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Research Paper
Enhancement of recombinant human serum albumin in transgenic rice cell culture system by cultivation strategy Yu-Kuo Liu1, Yu-Teng Li2, Ching-Fan Lu1 and Li-Fen Huang2 1 2
Graduate Institute of Biochemical and Biomedical Engineering, Chang Gung University, Kwei-Shan, Taoyuan County 320, Taiwan, ROC Graduate School of Biotechnology and Bioengineering, Yuan Ze University, 135 Yuan-Tung Road, Zhongli, Taoyuan County 320, Taiwan, ROC
Fusion of the sugar-starvation-induced aAmy3 promoter with its signal peptide has enabled secretion of recombinant human serum albumin (rHSA) into the culture medium. To simplify the production process and increase the rHSA yield in rice suspension cells, a one-step strategem without medium change was adopted. The yield of rHSA was increased sixfold by this one-step approach compared with the two-step recombinant protein process, in which a change of the culture medium to sugar-free medium is required. The one-step strategem was applied to check repeated cycle of rHSA production, and the production of rHSA was also higher in each cycle in the one-step, as opposed to the two-step, production process. The use of the one-step process resulted in fewer damaged cells during the cell sugar starvation phase for recombinant protein production. Furthermore, we scaled up the rHSA production in a 2-L airlift and a 2-L stirred tank bioreactor by the one-step approach, and concluded that rHSA can be enriched to 45 mg L1 in plant culture commonly used MS medium by the airlift-type bioreactor. Our results suggest that rHSA production can be enriched by this optimized cultivation strategem.
Introduction Pharmaceutical recombinant proteins in the market are expanding rapidly in recent decades, and they have been produced in various host cell types, which differ in terms of their biosafety, biological activity and protein stability. Like mammalian cells, plant cells are capable of protein posttranslational modification, such as glycosylation, but without the risks associated with human/animal pathogens in mammalian cell cultures. Taking advantage of this absence of risk, plant cells have been used to produce complex therapeutic proteins that are required for biosafety, bioactivity and stability. Rice (Oryza sativa), a crop with the third highest level of cultivation globally, is considered a model plant whose genome has been sequenced, and the technologies for gene transformation and tissue culture have been well developed [1,2]. Its hypoallergenic property also makes rice become an excellent host cell Corresponding authors: Liu, Y.-K. ([email protected]), Huang, L.-F. ([email protected]) www.elsevier.com/locate/nbt
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for recombinant protein production [3,4]. However, the presence of genetically modified plants in the field has raised public concerns and is regulated strictly by governments. A rice suspension cell system is an alternative option to limit public concern about genetically modified organisms, and it has been developed for the efficient scaling up of recombinant protein production, such as for pharmaceutical proteins [5–12], vaccines [13,14], or antibodies [3,15]. A platform for recombinant protein production in rice suspension cells has been established using a rice a-amylase gene promoter, RAmy3Dp/aAmy3p, the activity of which is induced by sugar starvation [16]; its signal peptide allows the secretion of recombinant proteins outside the cells. These properties motivated the development of a two-step process for producing recombinant protein by using RAmy3Dp/aAmy3p. In the first step, the transgenic rice cells are cultured in sucrose containing medium to increase the cell number and maintain cell viability and cell activity. In the second step, the sucrose-rich medium is removed from the amplified cells, and the sugar-free medium is added to http://dx.doi.org/10.1016/j.nbt.2015.03.001 1871-6784/ß 2015 Elsevier B.V. All rights reserved.
New Biotechnology Volume 32, Number 3 May 2015
Materials and methods Plant material The genetic background of rice (O. sativa L) cell line used in this study is Tainung No. 67. Rice grains were dehulled, sterilized with 2.4% sodium hypochlorite for 1 hour, washed extensively with sterile water, and placed on N6 agar medium [29] containing 3% sucrose and 10 mM 2,4-dichlorophenoxyacetic acid (2,4-D) (N6D) at 288C in the dark to induce calli. Calli were subcultured in liquid Murashige and Skoog (MS) medium [30] containing 3% sucrose and 10 mM 2,4-D at 288C in the dark to establish suspension-
cultured cells. The resultant suspension-cultured cells were agitated on a reciprocal shaker at 120 rpm and incubated at 288C in the dark.
Rice transformation The plasmid pA3HSA, which contains an aAmy3 promoter – signal peptide – HSA fusion construct, was generated in our previously reported work [7]. Agrobacterium tumefaciens strain EHA105 [31] was used as a host stain for pA3HSA by electroporation, and rice calli were transformed as described previously [32]. Selection of transformed calli was performed on N6 agar medium with hygromycin (50 mg L1).
Characterization of the transgenic cell lines The presence of the transgene insertion in rice calli was analyzed by genomic DNA PCR. Genomic DNA was isolated from putative transgenic calli and then subjected to PCR by using primers HSA-F (50 -GGGCATGTTTTTGTATGAAT-30 ) and HSA-R (50 -TTATAAGCCTAAGGCAGCTT-30 ).
RT-PCR Total RNA was isolated from rice transgenic cell lines using Trizol (Invitrogen, Carlsbad, CA, USA) and treated with RNase-free DNase. First-strand cDNA was synthesized by SuperScript III Reverse Transcriptase (Invitrogen) with an oligo-dT primer. A 20-fold dilution of the first-strand cDNA was subjected to PCR with gene-specific primers; the HSA-F and HSA-R were used for HSA; the Amy3F (50 -TACAGCGTCTGGGAGAAGGGGTC-30 ) and Amy3R (50 -TGCCCCGCAATTAACCTAGAGGC-30 ) were used for aAmy3; the Actin-F (50 -CTGATGGACAGGTTATCACC-30 ) and Actin-R (50 -CAGGTAGCAATAGGTATTACAG-30 ) were used for Act1.
