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Production of baculoviruses and expression of green fluorescent protein in immobilised Sf21 insect cell cultivation
Biochemical Engineering Journal 29 (2006) 55–61
Production of baculoviruses and expression of green fluorescent protein in immobilised Sf21 insect cell cultivation Jeong Hwa Son a , Rainer Buchholz b , Jung-Keug Park c , Sung Koo Kim a,∗ a
c
Department of Biotechnology & Bioengineering, Pukyong National University, 599-1, Daeyeon 3-dong, Nam-gu, Busan 608-737, Republic of Korea b Department of Bioprocess Engineering, Friedrich-Alexander University, Erlangen-Nuremberg, Germany Department of Chemical and Biochemical Engineering, Dongguk University, Seoul 100-715, Republic of Korea Received 15 October 2004; accepted 19 February 2005
Abstract Spodoptera frugiperda (Sf21) insect cells were grown in the microspheres that were prepared using sodium-cellulosesulfate (NaCS) and polydiallyldimethylammoniumchloride (PDADMAC). The highest Sf21 cell density was 1 × 108 cells/ml in the microshperes. The immobilised Sf21 cells were infected with Autograpa californica multiple nuclear polyhedrosis virus (AcMNPV) at a “theoretical” MOI of 1.0 and a TOI of 3.9 × 107 cells/ml in the microspheres. The AcMNPV polyhedral inclusion bodies (PIBs) titer reached was 1.76 × 1010 PIBs/ml in the microspheres, which was 58 times higher than that (2.75 × 107 PIBs/ml) in a suspension condition. The immobilised Sf21 cells were infected with a recombinant baculovirus, Ac-omega-GFP baculovirus, and the highest concentration of green fluorescent protein (GFP) produced was 0.0048 mg/ml in suspension culture and 0.159 mg/ml in the microspheres. © 2005 Published by Elsevier B.V. Keywords: Immobilisation; NaCS; PDADMAC; Ac-omega-GFP baculovirus; PIBs; GFP
1. Introduction Baculovirus-insect cell culture system has been used as an attractive tool to produce wild type baculoviruses for the use as a biopesticide as well as genetically engineered baculoviruses for the expression of recombinant proteins of medical and pharmaceutical importance, which system has several advantages such as high expression levels, limitless size of the expressing of the protein, post translational modifications, simultaneous expression of multiple genes and safety for vertebrates [1,2]. Most common recombinant baculoviruses (baculovirus expression vector, BEV) can be created by replacing the polyhedrin gene with a target gene under the transcriptional control of the polyhedrin promoter of the Autographa californica nuclear polyhedrosis virus (AcNPV).
∗
Corresponding author. Tel.: +82 51 620 6188; fax: +82 51 620 6180. E-mail address: [email protected] (S.K. Kim).
1369-703X/$ – see front matter © 2005 Published by Elsevier B.V. doi:10.1016/j.bej.2005.02.029
Then, the desired proteins are expressed during the very late stages of infection in insect cell culture [3]. Although insect cell culture has been usually employed for high production level, only a few desired products have been produced [1–3]. The main problem is associated with a scale-up of virus infected insect cell culture that is a desirable procedure for the commercial production. At large cultures, the common methods for supply oxygen to the cells such as sparging and agitation can damage the cells that are extremely shear-sensitive. In addition, the oxygen demand of insect cells is higher than that of mammalian cells, and oxygen uptake increase upon virus infection [2]. As well as, due to the lytic behavior of the baculovirus infected cells, it is difficult to achieve high cell densities and high production levels. Cell immobilisation system has been shown as a solution to overcome these disadvantages [4–7]. Immobilised insect cells can be protected from any shear stresses, while oxygen and nutrients are sufficiently supplied to the cells, and the cells can be remained in the microspheres completely dur-
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J.H. Son et al. / Biochemical Engineering Journal 29 (2006) 55–61
ing medium exchange. Hence, it is possible to reach higher cell densities and product concentrations as compared to in suspension culture. Green fluorescent protein (GFP) isolated from jellyfish Aequaorea victoria is a protein consisting of 238 amino acids, with a molecular weight of 27 kDa [8]. The protein has a maximal absorption peak at 395 nm and a minor peak at 470 nm with an emission peak at 509 nm. Its fluorescence is stable, independent, and requires no substrate, cofactor, or additional proteins for illuminating green light, therefore, GFP is highly attractive as a visual marker for gene expression studies [9–12]. In this study, Sf21 insect cells were infected with wild type A. californica multiple nuclear polyhedrosis virus (AcMNPV) and a recombinant Ac-omega-GFP baculovirus, and the productions of polyhedral inclusion bodies (PIBs) and green fluorescent proteins were evaluated in suspension and immobilised culture system.
