of factorial experiments are provided in the statistical textbook of Montgomery [9]. Fractional factorial design and central composite design data were regressed and analyzed by running the glm and rsreg procedures in SAS software (Cary, NC, USA). The linear-regression coefficients of fractional factorial design can be applied in selection of important
2315
medium components. In the screening tests, the magnitude and sign of the regression constants can be used to identify the significance of the variables on responses such as cell density and M-CSF production. If the coefficient is relatively large, it has more significant effects on the response as compared to the small one does. Furthermore, the variable with positive fitted constant is helpful to the response and that one with negative coefficient has inhibitory effects on the response. The three-dimension and contour plots were generated by Sigmaplot package (Systat Co., Point Richmond, USA).
2.4. Chemicals Insulin, meat peptone, Pluronic F68, bovine serum albumin (BSA), linoleic acid–albumin, methotrexate, and dextran sulfate (Average MW 5000 by low angle laser light scattering method) were purchased from Sigma–Aldrich. Yeast extract and SerEx were obtained from Difco (Sparks, MD, USA) and PAA Laboratories (Linz, Austria), respectively.
3. Results and discussion 3.1. Additives screening Based on the results of preliminary experiments and literature review, seven ingredients including insulin, meat peptone, yeast extract, SerEx, BSA, linoleic acid–BSA, and dextran sulfate were selected to evaluate their stimulatory effects on CHO cells under serum-free conditions. Bovine serum albumin (BSA) has many physical functions including acting as a carrier for fatty acids, trace mineral, and functioning as a detoxifier for H2O2. Exogenous lipids or their precursors such as linoleic acid were required for cell proliferation. Insulin is an important supplement in a serum-free medium. It can stimulate the proliferation of animal cells [10]. SerEx, containing growth factors and hormones, is completely defined and free of animal derived components. Dextran sulfate with MW 12,500 was reported to be an effective agent for inducing single cell cultures and enhancing recombinant protein production of insect cells under serum-free conditions [11]. Therefore, insulin, BSA, linoleic acid–BSA, and SerEx were included in this screening test. Other additives such as meat peptone, yeast extract, and dextran sulfate were also considered. Meat peptone was adopted owing to its potential role in promoting cell growth and specific antibody production [6,12]. Yeast extract can partially replace serum in culture of BHK-21 cells [13] and can be a low-cost additive to serum-free medium of CHO cells [14]. The fractional factorial design was applied to test the stimulatory effects of seven ingredients on cell growth and MCSF production in this study. From the principle of the sparsity of effects [9], a system or process is likely to be driven primarily by some main and low-order interaction effects. Effects of interaction were neglected in our first-order linear model to simplify and accelerate the screening process. However, the interaction between the selected key components will be discussed in the following optimization Section 3.2. The 273 fractional factorial design could efficiently test 7 compounds with 16 trials and preserve enough degrees of freedom to evaluate performance of these supplements. The concentration of each component and results of cell growth, M-CSF production and specific M-CSF productivity are demonstrated in Table 1. Regression results of fractional factorial design (Table 2) indicated that addition of insulin and
2316
C.-H. Liu, T.-Y. Chang / Process Biochemistry 41 (2006) 2314–2319
Table 1 Matrix and results of 273 fractional factorial design
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
A
B
C
D
E
F
G
M-CSF production (ng/ml)
Cell growth (105 cells/ml)
Specific M-CSF production rate (ng/105 cells/day)
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
204 475 138 556 213 792 231 667 688 776 600 729 870 806 563 656
0.80 3.71 1.11 6.33 1.21 7.76 1.43 5.42 5.82 7.40 5.61 7.64 5.01 5.93 8.55 7.11
64 32 31 22 44 26 40 31 30 26 27 24 43 34 16 23
A: insulin, 5 mg/L; B: meat peptone, 2.5 mg/ml; C: yeast extract, 2.5 mg/ml; D: SerEx, 1%; E: BSA, 1 mg/ml; F: linoleic acid–albumin, 1 mg/ml; G: dextran sulfate, 500 mg/ml. 1: addition, 1: no addition. Cell was cultured in DMEM/F12 supplemented with indicated ingredients for 4 days.