Protein gel blot analysis To obtain total soluble secretory proteins from rice suspension cells, the rice cultured medium was filtered through Whatman No. 1 filter paper to remove cells, and then centrifuged at 18,000 g at 48C for 15 min to obtain the supernatant. The concentration of protein in the supernatant was measured using the Bio-Rad Protein Assay reagent (Bio-Rad, Hercules, CA). Western blotting was performed as described previously [7]. The rabbit antiserum to HSA (ICN Pharmaceuticals, Costa Mesa, CA), applied as the primary antibody, was diluted 1000-fold before use. Then, a 2000-fold dilution of horseradish peroxidase conjugated goat secondary antibody against rabbit IgG (Pierce, Rockford, IL) was diluted before use. Chemiluminescent signals emitted from the complex of primary and secondary antibodies were detected by using the Lumi-light Western blotting substrate (Roche, Basel, Switzerland).
Enzyme-linked immunosorbent assay (ELISA) The yields of rHSA secreted from transgenic rice cells into cultured medium were measured by a sandwich ELISA method as described previously [7]. Each well in the microtiter plates was coated with 10 mg/mL goat antiserum to HSA at 258C for 4 hours. Then, 50 mL of each cell-cultured medium sample was added to individual wells and incubated at 378C for 1 hour. Subsequently, 10 mg/mL rabbit anti-HSA polyclonal antibodies (ICN Pharmaceuticals) was added to the wells and incubated at 378C for 1 hour. Peroxidase-conjugated anti-rabbit IgG antibodies were diluted 2000-fold and apwww.elsevier.com/locate/nbt
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produce recombinant protein. Several recombinant proteins have been produced successfully and secreted into the culture medium from these sugar-starved transgenic rice cells by using this two-step process [7,9,10,14,15,17–20]. However, as described above, exposure to two types of culture medium is required in the two-step production procedure, which increases both the cost of the process and the risk of contamination when changing the medium. Previously, we reported that the production of a recombinant protein, mouse granulocyte-macrophage colony-stimulating factor (rmGM-CSF), could be achieved in one step by using a cultivation strategy that does not require a change of medium [11]. Transgenic rice suspension cells were cultured in N6D medium with 3% sucrose and rmGM-CSF was produced upon sucrose depletion in the medium. The level of rmGM-CSF production was doubled compared with that in the two-step production process [11]. Furthermore, rmGM-CSF production was scaled up to bioreactor levels by using this cultivation strategy [11]. However, more successful examples of such production are required to determine whether other recombinant proteins or other common plant growth media can also benefit from this cultivation strategy. Human serum albumin (HSA) is a single unglycosylated protein that contains 585 amino acid residues and forms 17 pairs of disulfide bonds. HSA is the most abundant blood plasma protein and plays important roles in the regulation of osmotic pressure and pH, as well as acting as a versatile carrier for hormones and numerous drugs [21]. HSA is frequently applied in medical treatments, such as surgery and hepatocirrhosis, and also widely used in biochemical applications, such as vaccine formulation and cell culture medium. The current major HSA source is human plasma, but its availability is insufficient to meet the increasing medical demand. Moreover, the use of human plasma is associated with a risk of disease transmission, such as hepatitis B, syphilis, and HIV [22]. In order to meet the increasing demand and assuage biosafety concerns, several platforms for producing recombinant protein, including rice suspension cells, have been developed for producing recombinant HSA (rHSA) [7,23–28]. In our previous work, we generated a transgenic rice cell line by Agrobacterium-mediated transformation, and produced rHSA in the culture medium by a two-step process [7]. In the study reported herein, we investigated the effects of various strategies for producing rHSA by culturing transgenic rice suspension cells in flasks. We also demonstrated that the yield of rHSA was increased sixfold when the culture medium was not changed after sucrose was exhausted naturally, compared with that upon changing to sucrose-free medium. Furthermore, we scaled up the production of rHSA successfully in a modified bioreactor by this optimized cultivation strategy.
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plied to the well. The samples were incubated at 378C for 1 hour, and ABTS substrate solution (Sigma, St. Louis, MO) was added to the wells. The optical density (OD) of each well was measured in an Epoch Multi-Volume Spectrophotometer System (BioTek, Winooski, VT) at 450 nm.
Determination of cell viability
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Cell viability was estimated by reduction of triphenyltetrazolium chloride (TTC), as described by Chen et al. [33]. The cell samples were incubated with TTC solution at room temperature in the dark for 12 hours. After ethanol extraction, OD 530 was measured with an Epoch Multi-Volume Spectrophotometer System (BioTek).
Determination of cell dry weight Cells were collected from the liquid culture medium, and immediately washed three times with distilled water. The dry weight was determined after drying at 808C until constant weight.
Culture of rice suspension cells in a bioreactor Rice suspension cells were grown and HSA was produced in a 3-L modified bubble-column bioreactor (MBCR) and a 3-L StirredTank bioreactor FB-3B (STR, Firstek Scientific, Taipei, Taiwan) with a single pitched blade (low-shear) impeller, respectively. The diameter of the inner cylinder of the MBCR was 10 cm and its height was 40 cm. Rice suspension cells were cultured in the STR and MBCR bioreactors at 288C (18C) in the dark. The bioreactors were filled with 2.0 L of sterilized MS medium, and inoculated at 3% (v/ v) with 7-day-old rice suspension cells. The aeration rate was controlled at 1.0 L/min in MBCR and 0.5 L/min in STR using an airflow meter (RK1150, Kojima, Kyoto, Japan). The agitation speed was controlled at 75 rpm in STR. The culture medium was refreshed with MS medium for every 8 days until cell density reached around 24% and the sucrose concentration of the medium and yield of HSA were determined.