2. Materials and methods
w/v) were prepared in 1% (w/v) NaCl solution and autoclaved (121 ◦ C, 10 min). The procedure of immobilisation is shown in Fig. 1. An encapsulation apparatus was used for immobilisation of Sf21 insect cells as described by H¨ubner et al. [5]. Briefly, the apparatus was connected to the storage bin containing the 3.5% NaCS solution and the cell solution through a silicone tube (D = 2 mm). The volume flow was adjusted to 200 ml/h. After dropping the mixed solution of NaCS and cells into the 2.2% PDADMAC solution, the mixture formed microspheres. The microspheres were stirred in the PDADMAC solution for 15 min, and washed extensively three times with the phosphate buffered saline (PBS: 0.2 g/l KCl, 0.2 g/l KH2 PO4 , 8 g/l NaCl, 1.15 g/l Na2 HPO4 , pH 7.4), then transferred into culture flasks for cultivation. The microspheres have about 10 m membrane gaps as shown in Fig. 1. These gaps were caused by the death of protruding cells from the capsule membrane due to the cytotoxicity of PDADMAC in the PDADMAC solution for the stirring time. Microspheres containing the cells (7.5 ml, wet volume) were cultured in 50 ml of SF900II serum free medium in a 500 ml shaker flask with four baffles at 80 rpm, 27 ◦ C and pH 6.2.
2.1. Suspension cell culture 2.3. Measurement of living cell density (MTT-test) Spodoptera frugiperda (Sf21) insect cells were cultured in SF900II serum free medium (GIBCOBRL). The culture condition was controlled at 27 ◦ C and pH 6.2. The viability of the cells was determined by a haemocytometer using 0.4% (w/v) trypan blue exclusion. 2.2. Cell immobilisation Sodium-cellulosesulfate (NaCS, 3.5%, w/v) and polydiallyldimethylammoniumchloride (PDADMAC, 2.2%,
One millilitre of cell solution from the suspension culture or 10 microspheres from the immobilised culture was daily taken out for the MTT-test [13]. Briefly, one part of MTT solution was mixed with four parts of sample solution, and incubated at 27 ◦ C and dark for 4 h, then, the microspheres were homogenized in sodium-dodecylsulfate (SDS, 20%, w/v). One millilitre of the homogenized solution or incubated cells with MTT solution were added to 4 ml of lysis buffer (405 ml iso-propanol, 20 ml 1N HCl, 75 ml SDS
Fig. 1. An encapsulation apparatus. The size of microspheres is about 3 mm in diameter. The microspheres have about 10 m membrane gaps due to the death of cells protruding from the membrane of spheres in the PDADMAC solution during the stirring time.
J.H. Son et al. / Biochemical Engineering Journal 29 (2006) 55–61
(20%, w/v)) and supersonicated. The debris was centrifuged, and the absorbance (λ = 570 nm) of supernatant was measured by a spectrophotometer. 2.4. AcMNP Virus infection of suspended and immobilised insect cells Suspended Sf21 cells were infected at a multiplicity of infection (MOI) of 1.0 by adding a virus stock solution (MOI of 0.1) of AcMNPV (A. californica multiple nuclear polyhedrosis virus, Pharmingen, Hamburg). The virus titers were determined by the end-point dilution assay [7]. The infected cells were cultured in 50 ml SF900II medium in a 500 ml culture flask at 80 rpm and 27 ◦ C. When the immobilised cells reached the stationary growth phase, the cells were infected at a “theoretical” MOI of 1.0 with the virus stock solution. The “theoretical” MOI of 1.0 was determined by the immobilised cell density that was measured by MTT-test as before viral infection [13]. The medium was changed every day after viral infection. 2.5. Counting the polyhedral inclusion bodies (PIBs) One ml of the sample in the suspension culture or five microshperes in the immobilised culture homogenized in 20% (w/v) SDS solution were centrifuged at 1300 × g for 15 min. The pellet was extensively washed in polyhedra lysis buffer (1.21 g/l Tris, 0.37 g/l EDTA, 0.72 g/l SDS) and supersonicated until cell membrane and debris were removed. Then, the sample was centrifuged again at 1300 × g for 15 min, and polyhedra lysis buffer was added. This process was repeated until only polyhedral inclusion bodies (PIBs) were remained in the buffer. PIBs in lysis buffer were observed as 2–5 m particles with 0.4% trypan blue exclusion under a light microscope and counted using a haemocytometer. 2.6. Ac-omega-GFP baculovirus stock Ac-omega-GFP baculovirus (polh− ) was used for Sf21 insect cell infection. The recombinant baculovirus has been obtained from Institute for Applied Microbiology, University of Agricultural Science, Vienna, Austria. The baculovirus vector was constructed by inserting GFP gene into the unique restriction site Sce-I of Ac-omega viral genome that is genetically engineered AcNPV (A. californica nuclear polyhedrosis virus) genome by introducing a unique restriction site downstream of the strong polyhedrin promoter (Ac-omega) to allow linearization with a specific endonuclease (Sce-Imeganuclease). The suspended cells were infected with Ac-omega-GFP baculoviruses at a multiplicity of infection (MOI) of 0.1. The infected cells were harvested at 96 h post infection (hpi), centrifuged at 50 × g for 10 min, and the supernatant were kept in the 4 ◦ C for using as a virus stock.