SerEx greatly facilitated cell growth of CHO cells at significant level of 0.01. Additionally, meat peptone, yeast extract, linoleic acid–BSA had stimulatory effects on cell growth. Whereas, BSA and dextran sulfate had inhibitory effects on cell growth in the tested concentration range. Regression results of M-CSF production (Table 2) indicated that addition of insulin and SerEx enhanced M-CSF production by CHO cells at significant level of 0.05. Besides, yeast extract had stimulatory effects on M-CSF production. Meat peptone, BSA, linoleic acid–BSA, and dextran sulfate had inhibitory effects on M-CSF production in the tested concentration range. Furthermore, product of the specific cellular productivity and the integrated viable cell density can determine the final volumetric production. Besides effects of tested components on cell growth, effects of tested components on specific M-CSF productivity were also evaluated. As shown in Table 1, poor cell growth often accompanied good specific M-CSF productivity. Different culture conditions such as hyperosmotic pressure, hypothermia and chemical were reported to inhibit specific growth rate in hybridoma and CHO cell cultures [15–17]. The enhanced recombinant protein productivity also accompanied the
decreased cell growth in these mammalian cells. The real mechanisms of decreased cell growth and enhanced productivity merit more investigation. In Table 2, insulin, meat peptone and linoleic acid were found to significantly (P < 0.05) inhibit specific M-CSF productivity by regression analysis. A marginal improvement of specific M-CSF productivity was achieved when yeast extract and BSA were supplemented in the basal medium (Table 2). However, it should be noted that the aim of our optimization was to maximize volumetric productivity of M-CSF. Therefore, the most important ingredients on M-CSF volumetric production, i.e. SerEx and insulin, were optimized to find their formulation by the following two central composite design. 3.2. Optimization experiments Based on the results of screening experiments, insulin and SerEx were finally selected as supplements for serum-free medium. Central composite design and response surface methodology were adopted as an efficient way to find an optimal culture medium for M-CSF production by CHO cells.
Table 2 Regression coefficients and significance analysis of a fractional factorial design for M-CSF production, specific M-CSF production and cell growth M-CSF production (ng/ml)
Constant Insulin Meat peptone Yeast extract SerEx BSA Linoleic acid–BSA Dextran sulfate *
P < 0.05.
Specific M-CSF production rate (ng/105 cells/day)
Cell growth (105 cells/ml)
Coefficients
Significance
Coefficient
Significance
Coefficient
Significance
560 122 43 40 151 11 12 32
0.000 0.013* 0.298 0.334 0.004* 0.784 0.753 0.427
5.05 1.36 0.35 0.25 1.58 0.48 0.61 0.41
0.000 0.007* 0.381 0.523 0.003* 0.236 0.143 0.308
32.06 4.81 5.31 0.06 4.19 0.06 4.81 0.56
0.000 0.038* 0.026* 0.975 0.063 0.975 0.038* 0.779
C.-H. Liu, T.-Y. Chang / Process Biochemistry 41 (2006) 2314–2319
2317
Table 3 Experimental design and results of the central composite designa
1 2 3 4 5 6 7 8 9(C) 10(C) 11(C) 12(C) 13(C) a
Insulin (mg/l)
SerEx (%)
Cell number S.D. (105 cells/ml)
M-CSF S.D. (ng/ml)
0.064 0.128 0.064 0.128 0.051 0.141 0.096 0.096 0.096 0.096 0.096 0.096 0.096
0.14 0.14 0.28 0.28 0.21 0.21 0.11 0.31 0.21 0.21 0.21 0.21 0.21
3.62 0.43 2.99 0.04 2.85 0.29 4.12 0.07 3.13 0.02 3.05 0.16 5.13 0.64 5.48 0.58 5.33 0.90 5.46 0.06 5.11 0.28 4.97 0.41 2.95 0.18
552 47 479 24 373 35 657 64 386 43 420 36 888 131 741 65 746 106 948 43 806 134 754 49 389 143
Standard deviation (S.D.) was determined in duplicate experiments.