Results and discussion Generation of transgenic rice cell lines expressing HSA Previously, a transgenic cell line expressing rHSA was generated and rHSA yield reached 76 mg L1 in a bioreactor with 50% cell density [7]. However, this cell line starts to form a very fine yellowwhite powder-like aggregate of cell clumps, then secretes less and less rHSA after years of continuous cell culture. Therefore, it is not suitable for testing the proposed cultivation strategy for increasing the yield of rHSA yield. To obtain rHSA-producing rice suspension cells with normal morphology and physiology, we regenerated transgenic cell lines harboring an HSA expression cassette, which contains the aAmy3 promoter-signal sequence and the HSA cDNA sequence, by the Agrobacterium-mediated transformation system. Several putative transgenic calli that were resistant to hygromycin were subjected to transgene analysis (Fig. 1). The line of H11 that produced higher levels of rHSA mRNA (Fig. 1) was selected to generate suspension-cultured cells for further investigation.
rHSA production in a transgenic rice cell line cultured by a twostage production process To monitor the level of production of rHSA proteins secreted from rice suspension cultured cells for the two-step production process, a transgenic rice suspension cell line was established in MS medium 330
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FIGURE 1
Generation of rHSA expression rice calli. Calli were starved in sugar-free MS medium for 72 hours and used for (a) DNA detection by genomic PCR, and (b) mRNA detection by RT-PCR. Specific primer sets were used in PCR for HSA, ActI, and aAmy3.
(the most commonly used plant culture medium) with 10 mM 2,4-D and 3% sucrose, then incubated in sugar-free medium at an initial cell density of 3% (cell volume/medium volume) for various periods of time. Cell viability dropped to 40% on day 1 after sugar starvation, and then decreased progressively until almost no living cells were left by day 6 (Fig. 2). Cell dry weight was used to represent cell growth, and it decreased slightly and remained at about 50% of the original level on day 6 after sugar starvation (Fig. 2). Secreted rHSA proteins accumulated steadily in the culture medium, peaked at around 2.5 mg L1 on day 6, and were maintained until day 10 after sugar starvation (Fig. 2c–e). These results indicate that the recombinant proteins were secreted from MS cultured sugar-starved rice cells.
Enhancement of recombinant protein productivity by cultivation strategy Previously, we showed that the production of rmGM-CSF was increased by using an approach that does not require change of medium when grown in N6D culture medium [11]. A similar cultivation strategem was used to determine whether it is also useful to increase the production of rHSA protein in cells cultured with MS medium. Cells of the H11 line were incubated in MS medium and the yield of secreted rHSA was monitored. Suspension cells were immersed at an initial density of 3% (v/v) in MS medium that contained 3% sucrose. Cell growth (Fig. 3a) and cell viability (Fig. 3b) were increased and maintained in MS containing sucrose, respectively, then decreased after the sucrose almost ran out on day 10 (Fig. 3c). Secreted rHSA was detectable on day 11, and the protein yield reached 15 mg L1 on day 14 (Fig. 3d). The yield of secreted rHSA increased sixfold when using the approach that does not require a change of medium, compared with 2.5 mg L1
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FIGURE 3
FIGURE 2
rHSA production in sucrose-free MS culture medium. The cell line H11 was cultured in sucrose-free medium for various lengths of time. Cells were collected for the determination of cell dry weight (a) and cell viability (b). Samples of culture media were collected to determine the concentration of rHSA by ELISA (c), silver staining (d), and Western blotting (e). The error bar represents the standard deviation from triplicate cultures. Commercial HSA, 30 ng, produced in E. coli (eHSA) was applied as a positive control in (d,e). The rHSA are indicated by black arrows.
Secreted rHSA proteins were enriched in the culture medium by the one-step cultivation strategem. Four different concentrations of suspension cells from the cell line H11 [3% (v/v), 6%, 12%, and 24%] were cultured in MS medium containing sucrose for various lengths of time. The cell dry weight (a) and viability (b) of the cells were determined after sample collection. In the meantime, the concentrations of sucrose (c) and rHSA proteins (d) in the culture medium were determined. The error bars represent the standard deviation from triplicate cultures.
(Fig. 2c) in the two-step process in which the original culture medium has to be replaced by sucrose-free medium. The yield of rHSA was also determined when initial cell densities of 6%, 12%, and 24% were used. Similar patterns of the changes in cell growth and cell viability were obtained as for an initial cell density of 3% (Fig. 3a,b). The times at which the sucrose ran out with cell www.elsevier.com/locate/nbt
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densities of 6%, 12%, and 24% were earlier than with 3% (Fig. 3c). Under cell densities of 24%, rHSA began to be produced on day 5 (Fig. 3d), when the rice cultured cells started to starve (Fig. 3c), and the yield reached around 25 mg L1 on day 7 (Fig. 3d). These results indicate that a greater amount of rHSA can be produced by increasing the cell density in this one-step cultivation strategy without a change of medium. Our data also support previous reports that a high yield of recombinant protein could be achieved by increasing the cell density in rice suspension cells [7,11]. On the other hand, the level of rHSA could be increased sixfold in MS medium by adopting the one-step approach without a change of medium. During the phase of recombinant protein production, the slope of decrease in cell viability in the one-step approach was clearly gentler than that
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in the two-step process. In other words, the rice cells were healthier in the one-step process, so more recombinant proteins were produced during the production phase.