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2.7. Ac-omega-GFP baculovirus infection to suspension and immobilised culture S. frugiperda (Sf21) insect cells were cultured in 125ml SF900II serum free medium (GIBCOBRL) in a 500 ml spinner flask at 65 rpm and 27 ◦ C. The suspended cells in a spinner flask were infected with the Ac-omega-GFP baculovirus stock at a MOI of 1.0 at the cell density of 1 × 106 cells/ml (over 90% of cell viability). The medium of infected cells was changed every 2 days by cell centrifugation (50 × g, 5 min). Immobilised Sf21 insect cells in the stationary phase were infected with the Ac-omega-GFP baculovirus at a “theoretical” MOI of 1.0, adding the virus stock solution, and the medium was changed every 2 days. 2.8. SDS-PAGE The samples from the suspended and immobilised culture infected with Ac-omega-GFP baculovirus were analyzed by sodium dodecyl sulfate-polyacrlyamide gel electrophoresis (SDS-PAGE) [14]. In the case of immobilised culture, five microspheres were taken out from the culture and ruptured in lysis buffer (50 mM glucose, 10 mM EDTA, 25 mM Tris–HCl (pH 8.0)) and supersonicated for 15 min, then centrifuged at 1500 × g for 5 min. One part of the supernatant and one part of the sample buffer were mixed, boiled at 100 ◦ C for 10 min, centrifuged at 1500 × g for 5 min. Then, the sample was separated onto 12% slab gels by electrophoresis, and the SDS gel was stained by Coomassie Brilliant Blue R250. 2.9. ELISA The GFP produced in the suspended and immobilised culture was quantified using direct ELISA. Suspension sample was supersonicated for 15 min and centrifuged (1500 × g, 15 min). The clear supernatant was coated onto a 96-well plate and the plate was covered with parafilm. After incubation for 2 h at 37 ◦ C, the excess sample solution was removed. Then 200 l blocking buffer (1%BSA in PBS) was added per well and the plate was incubated at room temperature for 2 h. After removing of blocking buffer, the plate was washed two times with 200 l washing buffer (0.05% Tween-20 in PBS) and 100 l horseradish peroxidase conjugated GFP antibody (1:1000, CLONTECH, CA, USA) was added in the plate. After incubation at room temperature with shaking for 1 h, the plate was washed three times with 200 l wash buffer per well, and 100 l TMB (tetramethylbenzidine) substrate solution was added in the plate. Then, the result quantified at 595 nm and the value was compared with a standard EGFP (EGFP: CLONTECH) calibration curve to calculate the GFP concentration. In immobilised culture, two microcapsules in 400 l lysis buffer (50 mM glucose, 10 mM EDTA, 25 mM Tris/HCl (pH 8.0)) were ruptured and supersonicated for 15 min. The sample was centrifuged at 1500 × g for 5 min, finally measured by ELISA as above.
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Fig. 2. Immobilised Sf21 cells. The cells entrapped in NaCS-PDADMAC membrane as indicated by arrows (A), microspheres containing live cells after MTT-test (B, b/w picture), and microspheres containing dead cells after MTT-test (C, b/w picture).
3. Results and discussion 3.1. Sf21 cell culture in immobilised condition Fig. 2 shows the microspheres entrapping Sf21 cells. A single cell and cell aggregates are visible inside the sphere (Fig. 2A). The colors of microspheres containing live cells was changed to dark purple by enzymatic catalysis reaction between MTT and dehydrogenase from mitochondria of the live cell (Fig. 2B), however, the colors of microshperes containing dead cells were not changed by MTT-test (Fig. 2C). The sphere size was about 3 mm in diameter. In the immobilised culture, the highest cell density was 1 × 108 cells/ml in the microspheres, which was about 32 times higher than that (3.2 × 106 cells/ml) in suspension culture. 3.2. The production of PIBs in the suspended and immobilised insect cell culture Suspended Sf21 cells were infected with AcMNPV, at the cell density of 1.0 × 106 cells/ml and the viability over 90% with a MOI of 1.0 as shown in Fig. 3A. After the virus infection, the infected cells produced extracellular virus particles (nonincluded viruses, NOVs) and occluded virus particles (polyhedral inclusion bodies, PIBs). At 48 hpi (hours post infection), PIBs were detected as 2–5 m in diameter under the microscope. The viability of infected cells significantly decreased within 96 hpi. The highest concentration of PIBs achieved was 2.75 × 107 PIBs/ml in 96 hpi (Fig. 3A). The immobilised Sf21 cells in the stationary phase were infected with AcMNPV at a theoretical MOI of 1.0 (Fig. 3B), PIBs produced in the microspheres reached the concentration above 1.0 × 109 PIBs/ml after 4 days post infection (dpi). Then, the highest PIBs concentration was 1.76 × 1010 PIBs/ml in the microspheres at 15 dpi. In addition, PIBs produced up to 1 × 107 PIBs/ml could be detected daily in the spent medium. The production of PIBs, in the suspended and immobilised cultures, is shown in Table 1. In the microspheres, the PIBs produced were 3.9 × 108 PIBs/ml per day. This value was higher than the PIBs concentration produced in the suspension culture for 4 days (2.75 × 107 PIBs/ml), and the PIBs
Fig. 3. Production of polyhedral inclusion bodies (PIBs) in suspended (A) and immobilised (B) Sf21 insect cell culture (A: , Sf21 cell viability; , PIBs; 䊉, live cells; , dead cells and line: AcMNPV infection (MOI = 1.0), B: , PIBs in the capsules; , PIBs in the medium; 䊉, cell density in the capsules and line: AcMNPV infection).