From preliminary experiment, the concentration interval studied in the final optimization was 0.051–0.141 mg/l insulin and 0.113–0.315% SerEx. The concentration for each supplement, central composite design matrix, and experimental results are shown in Table 3. Five repeats are included at the center of the design, and the total number of test runs for this design is 13. The full second-order polynomial model for MCSF production obtained by the rsreg procedure of SAS software are shown in the following equation: M-CSF ¼ 808:82 þ 32:64 insulin 26:11 SerEx 227:40 insulin2 þ 89:25 insulin SerEx 20:67 SerEx2 (1)
Fig. 2. Contour plot of M-CSF production by addition of insulin and SerEx from the response surface model (Eq. (1)).
respectively. The F-value indicated that the second-order response surface model was significant at 0.5% level. Accordingly, M-CSF production by CHO cells is well represented by the second order model. Besides, the fact that F-value of the lack of fit test was insignificant (F-value = 1.26, probability > 0.4008) indicated that the model was not influenced by the error of lack of fit. Canonical analysis provided by SAS software is a mathematical procedure applied to simplify a second-order polynomial model and find the extreme value of the response surface model. The results of canonical analysis demonstrated that the response surface had a maximum M-CSF production at the composition of 0.093 mg/l of insulin and 0.15% of SerEx. The maximum M-CSF
2
The F-value and determination coefficient (R ) for the full quadratic equation for M-CSF production are 10.83 and 0.8856,
Fig. 1. Response surface of M-CSF production by addition of insulin and SerEx from the response surface model (Eq. (1)).
Fig. 3. Effect of insulin and SerEx concentration on M-CSF production. The short dash line shows the theoretical prediction calculated from the response surface model (Eq. (1)). (~) Experimental data and (*) medium composition.
2318
C.-H. Liu, T.-Y. Chang / Process Biochemistry 41 (2006) 2314–2319
Fig. 4. Trends of M-CSF production in different media, including DMEM/F12IS, HyQ, and DMEM/F12 + 5% FBS. Results were expressed as means of duplicate samples and coefficient of variation was less than 10%.
production predicted by the model is 817 ng/ml of M-CSF. The developed serum-free medium, including DMEM/F12, 0.093 mg/l of insulin, and 0.15% of SerEx, was termed as DMEM/F12-IS. The influence of insulin and SerEx on M-CSF production was visualized by virtue of three-dimension plot (Fig. 1) and contour plot (Fig. 2). A set of experiments with different concentrations of insulin and SerEx were performed to check how experimental data fit the data predicted by the model. As shown in Fig. 3, an experimental value of 1165 ng/ ml M-CSF was obtained at the optimal medium composition. Good agreement was shown to exist between the experimental points and the values predicted by the response model. In order to make a comparison between the DMEM/F12-IS, HyQ, and DMEM/F12 + 5% FBS in regard to M-CSF production, CHO cells were cultivated in three media for 6 days (Fig. 4). Interestingly, M-CSF production did not increase after day 4 in DMEM/F12-IS medium, which was different from the trend in HyQ and DMEM/F12 + 5% FBS cultures. The cease of M-CSF production was related to the viable cell growth. CHO cells
stopped growing after 4-day culture in DMEM/F12-IS medium as shown in Fig. 5. Whereas the formulation of DMEM/F12-IS medium was designed according to 4-day culture results, the formulated supplements were only enough for CHO cells to grow in a 4-day period. Accordingly, cease of growth and MCSF production might be due to the limitation of key ingredients (insulin and SerEx) after the elongated cultivation. The average specific M-CSF production rates for HyQ, DMEM/F12-IS and DMEM/F12 + 5% FBS were 46, 55 and 56 ng M-CSF/105cells/day, respectively. Although CHO cells in DMEM/F12-IS had high specific M-CSF production rates, the cell density was lowest among three tested media. Consequently, product of specific M-CSF production rates and viable cell concentration, i.e. M-CSF volumetric production in this optimized DMEM/F12-IS, was 91% of that in DMEM/F12 + 5% FBS and 83% of commercial HyQ medium after 4-day culture. The better M-CSF production in HyQ medium may be due to the CHO cells were already adapted to grow in HyQ medium. Further adaptation of the CHO cells in the new developed medium was needed for the better growth and M-CSF production. Taken together, pre-weaning strategy combined with experimental designs could simplify and accelerate the procedures for serum-free medium optimization. 4. Conclusion We combined pre-weaning strategy and central composite design to successfully develop the serum-free medium for MCSF by Chinese hamster ovary (CHO) cells. The optimized serum-free medium (DMEM/F12-IS) included the following compositions: DMEM/F12, 0.093 mg/l of insulin, and 0.15% of SerEx. An M-CSF titer in this DMEM/F12-IS medium was 91% of that in DMEM/F12 + 5% FBS. The protocol presented here could be applied to develop serum-free medium for other mammalian and insect cells with reduced effort. Acknowledgements This work was supported by grants NSC 94-2214-E-182-008 and CMRPD 32051. References
Fig. 5. Trends of cell growth in different media, including DMEM/F12-IS, HyQ, and DMEM/F12 + 5% FBS. Results were expressed as means of duplicate samples and coefficient of variation was less than 15%.