Production of rHSA under repeated cycles of the approach without change of medium When recombinant rHSA proteins were secreted from rice suspension cells during starvation, cell viability decreased. To increase the productivity of rHSA in the same batch of suspension cultured rice cells, we examined the stability of rHSA production in repeated cycles of the cultivation approach without change of medium. The HSA cell line was cultured at a cell density of 24% in sucrose-containing MS medium for 5 days. Then the medium was harvested. This procedure comprised one cycle. The cycle was
FIGURE 4
Stable production of rHSA under repeated cycles of the two cultivation strategies. (a) In the one-step approach with medium change, 12 mL of H11 suspension cells was cultured in 50 mL of sucrose-containing MS medium for 5days, which was then replaced with fresh sucrose-containing MS medium every 5 days for three cycles (in a period of 20 days). (b) In the two-stage process, 12 mL of H11 suspension cells was cultured in 50 mL of sucrose-containing MS medium for 3days, which was replaced with 50 mL of sucrose-free MS medium for 3 days. The cycles of 3-day growth phase and 3-day production phase were repeated three times (in a period of 21 days). Cells were collected for the determination of cell volume and viability, and media were collected for determination of the yields of secreted rHSA by ELISA. Error bars indicate standard deviation from triplicate cultures. For cell viability, the capability of TTC reduction in cells cultured for 5 days in sucrosecontaining medium in the first cycle is expressed as 100%. 332
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TABLE 1
Comparison of the two cultivation strategies for recombinant HSA production in shake-flask cultures One-stage cultivation strategy Cycle 1
Cycle 2
Cycle 3
Two-stage cultivation strategy Cycle 4
Cycle 1
Cycle 2
Cycle 3
Cycle 4
Culture medium used (L)
1
1
1
1
2
2
2
2
Running time in each cycle (day)
5
5
5
5
6
6
6
6
23.6
25.2
11.3
4.0
4.6
Yield (mg L )
8.6
Total running time (day) Total rHSA production (mg)
22.4 20
4.0 24
79.8
24.0 Research Paper
1
repeated four times. In each cycle, the sucrose level declined to almost the limit of detection on day 5. The cell viability declined to around 50% of the original level, but recovered to almost 80% of the original level when the MS medium was renewed (Fig. 4a). Cell volume, which was used to represent cell growth, increased continually throughout the four cycles (Fig. 4a). The concentration of rHSA in the culture medium on day 5 after MS medium renewed was determined as 8 mg/L for the first cycle. It increased to around 20–25 mg/L in cycles 2–4 (Fig. 4a). A parallel experiment was performed to monitor the rHSA production in repeated cycles of the two-step process. The HSA cell line was cultured at a cell density of 24% in sucrose-containing MS medium for 3 days. Then, the medium was changed to sucrosefree MS medium for 3 days. The medium was collected for four cycles. As shown by the results in Fig. 4b, the cell viability declined to 30–40% after the shift to sucrose-free medium, but then recovered to around 80% of the original level when the medium was replaced with sucrose-containing medium. Cell volume increased throughout the four cycles (Fig. 4b), which indicated that the cells were growing during the repeated cycles of the two-step process. The concentration of rHSA in the culture medium on day 6 was 11 mg/L for the first cycle. It decreased to around 4 mg/L in cycles 2–4 (Fig. 4b). By comparison of these two cultivation strategies with repeated cycles, cell viability was shown to be higher in each cycle when using the one-step approach without change of medium than when using the two-step process. This finding is consistent with the results described above. As a consequence of the increase in cell viability, rHSA production was also higher in each cycle in the one-step approach than in the two-step approach. The total yield of rHSA was 74.19 mg L1 in the one-step approach after four cycles, which is three times greater than in the two-step approach (Table 1).
Production of rHSA in a bioreactor by the approach without change of medium Scaled-up tests of rHSA production were performed in a 2-L airlift and a stirred tank bioreactor. Batch cultures were repeated until the sedimentation volume of the suspension cells reached 24% of the working volume in each bioreactor (A sedimentation volume of cells reached approximately 0.48 L in a working volume of 2 L). The culture medium in the bioreactor was then refreshed, and sucrose concentration and rHSA accumulation in the medium were both monitored. Although the cell viability decreased progressively to 8% of the original incubation time on day 12 in the airlift bioreactor, the cell volume was maintained at 24% (Fig. 5).
FIGURE 5
Scaled-up production of rHSA in either an airlift-type bioreactor or a stirred tank bioreactor. Suspended cells from line H11 were cultured in bioreactors with sucrose-containing MS medium to reach 24% cell sedimentation volume. During the cell enrichment stage, cell volume, cell viability, and sucrose content were monitored (from day 8 to 0). After replacement of the medium with MS medium, cell volume, cell viability, sucrose content, and rHSA concentration in the bioreactors were determined during the culture period (day 0–14). The error bar represents the standard deviation from triplicate measurements. www.elsevier.com/locate/nbt
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When the sucrose concentration dropped to 10 g L1 on day 4 (Fig. 5), rHSA could be detected in the culture medium. The level of rHSA reached a maximum (around 45 mg L1) on day 6 (Fig. 5). On the other hand, the cell viability was decreased to 80% of that at the original incubation time, when the volume of cell sedimentation reached 24% in the stirred tank bioreactor. After replacement of the medium with sucrose-containing MS medium, the cell viability in the stirred tank bioreactor decreased progressively (Fig. 5) slightly faster than with the airlift bioreactor, and the cell volume in the stirred tank bioreactor was also decreased progressively after day 2 (Fig. 5). The concentration of sucrose dropped to zero on day 8 (Fig. 5), which involved a 2-day delay compared with that for the airlift bioreactor. The rHSA yield was 5 mg L1 in the culture medium on day 8 and mainly showed similar levels during the sugar starvation period (Fig. 5). Cell viability clearly decreased in the stirred tank bioreactor, which suggested that shear force may have damaged the suspended rice cells, thereby decreasing the rHSA yield. In light of these observations, the airlift-type bioreactor is indicated for use in the rice suspension cultured cell system to produce the recombinant protein rHSA.
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Conclusion In the study reported herein, we developed a platform for producing recombinant rHSA using the rice suspension cultured cell system and showed that more rHSA can be produced when using the one-step cultivation strategem without a change of medium. The HSA transgene was transformed directly into the rice genome through Agrobacterium-mediated transformation. The rHSA was produced and secreted from MS-cultured rice suspension cells. The production of rHSA can be increased sixfold by the one-step cultivation strategem, which might produce fewer damaged cells during the cell sugar starvation phase for the production of recombinant protein. Furthermore, we scaled up the production of rHSA in a 2-L airlift and a 2-L stirred tank bioreactor by using the one-step cultivation strategy. We conclude that rHSA can be enriched in MS medium, which is the type most commonly used for plant culture, by using an airlift-type bioreactor.
Acknowledgement This work was supported by grants from the National Science Council (97-2317-B-182-001 to Yu-Kao Liu and 99-2313-B-155001-MY3 to Li-Fen Huang) of the Republic of China.