J.H. Son et al. / Biochemical Engineering Journal 29 (2006) 55–61 Table 1 Production of polyhedral inclusion bodies (PIBs) in suspension and immobilised Sf21 insect cell cultivation Value
Suspension cultivation
Immobilised cultivation
TOI (106 cells/ml)a PIBs/ml and production time (day)
1 2.75 × 107 (4)b
30 3.9 × 108 (1)c 1.76 × 1010 (15)d
Relative PIBs productivity Specific PIB productivity in 4 dpi (PIBs/cell) Relative yield of PIBs (PIBs/cell)
1e 27.5
58 52
1f
1.9
a
Time of infection (TOI: cell density when virus infection). The density of baculoviruses (PIBs/ml) produced for 4 days. c The density of baculoviruses (PIBs/ml) produced for 1 day. d The density of baculoviruses (PIBs/ml) produced for 15 days. e Relative PIBs productivity (2.75 × 107 ) in the suspension condition as set to 1 to compared with the value in the immobilised condition. f Relative yield of PIBs (27.5) in the suspension condition as set to 1 to compared with the value in the immobilised condition. b
production term in the suspension culture was just 5 dpi (Fig. 3A), which was terminated due to the death of infected cells. Therefore, the relative PIBs productivity in the immobilised cell culture was 58 times higher as compared to that in the suspension cultivation. 3.3. GFP analysis by SDS-PAGE Suspended and immobilised Sf21 cells were infected with Ac-omega-GFP baculovirus. Then, GFP light from the cultures could be detected from 24 hpi under a fluorescence microscope (492 nm). Fig. 4 shows SDS-PAGE results of the suspension culture (Fig. 4A) and the immobilised culture (Fig. 4B) infected by Ac-omega-GFP baculovirus. In Fig. 4A, GFP was expressed with the molecular weight of 27 kD at 96 hpi (lane no. 3) and 144 hpi (lane no. 4), but not 24 hpi (lane no. 2) because GFP was expressed under the polyhedrin promoter control at very late in the virus infection cycle. The protein content of one lane on the gel was corresponded to the extract of 6 × 104 cells.
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In the immobilised culture (Fig. 4B), GFP expressed could be observed from 72 hpi (lane no. 3). The protein content of one lane was corresponded to the extract of about 2.4 × 104 cells. In addition, the GFP from the cultured medium in the immobilised culture was not detected (lane no. 4). 3.4. Quantification of GFP in the suspended and immobilised culture Fig. 5A shows Sf21 insect cell culture in a 500 ml spinner flask in a perfusion batch system. The highest noninfected Sf21 insect cell density obtained was 1.2 × 106 cells/ml. After the infection of Ac-omega-GFP baculovirus, the medium was changed every 2 days, however, the infected cell density decreased significantly. The concentration of the GFP produced in the suspension culture was 0.0012 mg/ml at 48 hpi, and the highest GFP production reached was 0.0048 mg/ml at 144 hpi (Fig. 5A). The suspension cultivation could not be performed after 168 hpi in spite of fresh culture condition by medium exchange every 2 days after virus infection. In the immobilised cell cultivation, the highest cell density achieved was 1 × 108 cells/ml in the microspheres before the virus infection as shown in Fig. 5B. After the virus infection, the immobilised cell density was maintained about 5 × 107 cells/ml in the microspheres throughout the cultivation period (510 hpi). After 510 hpi, the immobilised culture was kept to without medium exchange, hence decreasing of the immobilised cell density after 4 days of culture. The concentration of GFP produced was 0.057 mg/ml in the microspheres at 120 hpi, and the highest GFP concentration achieved was 0.159 mg/ml at 300 hpi in the microspheres (Fig. 5B). This concentration was about 33 times higher than that in the suspension culture. In addition, 0.2–0.43 g/ml of GFP in the spent medium was constantly detected every 2 days. In the immobilised culture system, trypan blue exclusion is not valid for the measurement of the viable cells because the cells are entrapped by capsule polymer, thus it is impossible to distinguish the dead and living cells. Another approach for measurement of viable cell density was performed after rup-
Fig. 4. Expression of green fluorescent protein from suspended (A) and immobilised culture (B) by SDS-PAGE.
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Fig. 5. Production of green fluorescent protein in suspended (A) and immobilised (B) culture. The medium was changed every 2 days after virus infection (A: 䊉, cell density; , GFP produced; ♦, specific GFP production; ↓, the time of medium perfusion, B: 䊉, the immobilised Sf21 cell density; , GFP produced; ♦, specific GFP production).