[1] Li G, Song YH, Wu KF, Lin YM, Cao ZY, Zheng GG. Clone and expression of mutant M-CSF and its receptor from human leukemic cell line J6-1. Leukemia Res 2002;26:377–82. [2] Freshney RI, Serum-free media. In culture of animal cells: a manual of basic technique, 4th ed., New York: Wiley; 2000. p. 105–20. [3] Castro PML, Hayter PM, Ison AP, Bull AP. Application of a statistical design to the optimization of culture medium for recombinant interferongamma production by Chinese hamster ovary cells. Appl Microbiol Biotechnol 1992;38:84–90. [4] Kim EJ, Kim NS, Lee GM. Development of a serum-free medium for the production of humanized antibody from Chinese hamster ovary cells using statistical design. In Vitro Cell Dev Biol Anim 1998;34:757–61. [5] Lee GM, Kim EJ, Kim NS, Yoon SK, Ahn YH, Song JY. Development of a serum-free medium for the production of erythropoietin by suspension culture of recombinant Chinese hamster ovary cells using a statistical design. J Biotechnol 1999;69:85–93.
C.-H. Liu, T.-Y. Chang / Process Biochemistry 41 (2006) 2314–2319 [6] Liu CH, Chu IM, Hwang SM. Factorial designs combined with the steepest ascent method to optimize serumfree media for CHO cells. Enzyme Microb Technol 2001;28:314–21. [7] Deshpande RR, Wittmann C, Heinzle E. Microplates with integrated oxygen sensing for medium optimization in animal cell culture. Cytotechnol 2004;46:1–8. [8] Kitano M. Serum-free media. In: Ho CS, Wang DIC, editors. Animal cell bioreactors. Boston: Butterworth–Heinemann Press; 1991. p. 73–106. [9] Montgomery DC. Introduction to factorial designs. In: In design and analysis of experiments. New York: Wiley; 1991. p. 197–56. [10] Barnes D, Sato G. Methods for growth of culture cells in serum-free medium. Anal Biochem 1980;102:255–70. [11] Dee KU, Shuler ML, Wood HA. Inducing single-cell suspension of BTITN5B1-4 insect cells: the use of sulfated polyanions to prevent cell aggregation and enhance recombinant protein production. Biotechnol Bioeng 1997;54:191–205. [12] Jan DCH, Jones SJ, Emery AN, Al-Rubeai M, Peptone. a low-cost growthpromoting nutrient for intensive animal cell culture. Cytotechnol 1994;16:17–26.
2319
[13] Saha SN, Sen AK. Use of yeast extract of partial replacement of serum for growth of foot-to-mouth virus in BHK-21 cells. Indian J Anim Sci 1995;65:13058. [14] Sung YH, Chung JY, Lee GM, Lim SW. Yeast hydrolysate as a low-cost additive to serum-free medium for the production of human thrombopoietin in suspension cultures of Chinese hamster ovary cells. Appl Microbiol Biotechnol 2004;63:527–36. [15] Ryu JS, Kim TK, Chung JY, Lee GM. Osmoprotective effect of glycine betaine on foreign protein production in hyperosmotic recombinant chinese hamster ovary cell cultures differs among cell lines. Biotechnol Bioeng 2000;70:167–75. [16] Fox SR, Patel UA, Yap MG, Wang DIC. Maximizing interferon gamma production by Chinese hamster ovary cells through temperature shift optimization: experimental and modeling. Biotechnol Bioeng 2004;85:177–84. [17] Li J, Sun X, Zhang Y. Improvement of hepatitis B surface antigen expression by dimethyl sulfoxide in the culture of recombinant Chinese hamster ovary cells. Process Biochem 2006;41: 317–322.