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Research Paper
Enhancement of recombinant human serum albumin in transgenic rice cell culture system by cultivation strategy Yu-Kuo Liu1, Yu-Teng Li2, Ching-Fan Lu1 and Li-Fen Huang2 1 2
Graduate Institute of Biochemical and Biomedical Engineering, Chang Gung University, Kwei-Shan, Taoyuan County 320, Taiwan, ROC Graduate School of Biotechnology and Bioengineering, Yuan Ze University, 135 Yuan-Tung Road, Zhongli, Taoyuan County 320, Taiwan, ROC
Fusion of the sugar-starvation-induced aAmy3 promoter with its signal peptide has enabled secretion of recombinant human serum albumin (rHSA) into the culture medium. To simplify the production process and increase the rHSA yield in rice suspension cells, a one-step strategem without medium change was adopted. The yield of rHSA was increased sixfold by this one-step approach compared with the two-step recombinant protein process, in which a change of the culture medium to sugar-free medium is required. The one-step strategem was applied to check repeated cycle of rHSA production, and the production of rHSA was also higher in each cycle in the one-step, as opposed to the two-step, production process. The use of the one-step process resulted in fewer damaged cells during the cell sugar starvation phase for recombinant protein production. Furthermore, we scaled up the rHSA production in a 2-L airlift and a 2-L stirred tank bioreactor by the one-step approach, and concluded that rHSA can be enriched to 45 mg L1 in plant culture commonly used MS medium by the airlift-type bioreactor. Our results suggest that rHSA production can be enriched by this optimized cultivation strategem.
Introduction Pharmaceutical recombinant proteins in the market are expanding rapidly in recent decades, and they have been produced in various host cell types, which differ in terms of their biosafety, biological activity and protein stability. Like mammalian cells, plant cells are capable of protein posttranslational modification, such as glycosylation, but without the risks associated with human/animal pathogens in mammalian cell cultures. Taking advantage of this absence of risk, plant cells have been used to produce complex therapeutic proteins that are required for biosafety, bioactivity and stability. Rice (Oryza sativa), a crop with the third highest level of cultivation globally, is considered a model plant whose genome has been sequenced, and the technologies for gene transformation and tissue culture have been well developed [1,2]. Its hypoallergenic property also makes rice become an excellent host cell Corresponding authors: Liu, Y.-K. ([email protected]), Huang, L.-F. ([email protected]) www.elsevier.com/locate/nbt
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for recombinant protein production [3,4]. However, the presence of genetically modified plants in the field has raised public concerns and is regulated strictly by governments. A rice suspension cell system is an alternative option to limit public concern about genetically modified organisms, and it has been developed for the efficient scaling up of recombinant protein production, such as for pharmaceutical proteins [5–12], vaccines [13,14], or antibodies [3,15]. A platform for recombinant protein production in rice suspension cells has been established using a rice a-amylase gene promoter, RAmy3Dp/aAmy3p, the activity of which is induced by sugar starvation [16]; its signal peptide allows the secretion of recombinant proteins outside the cells. These properties motivated the development of a two-step process for producing recombinant protein by using RAmy3Dp/aAmy3p. In the first step, the transgenic rice cells are cultured in sucrose containing medium to increase the cell number and maintain cell viability and cell activity. In the second step, the sucrose-rich medium is removed from the amplified cells, and the sugar-free medium is added to http://dx.doi.org/10.1016/j.nbt.2015.03.001 1871-6784/ß 2015 Elsevier B.V. All rights reserved.
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Materials and methods Plant material The genetic background of rice (O. sativa L) cell line used in this study is Tainung No. 67. Rice grains were dehulled, sterilized with 2.4% sodium hypochlorite for 1 hour, washed extensively with sterile water, and placed on N6 agar medium [29] containing 3% sucrose and 10 mM 2,4-dichlorophenoxyacetic acid (2,4-D) (N6D) at 288C in the dark to induce calli. Calli were subcultured in liquid Murashige and Skoog (MS) medium [30] containing 3% sucrose and 10 mM 2,4-D at 288C in the dark to establish suspension-
cultured cells. The resultant suspension-cultured cells were agitated on a reciprocal shaker at 120 rpm and incubated at 288C in the dark.
Rice transformation The plasmid pA3HSA, which contains an aAmy3 promoter – signal peptide – HSA fusion construct, was generated in our previously reported work [7]. Agrobacterium tumefaciens strain EHA105 [31] was used as a host stain for pA3HSA by electroporation, and rice calli were transformed as described previously [32]. Selection of transformed calli was performed on N6 agar medium with hygromycin (50 mg L1).
Characterization of the transgenic cell lines The presence of the transgene insertion in rice calli was analyzed by genomic DNA PCR. Genomic DNA was isolated from putative transgenic calli and then subjected to PCR by using primers HSA-F (50 -GGGCATGTTTTTGTATGAAT-30 ) and HSA-R (50 -TTATAAGCCTAAGGCAGCTT-30 ).
RT-PCR Total RNA was isolated from rice transgenic cell lines using Trizol (Invitrogen, Carlsbad, CA, USA) and treated with RNase-free DNase. First-strand cDNA was synthesized by SuperScript III Reverse Transcriptase (Invitrogen) with an oligo-dT primer. A 20-fold dilution of the first-strand cDNA was subjected to PCR with gene-specific primers; the HSA-F and HSA-R were used for HSA; the Amy3F (50 -TACAGCGTCTGGGAGAAGGGGTC-30 ) and Amy3R (50 -TGCCCCGCAATTAACCTAGAGGC-30 ) were used for aAmy3; the Actin-F (50 -CTGATGGACAGGTTATCACC-30 ) and Actin-R (50 -CAGGTAGCAATAGGTATTACAG-30 ) were used for Act1.