turing capsules gently and the cell suspension washed with fresh medium was measured using trypan blue exclusion. Then, the cell concentration was compared to the values of MTT-test. The deviation between trypan blue exclusion and MTT-test of immobilised cell density was about 30%. However, many viable cells in capsules might die in the step of capsule rupturing for measurement of trypan blue exclusion. Practically, the calibration results of MTT-test between suspension and immobilised culture were about the same in the previous work [13]. This indicated that the reaction of MTTchemical in a capsule was performed was just viable cells in the microspheres. Thus the MTT-test in immobilised cultures was used as a simple method for the measurement of immobilised insect cell density in this study. The volumetric PIBs (PIBs/ml) production in the microspheres was 58 times higher as compared to that in the suspension culture. The specific PIBs production in the microspheres compared to in the suspension culture is shown in Table 1. In the suspension culture, one infected cell produced 27.5 PIBs, whereas, one immobilised cell produced 52 PIBs. When the PIB productivity per cell (PIBs/cell) in the suspension culture was set to 1, the relative PIB productivity
(PIBs/cell) in the microspheres was a little higher of 1.9 times. In the suspension culture, the PIB productivity per cell can be considered as an optimal production value in this study with high cell viability (over 90%) and replacement of the medium just before virus infection. However, the specific productivity in the suspension culture was lower than that in the microspheres, because in the suspension culture it is very likely that some cells died before they were able to accumulate the maximum PIB number inside the cell. In the previous works, high insect cell densities and production of wild type baculoviruses were obtained. Sf9 cells in NaCSPDADMAC microspheres were successfully cultured for a long time (13 weeks) maintaining high cell density [5] and Choristoneura fumiferana (Cf-2C1) cells were immobilised using NaCS-PDADMAC polymer and the immobilised cells after CfMNP virus infection produced 83 times higher CfMNPV PIBs as compared to that in the suspension condition [13]. In this study, the recombinant protein, GFP, was produced in immobilised Sf21 insect cell culture. The specific GFP production was little higher (11 ng/106 cells h) in the suspension culture rather than that (10 ng/106 cells h) in the immobilised culture as shown in Fig. 5. The total GFP produced amount in the immobilised culture was, however, 1.193 mg in 7.5 ml microspheres, which value was about two times higher than that (0.6 mg in 125 ml) in the suspension culture. In the immobilised condition, the high cell density could be maintained for a long time, thus the virus infection was performed at a high TOI (time of infection). Therefore, a higher cell density and production yields of wild type PIBs as well as recombinant proteins in the immobilised condition with the relative high TOI could be achieved as compared to that in the suspension culture. This indicates that a high TOI does not necessarily lead to a decrease of the productivity. It seems to be more important to maintain the optimal culture conditions with minimal biological and physical stresses to the cells during producing the desired products [15,16].
Acknowledgments This research was supported by a grant (P-2004-02) from Marine Bioprocess Research Center of the Marine Bio 21 center funded by the Ministry of Maritime Affairs & Fisheries, Republic of Korea. Dr. Jeong Hwa Son is financially supported by a fund from the Brain Korea 21 project of the Ministry of Education, Korea.
References [1] L.K. Miller, The Baculoviruses, Plenum Press, New York/London, 1997. [2] M.L. Shuler, H.A. Wood (Eds.), Baculovirus Expression Systems and Biopesiticides, Wiley-Liss, New York, 1995. [3] D.R. O’Reilly, L.K. Miller, V.A. Luckow, Baculovirus Expression Vectors, W.H. Freemann and Company, New York, 1992.
J.H. Son et al. / Biochemical Engineering Journal 29 (2006) 55–61 [4] T. Wang, I. Lacik, M. Brissova, A.V. Anikumar, An encapsulation system for the immunoisolation of pancreatic islets, Nat. Biotechnol. 15 (1997) 358–362. [5] H. H¨ubner, R. Buchholz, High density cultivation of insect cells in microhollow spheres, in: Invertebrate Cell Culture/Novel directions and Biotechnology Applications, Science Publishers Inc., Enfield, NH, 1997, pp. 123–129. [6] G.A. King, A.J. Daugulis, P. Faulkner, D. Bayly, M.F.A. Goosen, Growth of baculovirus-infected cells in microcapsules to a high cell and virus density, Biotechnol. Lett. 10 (1988) 683–688. [7] G.A. King, A.J. Daugulis, P. Faulkner, D. Bayly, M.F.A. Goosen, Alginate concentration: a key factor in growth of temperaturesensitive baculovirus-infected insect cells in microcapsules, Biotechnol. Bioeng. 34 (1989) 1085–1091. [8] M. Chalfie, Y. Tu, G. Euskirchen, W.W. Ward, D.C. Prasher, Green fluorescent protein as a marker for gene expression, Science 263 (1994) 802–805. [9] H.J. Cha, N.G. Dalal, M.Q. Pham, V.N. Vakharia, G. Rao, W.E. Bentley, Insect larval expression process is optimized by generating fusions with green fluorescent protein, Biotechnol. Bioeng. 65 (1999) 316–324.
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[10] C. Oker-Blom, A. Orellana, K. Keinanen, Highly efficient production of GFP and its derivatives in insect cells for visual in vitro applications, FEBS Lett. 389 (1996) 238–243. [11] K.J. Oparka, A.G. Roberts, S.S. Cruz, P. Boevink, D.A.M. Prior, A. Smallcombe, Using GFP to study virus invasion and spread in plant tissues, Nature 388 (1997) 401–402. [12] D. Trouet, B. Nilius, T. Voets, G. Droogmans, J. Eggermont, Use of a bicistronic GFP-expression vector to characterise ion channels after transfection in mammalian cells, Pfluegers Arch. Eur. J. Physiol. 434 (1997) 632–638. [13] J.H. Son, R. Buchhloz, S.K. Kim, An immobilized culture of Choristoneura fumiferana cells for high production of CfMNPV, J. Microbiol. Biotechnol. 14 (2004) 395–400. [14] U.K. Laemmli, Cleavage of structural proteins during assembly of the heads of bacteriophage, Nature 227 (1970) 680–685. [15] P.E. Cruz, A. Cunha, C.C. Peixoto, J. Clemente, J.L. Moreira, M.J.T. Carrondo, Optimization of the production of virus-like particles in insect cells, Biotechnol. Bioeng. 60 (1998) 408–418. [16] T. Gotoh, G. Mochizuki, K.I. Kikuchi, Perfluorocarbon-mediated aeration applied to recombinant protein production by virus-infected insect cells, Biochem. Eng. J. 7 (2001) 69–78.