Protein gel blot analysis To obtain total soluble secretory proteins from rice suspension cells, the rice cultured medium was filtered through Whatman No. 1 filter paper to remove cells, and then centrifuged at 18,000 g at 48C for 15 min to obtain the supernatant. The concentration of protein in the supernatant was measured using the Bio-Rad Protein Assay reagent (Bio-Rad, Hercules, CA). Western blotting was performed as described previously [7]. The rabbit antiserum to HSA (ICN Pharmaceuticals, Costa Mesa, CA), applied as the primary antibody, was diluted 1000-fold before use. Then, a 2000-fold dilution of horseradish peroxidase conjugated goat secondary antibody against rabbit IgG (Pierce, Rockford, IL) was diluted before use. Chemiluminescent signals emitted from the complex of primary and secondary antibodies were detected by using the Lumi-light Western blotting substrate (Roche, Basel, Switzerland).
Enzyme-linked immunosorbent assay (ELISA) The yields of rHSA secreted from transgenic rice cells into cultured medium were measured by a sandwich ELISA method as described previously [7]. Each well in the microtiter plates was coated with 10 mg/mL goat antiserum to HSA at 258C for 4 hours. Then, 50 mL of each cell-cultured medium sample was added to individual wells and incubated at 378C for 1 hour. Subsequently, 10 mg/mL rabbit anti-HSA polyclonal antibodies (ICN Pharmaceuticals) was added to the wells and incubated at 378C for 1 hour. Peroxidase-conjugated anti-rabbit IgG antibodies were diluted 2000-fold and apwww.elsevier.com/locate/nbt
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produce recombinant protein. Several recombinant proteins have been produced successfully and secreted into the culture medium from these sugar-starved transgenic rice cells by using this two-step process [7,9,10,14,15,17–20]. However, as described above, exposure to two types of culture medium is required in the two-step production procedure, which increases both the cost of the process and the risk of contamination when changing the medium. Previously, we reported that the production of a recombinant protein, mouse granulocyte-macrophage colony-stimulating factor (rmGM-CSF), could be achieved in one step by using a cultivation strategy that does not require a change of medium [11]. Transgenic rice suspension cells were cultured in N6D medium with 3% sucrose and rmGM-CSF was produced upon sucrose depletion in the medium. The level of rmGM-CSF production was doubled compared with that in the two-step production process [11]. Furthermore, rmGM-CSF production was scaled up to bioreactor levels by using this cultivation strategy [11]. However, more successful examples of such production are required to determine whether other recombinant proteins or other common plant growth media can also benefit from this cultivation strategy. Human serum albumin (HSA) is a single unglycosylated protein that contains 585 amino acid residues and forms 17 pairs of disulfide bonds. HSA is the most abundant blood plasma protein and plays important roles in the regulation of osmotic pressure and pH, as well as acting as a versatile carrier for hormones and numerous drugs [21]. HSA is frequently applied in medical treatments, such as surgery and hepatocirrhosis, and also widely used in biochemical applications, such as vaccine formulation and cell culture medium. The current major HSA source is human plasma, but its availability is insufficient to meet the increasing medical demand. Moreover, the use of human plasma is associated with a risk of disease transmission, such as hepatitis B, syphilis, and HIV [22]. In order to meet the increasing demand and assuage biosafety concerns, several platforms for producing recombinant protein, including rice suspension cells, have been developed for producing recombinant HSA (rHSA) [7,23–28]. In our previous work, we generated a transgenic rice cell line by Agrobacterium-mediated transformation, and produced rHSA in the culture medium by a two-step process [7]. In the study reported herein, we investigated the effects of various strategies for producing rHSA by culturing transgenic rice suspension cells in flasks. We also demonstrated that the yield of rHSA was increased sixfold when the culture medium was not changed after sucrose was exhausted naturally, compared with that upon changing to sucrose-free medium. Furthermore, we scaled up the production of rHSA successfully in a modified bioreactor by this optimized cultivation strategy.
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plied to the well. The samples were incubated at 378C for 1 hour, and ABTS substrate solution (Sigma, St. Louis, MO) was added to the wells. The optical density (OD) of each well was measured in an Epoch Multi-Volume Spectrophotometer System (BioTek, Winooski, VT) at 450 nm.
Determination of cell viability
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Cell viability was estimated by reduction of triphenyltetrazolium chloride (TTC), as described by Chen et al. [33]. The cell samples were incubated with TTC solution at room temperature in the dark for 12 hours. After ethanol extraction, OD 530 was measured with an Epoch Multi-Volume Spectrophotometer System (BioTek).
Determination of cell dry weight Cells were collected from the liquid culture medium, and immediately washed three times with distilled water. The dry weight was determined after drying at 808C until constant weight.
Culture of rice suspension cells in a bioreactor Rice suspension cells were grown and HSA was produced in a 3-L modified bubble-column bioreactor (MBCR) and a 3-L StirredTank bioreactor FB-3B (STR, Firstek Scientific, Taipei, Taiwan) with a single pitched blade (low-shear) impeller, respectively. The diameter of the inner cylinder of the MBCR was 10 cm and its height was 40 cm. Rice suspension cells were cultured in the STR and MBCR bioreactors at 288C (18C) in the dark. The bioreactors were filled with 2.0 L of sterilized MS medium, and inoculated at 3% (v/ v) with 7-day-old rice suspension cells. The aeration rate was controlled at 1.0 L/min in MBCR and 0.5 L/min in STR using an airflow meter (RK1150, Kojima, Kyoto, Japan). The agitation speed was controlled at 75 rpm in STR. The culture medium was refreshed with MS medium for every 8 days until cell density reached around 24% and the sucrose concentration of the medium and yield of HSA were determined.
Results and discussion Generation of transgenic rice cell lines expressing HSA Previously, a transgenic cell line expressing rHSA was generated and rHSA yield reached 76 mg L1 in a bioreactor with 50% cell density [7]. However, this cell line starts to form a very fine yellowwhite powder-like aggregate of cell clumps, then secretes less and less rHSA after years of continuous cell culture. Therefore, it is not suitable for testing the proposed cultivation strategy for increasing the yield of rHSA yield. To obtain rHSA-producing rice suspension cells with normal morphology and physiology, we regenerated transgenic cell lines harboring an HSA expression cassette, which contains the aAmy3 promoter-signal sequence and the HSA cDNA sequence, by the Agrobacterium-mediated transformation system. Several putative transgenic calli that were resistant to hygromycin were subjected to transgene analysis (Fig. 1). The line of H11 that produced higher levels of rHSA mRNA (Fig. 1) was selected to generate suspension-cultured cells for further investigation.
rHSA production in a transgenic rice cell line cultured by a twostage production process To monitor the level of production of rHSA proteins secreted from rice suspension cultured cells for the two-step production process, a transgenic rice suspension cell line was established in MS medium 330
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FIGURE 1
Generation of rHSA expression rice calli. Calli were starved in sugar-free MS medium for 72 hours and used for (a) DNA detection by genomic PCR, and (b) mRNA detection by RT-PCR. Specific primer sets were used in PCR for HSA, ActI, and aAmy3.
(the most commonly used plant culture medium) with 10 mM 2,4-D and 3% sucrose, then incubated in sugar-free medium at an initial cell density of 3% (cell volume/medium volume) for various periods of time. Cell viability dropped to 40% on day 1 after sugar starvation, and then decreased progressively until almost no living cells were left by day 6 (Fig. 2). Cell dry weight was used to represent cell growth, and it decreased slightly and remained at about 50% of the original level on day 6 after sugar starvation (Fig. 2). Secreted rHSA proteins accumulated steadily in the culture medium, peaked at around 2.5 mg L1 on day 6, and were maintained until day 10 after sugar starvation (Fig. 2c–e). These results indicate that the recombinant proteins were secreted from MS cultured sugar-starved rice cells.
Enhancement of recombinant protein productivity by cultivation strategy Previously, we showed that the production of rmGM-CSF was increased by using an approach that does not require change of medium when grown in N6D culture medium [11]. A similar cultivation strategem was used to determine whether it is also useful to increase the production of rHSA protein in cells cultured with MS medium. Cells of the H11 line were incubated in MS medium and the yield of secreted rHSA was monitored. Suspension cells were immersed at an initial density of 3% (v/v) in MS medium that contained 3% sucrose. Cell growth (Fig. 3a) and cell viability (Fig. 3b) were increased and maintained in MS containing sucrose, respectively, then decreased after the sucrose almost ran out on day 10 (Fig. 3c). Secreted rHSA was detectable on day 11, and the protein yield reached 15 mg L1 on day 14 (Fig. 3d). The yield of secreted rHSA increased sixfold when using the approach that does not require a change of medium, compared with 2.5 mg L1
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FIGURE 3
FIGURE 2
rHSA production in sucrose-free MS culture medium. The cell line H11 was cultured in sucrose-free medium for various lengths of time. Cells were collected for the determination of cell dry weight (a) and cell viability (b). Samples of culture media were collected to determine the concentration of rHSA by ELISA (c), silver staining (d), and Western blotting (e). The error bar represents the standard deviation from triplicate cultures. Commercial HSA, 30 ng, produced in E. coli (eHSA) was applied as a positive control in (d,e). The rHSA are indicated by black arrows.
Secreted rHSA proteins were enriched in the culture medium by the one-step cultivation strategem. Four different concentrations of suspension cells from the cell line H11 [3% (v/v), 6%, 12%, and 24%] were cultured in MS medium containing sucrose for various lengths of time. The cell dry weight (a) and viability (b) of the cells were determined after sample collection. In the meantime, the concentrations of sucrose (c) and rHSA proteins (d) in the culture medium were determined. The error bars represent the standard deviation from triplicate cultures.
(Fig. 2c) in the two-step process in which the original culture medium has to be replaced by sucrose-free medium. The yield of rHSA was also determined when initial cell densities of 6%, 12%, and 24% were used. Similar patterns of the changes in cell growth and cell viability were obtained as for an initial cell density of 3% (Fig. 3a,b). The times at which the sucrose ran out with cell www.elsevier.com/locate/nbt
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densities of 6%, 12%, and 24% were earlier than with 3% (Fig. 3c). Under cell densities of 24%, rHSA began to be produced on day 5 (Fig. 3d), when the rice cultured cells started to starve (Fig. 3c), and the yield reached around 25 mg L1 on day 7 (Fig. 3d). These results indicate that a greater amount of rHSA can be produced by increasing the cell density in this one-step cultivation strategy without a change of medium. Our data also support previous reports that a high yield of recombinant protein could be achieved by increasing the cell density in rice suspension cells [7,11]. On the other hand, the level of rHSA could be increased sixfold in MS medium by adopting the one-step approach without a change of medium. During the phase of recombinant protein production, the slope of decrease in cell viability in the one-step approach was clearly gentler than that
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in the two-step process. In other words, the rice cells were healthier in the one-step process, so more recombinant proteins were produced during the production phase.