Production of baculoviruses and expression of green fluorescent protein in immobilised Sf21 insect cell cultivation Jeong Hwa Son a , Rainer Buchholz b , Jung-Keug Park c , Sung Koo Kim a,∗ a
c
Department of Biotechnology & Bioengineering, Pukyong National University, 599-1, Daeyeon 3-dong, Nam-gu, Busan 608-737, Republic of Korea b Department of Bioprocess Engineering, Friedrich-Alexander University, Erlangen-Nuremberg, Germany Department of Chemical and Biochemical Engineering, Dongguk University, Seoul 100-715, Republic of Korea Received 15 October 2004; accepted 19 February 2005
Abstract Spodoptera frugiperda (Sf21) insect cells were grown in the microspheres that were prepared using sodium-cellulosesulfate (NaCS) and polydiallyldimethylammoniumchloride (PDADMAC). The highest Sf21 cell density was 1 × 108 cells/ml in the microshperes. The immobilised Sf21 cells were infected with Autograpa californica multiple nuclear polyhedrosis virus (AcMNPV) at a “theoretical” MOI of 1.0 and a TOI of 3.9 × 107 cells/ml in the microspheres. The AcMNPV polyhedral inclusion bodies (PIBs) titer reached was 1.76 × 1010 PIBs/ml in the microspheres, which was 58 times higher than that (2.75 × 107 PIBs/ml) in a suspension condition. The immobilised Sf21 cells were infected with a recombinant baculovirus, Ac-omega-GFP baculovirus, and the highest concentration of green fluorescent protein (GFP) produced was 0.0048 mg/ml in suspension culture and 0.159 mg/ml in the microspheres. © 2005 Published by Elsevier B.V. Keywords: Immobilisation; NaCS; PDADMAC; Ac-omega-GFP baculovirus; PIBs; GFP
1. Introduction Baculovirus-insect cell culture system has been used as an attractive tool to produce wild type baculoviruses for the use as a biopesticide as well as genetically engineered baculoviruses for the expression of recombinant proteins of medical and pharmaceutical importance, which system has several advantages such as high expression levels, limitless size of the expressing of the protein, post translational modifications, simultaneous expression of multiple genes and safety for vertebrates [1,2]. Most common recombinant baculoviruses (baculovirus expression vector, BEV) can be created by replacing the polyhedrin gene with a target gene under the transcriptional control of the polyhedrin promoter of the Autographa californica nuclear polyhedrosis virus (AcNPV).
∗
Corresponding author. Tel.: +82 51 620 6188; fax: +82 51 620 6180. E-mail address: [email protected] (S.K. Kim).
1369-703X/$ – see front matter © 2005 Published by Elsevier B.V. doi:10.1016/j.bej.2005.02.029
Then, the desired proteins are expressed during the very late stages of infection in insect cell culture [3]. Although insect cell culture has been usually employed for high production level, only a few desired products have been produced [1–3]. The main problem is associated with a scale-up of virus infected insect cell culture that is a desirable procedure for the commercial production. At large cultures, the common methods for supply oxygen to the cells such as sparging and agitation can damage the cells that are extremely shear-sensitive. In addition, the oxygen demand of insect cells is higher than that of mammalian cells, and oxygen uptake increase upon virus infection [2]. As well as, due to the lytic behavior of the baculovirus infected cells, it is difficult to achieve high cell densities and high production levels. Cell immobilisation system has been shown as a solution to overcome these disadvantages [4–7]. Immobilised insect cells can be protected from any shear stresses, while oxygen and nutrients are sufficiently supplied to the cells, and the cells can be remained in the microspheres completely dur-
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J.H. Son et al. / Biochemical Engineering Journal 29 (2006) 55–61
ing medium exchange. Hence, it is possible to reach higher cell densities and product concentrations as compared to in suspension culture. Green fluorescent protein (GFP) isolated from jellyfish Aequaorea victoria is a protein consisting of 238 amino acids, with a molecular weight of 27 kDa [8]. The protein has a maximal absorption peak at 395 nm and a minor peak at 470 nm with an emission peak at 509 nm. Its fluorescence is stable, independent, and requires no substrate, cofactor, or additional proteins for illuminating green light, therefore, GFP is highly attractive as a visual marker for gene expression studies [9–12]. In this study, Sf21 insect cells were infected with wild type A. californica multiple nuclear polyhedrosis virus (AcMNPV) and a recombinant Ac-omega-GFP baculovirus, and the productions of polyhedral inclusion bodies (PIBs) and green fluorescent proteins were evaluated in suspension and immobilised culture system.