Production of rHSA under repeated cycles of the approach without change of medium When recombinant rHSA proteins were secreted from rice suspension cells during starvation, cell viability decreased. To increase the productivity of rHSA in the same batch of suspension cultured rice cells, we examined the stability of rHSA production in repeated cycles of the cultivation approach without change of medium. The HSA cell line was cultured at a cell density of 24% in sucrose-containing MS medium for 5 days. Then the medium was harvested. This procedure comprised one cycle. The cycle was
FIGURE 4
Stable production of rHSA under repeated cycles of the two cultivation strategies. (a) In the one-step approach with medium change, 12 mL of H11 suspension cells was cultured in 50 mL of sucrose-containing MS medium for 5days, which was then replaced with fresh sucrose-containing MS medium every 5 days for three cycles (in a period of 20 days). (b) In the two-stage process, 12 mL of H11 suspension cells was cultured in 50 mL of sucrose-containing MS medium for 3days, which was replaced with 50 mL of sucrose-free MS medium for 3 days. The cycles of 3-day growth phase and 3-day production phase were repeated three times (in a period of 21 days). Cells were collected for the determination of cell volume and viability, and media were collected for determination of the yields of secreted rHSA by ELISA. Error bars indicate standard deviation from triplicate cultures. For cell viability, the capability of TTC reduction in cells cultured for 5 days in sucrosecontaining medium in the first cycle is expressed as 100%. 332
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TABLE 1
Comparison of the two cultivation strategies for recombinant HSA production in shake-flask cultures One-stage cultivation strategy Cycle 1
Cycle 2
Cycle 3
Two-stage cultivation strategy Cycle 4
Cycle 1
Cycle 2
Cycle 3
Cycle 4
Culture medium used (L)
1
1
1
1
2
2
2
2
Running time in each cycle (day)
5
5
5
5
6
6
6
6
23.6
25.2
11.3
4.0
4.6
Yield (mg L )
8.6
Total running time (day) Total rHSA production (mg)
22.4 20
4.0 24
79.8
24.0 Research Paper
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repeated four times. In each cycle, the sucrose level declined to almost the limit of detection on day 5. The cell viability declined to around 50% of the original level, but recovered to almost 80% of the original level when the MS medium was renewed (Fig. 4a). Cell volume, which was used to represent cell growth, increased continually throughout the four cycles (Fig. 4a). The concentration of rHSA in the culture medium on day 5 after MS medium renewed was determined as 8 mg/L for the first cycle. It increased to around 20–25 mg/L in cycles 2–4 (Fig. 4a). A parallel experiment was performed to monitor the rHSA production in repeated cycles of the two-step process. The HSA cell line was cultured at a cell density of 24% in sucrose-containing MS medium for 3 days. Then, the medium was changed to sucrosefree MS medium for 3 days. The medium was collected for four cycles. As shown by the results in Fig. 4b, the cell viability declined to 30–40% after the shift to sucrose-free medium, but then recovered to around 80% of the original level when the medium was replaced with sucrose-containing medium. Cell volume increased throughout the four cycles (Fig. 4b), which indicated that the cells were growing during the repeated cycles of the two-step process. The concentration of rHSA in the culture medium on day 6 was 11 mg/L for the first cycle. It decreased to around 4 mg/L in cycles 2–4 (Fig. 4b). By comparison of these two cultivation strategies with repeated cycles, cell viability was shown to be higher in each cycle when using the one-step approach without change of medium than when using the two-step process. This finding is consistent with the results described above. As a consequence of the increase in cell viability, rHSA production was also higher in each cycle in the one-step approach than in the two-step approach. The total yield of rHSA was 74.19 mg L1 in the one-step approach after four cycles, which is three times greater than in the two-step approach (Table 1).
Production of rHSA in a bioreactor by the approach without change of medium Scaled-up tests of rHSA production were performed in a 2-L airlift and a stirred tank bioreactor. Batch cultures were repeated until the sedimentation volume of the suspension cells reached 24% of the working volume in each bioreactor (A sedimentation volume of cells reached approximately 0.48 L in a working volume of 2 L). The culture medium in the bioreactor was then refreshed, and sucrose concentration and rHSA accumulation in the medium were both monitored. Although the cell viability decreased progressively to 8% of the original incubation time on day 12 in the airlift bioreactor, the cell volume was maintained at 24% (Fig. 5).
FIGURE 5
Scaled-up production of rHSA in either an airlift-type bioreactor or a stirred tank bioreactor. Suspended cells from line H11 were cultured in bioreactors with sucrose-containing MS medium to reach 24% cell sedimentation volume. During the cell enrichment stage, cell volume, cell viability, and sucrose content were monitored (from day 8 to 0). After replacement of the medium with MS medium, cell volume, cell viability, sucrose content, and rHSA concentration in the bioreactors were determined during the culture period (day 0–14). The error bar represents the standard deviation from triplicate measurements. www.elsevier.com/locate/nbt
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When the sucrose concentration dropped to 10 g L1 on day 4 (Fig. 5), rHSA could be detected in the culture medium. The level of rHSA reached a maximum (around 45 mg L1) on day 6 (Fig. 5). On the other hand, the cell viability was decreased to 80% of that at the original incubation time, when the volume of cell sedimentation reached 24% in the stirred tank bioreactor. After replacement of the medium with sucrose-containing MS medium, the cell viability in the stirred tank bioreactor decreased progressively (Fig. 5) slightly faster than with the airlift bioreactor, and the cell volume in the stirred tank bioreactor was also decreased progressively after day 2 (Fig. 5). The concentration of sucrose dropped to zero on day 8 (Fig. 5), which involved a 2-day delay compared with that for the airlift bioreactor. The rHSA yield was 5 mg L1 in the culture medium on day 8 and mainly showed similar levels during the sugar starvation period (Fig. 5). Cell viability clearly decreased in the stirred tank bioreactor, which suggested that shear force may have damaged the suspended rice cells, thereby decreasing the rHSA yield. In light of these observations, the airlift-type bioreactor is indicated for use in the rice suspension cultured cell system to produce the recombinant protein rHSA.
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Conclusion In the study reported herein, we developed a platform for producing recombinant rHSA using the rice suspension cultured cell system and showed that more rHSA can be produced when using the one-step cultivation strategem without a change of medium. The HSA transgene was transformed directly into the rice genome through Agrobacterium-mediated transformation. The rHSA was produced and secreted from MS-cultured rice suspension cells. The production of rHSA can be increased sixfold by the one-step cultivation strategem, which might produce fewer damaged cells during the cell sugar starvation phase for the production of recombinant protein. Furthermore, we scaled up the production of rHSA in a 2-L airlift and a 2-L stirred tank bioreactor by using the one-step cultivation strategy. We conclude that rHSA can be enriched in MS medium, which is the type most commonly used for plant culture, by using an airlift-type bioreactor.
Acknowledgement This work was supported by grants from the National Science Council (97-2317-B-182-001 to Yu-Kao Liu and 99-2313-B-155001-MY3 to Li-Fen Huang) of the Republic of China.
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