2. Materials and methods
w/v) were prepared in 1% (w/v) NaCl solution and autoclaved (121 ◦ C, 10 min). The procedure of immobilisation is shown in Fig. 1. An encapsulation apparatus was used for immobilisation of Sf21 insect cells as described by H¨ubner et al. [5]. Briefly, the apparatus was connected to the storage bin containing the 3.5% NaCS solution and the cell solution through a silicone tube (D = 2 mm). The volume flow was adjusted to 200 ml/h. After dropping the mixed solution of NaCS and cells into the 2.2% PDADMAC solution, the mixture formed microspheres. The microspheres were stirred in the PDADMAC solution for 15 min, and washed extensively three times with the phosphate buffered saline (PBS: 0.2 g/l KCl, 0.2 g/l KH2 PO4 , 8 g/l NaCl, 1.15 g/l Na2 HPO4 , pH 7.4), then transferred into culture flasks for cultivation. The microspheres have about 10 m membrane gaps as shown in Fig. 1. These gaps were caused by the death of protruding cells from the capsule membrane due to the cytotoxicity of PDADMAC in the PDADMAC solution for the stirring time. Microspheres containing the cells (7.5 ml, wet volume) were cultured in 50 ml of SF900II serum free medium in a 500 ml shaker flask with four baffles at 80 rpm, 27 ◦ C and pH 6.2.
2.1. Suspension cell culture 2.3. Measurement of living cell density (MTT-test) Spodoptera frugiperda (Sf21) insect cells were cultured in SF900II serum free medium (GIBCOBRL). The culture condition was controlled at 27 ◦ C and pH 6.2. The viability of the cells was determined by a haemocytometer using 0.4% (w/v) trypan blue exclusion. 2.2. Cell immobilisation Sodium-cellulosesulfate (NaCS, 3.5%, w/v) and polydiallyldimethylammoniumchloride (PDADMAC, 2.2%,
One millilitre of cell solution from the suspension culture or 10 microspheres from the immobilised culture was daily taken out for the MTT-test [13]. Briefly, one part of MTT solution was mixed with four parts of sample solution, and incubated at 27 ◦ C and dark for 4 h, then, the microspheres were homogenized in sodium-dodecylsulfate (SDS, 20%, w/v). One millilitre of the homogenized solution or incubated cells with MTT solution were added to 4 ml of lysis buffer (405 ml iso-propanol, 20 ml 1N HCl, 75 ml SDS
Fig. 1. An encapsulation apparatus. The size of microspheres is about 3 mm in diameter. The microspheres have about 10 m membrane gaps due to the death of cells protruding from the membrane of spheres in the PDADMAC solution during the stirring time.
J.H. Son et al. / Biochemical Engineering Journal 29 (2006) 55–61
(20%, w/v)) and supersonicated. The debris was centrifuged, and the absorbance (λ = 570 nm) of supernatant was measured by a spectrophotometer. 2.4. AcMNP Virus infection of suspended and immobilised insect cells Suspended Sf21 cells were infected at a multiplicity of infection (MOI) of 1.0 by adding a virus stock solution (MOI of 0.1) of AcMNPV (A. californica multiple nuclear polyhedrosis virus, Pharmingen, Hamburg). The virus titers were determined by the end-point dilution assay [7]. The infected cells were cultured in 50 ml SF900II medium in a 500 ml culture flask at 80 rpm and 27 ◦ C. When the immobilised cells reached the stationary growth phase, the cells were infected at a “theoretical” MOI of 1.0 with the virus stock solution. The “theoretical” MOI of 1.0 was determined by the immobilised cell density that was measured by MTT-test as before viral infection [13]. The medium was changed every day after viral infection. 2.5. Counting the polyhedral inclusion bodies (PIBs) One ml of the sample in the suspension culture or five microshperes in the immobilised culture homogenized in 20% (w/v) SDS solution were centrifuged at 1300 × g for 15 min. The pellet was extensively washed in polyhedra lysis buffer (1.21 g/l Tris, 0.37 g/l EDTA, 0.72 g/l SDS) and supersonicated until cell membrane and debris were removed. Then, the sample was centrifuged again at 1300 × g for 15 min, and polyhedra lysis buffer was added. This process was repeated until only polyhedral inclusion bodies (PIBs) were remained in the buffer. PIBs in lysis buffer were observed as 2–5 m particles with 0.4% trypan blue exclusion under a light microscope and counted using a haemocytometer. 2.6. Ac-omega-GFP baculovirus stock Ac-omega-GFP baculovirus (polh− ) was used for Sf21 insect cell infection. The recombinant baculovirus has been obtained from Institute for Applied Microbiology, University of Agricultural Science, Vienna, Austria. The baculovirus vector was constructed by inserting GFP gene into the unique restriction site Sce-I of Ac-omega viral genome that is genetically engineered AcNPV (A. californica nuclear polyhedrosis virus) genome by introducing a unique restriction site downstream of the strong polyhedrin promoter (Ac-omega) to allow linearization with a specific endonuclease (Sce-Imeganuclease). The suspended cells were infected with Ac-omega-GFP baculoviruses at a multiplicity of infection (MOI) of 0.1. The infected cells were harvested at 96 h post infection (hpi), centrifuged at 50 × g for 10 min, and the supernatant were kept in the 4 ◦ C for using as a virus stock.
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2.7. Ac-omega-GFP baculovirus infection to suspension and immobilised culture S. frugiperda (Sf21) insect cells were cultured in 125ml SF900II serum free medium (GIBCOBRL) in a 500 ml spinner flask at 65 rpm and 27 ◦ C. The suspended cells in a spinner flask were infected with the Ac-omega-GFP baculovirus stock at a MOI of 1.0 at the cell density of 1 × 106 cells/ml (over 90% of cell viability). The medium of infected cells was changed every 2 days by cell centrifugation (50 × g, 5 min). Immobilised Sf21 insect cells in the stationary phase were infected with the Ac-omega-GFP baculovirus at a “theoretical” MOI of 1.0, adding the virus stock solution, and the medium was changed every 2 days. 2.8. SDS-PAGE The samples from the suspended and immobilised culture infected with Ac-omega-GFP baculovirus were analyzed by sodium dodecyl sulfate-polyacrlyamide gel electrophoresis (SDS-PAGE) [14]. In the case of immobilised culture, five microspheres were taken out from the culture and ruptured in lysis buffer (50 mM glucose, 10 mM EDTA, 25 mM Tris–HCl (pH 8.0)) and supersonicated for 15 min, then centrifuged at 1500 × g for 5 min. One part of the supernatant and one part of the sample buffer were mixed, boiled at 100 ◦ C for 10 min, centrifuged at 1500 × g for 5 min. Then, the sample was separated onto 12% slab gels by electrophoresis, and the SDS gel was stained by Coomassie Brilliant Blue R250. 2.9. ELISA The GFP produced in the suspended and immobilised culture was quantified using direct ELISA. Suspension sample was supersonicated for 15 min and centrifuged (1500 × g, 15 min). The clear supernatant was coated onto a 96-well plate and the plate was covered with parafilm. After incubation for 2 h at 37 ◦ C, the excess sample solution was removed. Then 200 l blocking buffer (1%BSA in PBS) was added per well and the plate was incubated at room temperature for 2 h. After removing of blocking buffer, the plate was washed two times with 200 l washing buffer (0.05% Tween-20 in PBS) and 100 l horseradish peroxidase conjugated GFP antibody (1:1000, CLONTECH, CA, USA) was added in the plate. After incubation at room temperature with shaking for 1 h, the plate was washed three times with 200 l wash buffer per well, and 100 l TMB (tetramethylbenzidine) substrate solution was added in the plate. Then, the result quantified at 595 nm and the value was compared with a standard EGFP (EGFP: CLONTECH) calibration curve to calculate the GFP concentration. In immobilised culture, two microcapsules in 400 l lysis buffer (50 mM glucose, 10 mM EDTA, 25 mM Tris/HCl (pH 8.0)) were ruptured and supersonicated for 15 min. The sample was centrifuged at 1500 × g for 5 min, finally measured by ELISA as above.
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Fig. 2. Immobilised Sf21 cells. The cells entrapped in NaCS-PDADMAC membrane as indicated by arrows (A), microspheres containing live cells after MTT-test (B, b/w picture), and microspheres containing dead cells after MTT-test (C, b/w picture).
3. Results and discussion 3.1. Sf21 cell culture in immobilised condition Fig. 2 shows the microspheres entrapping Sf21 cells. A single cell and cell aggregates are visible inside the sphere (Fig. 2A). The colors of microspheres containing live cells was changed to dark purple by enzymatic catalysis reaction between MTT and dehydrogenase from mitochondria of the live cell (Fig. 2B), however, the colors of microshperes containing dead cells were not changed by MTT-test (Fig. 2C). The sphere size was about 3 mm in diameter. In the immobilised culture, the highest cell density was 1 × 108 cells/ml in the microspheres, which was about 32 times higher than that (3.2 × 106 cells/ml) in suspension culture. 3.2. The production of PIBs in the suspended and immobilised insect cell culture Suspended Sf21 cells were infected with AcMNPV, at the cell density of 1.0 × 106 cells/ml and the viability over 90% with a MOI of 1.0 as shown in Fig. 3A. After the virus infection, the infected cells produced extracellular virus particles (nonincluded viruses, NOVs) and occluded virus particles (polyhedral inclusion bodies, PIBs). At 48 hpi (hours post infection), PIBs were detected as 2–5 m in diameter under the microscope. The viability of infected cells significantly decreased within 96 hpi. The highest concentration of PIBs achieved was 2.75 × 107 PIBs/ml in 96 hpi (Fig. 3A). The immobilised Sf21 cells in the stationary phase were infected with AcMNPV at a theoretical MOI of 1.0 (Fig. 3B), PIBs produced in the microspheres reached the concentration above 1.0 × 109 PIBs/ml after 4 days post infection (dpi). Then, the highest PIBs concentration was 1.76 × 1010 PIBs/ml in the microspheres at 15 dpi. In addition, PIBs produced up to 1 × 107 PIBs/ml could be detected daily in the spent medium. The production of PIBs, in the suspended and immobilised cultures, is shown in Table 1. In the microspheres, the PIBs produced were 3.9 × 108 PIBs/ml per day. This value was higher than the PIBs concentration produced in the suspension culture for 4 days (2.75 × 107 PIBs/ml), and the PIBs
Fig. 3. Production of polyhedral inclusion bodies (PIBs) in suspended (A) and immobilised (B) Sf21 insect cell culture (A: , Sf21 cell viability; , PIBs; 䊉, live cells; , dead cells and line: AcMNPV infection (MOI = 1.0), B: , PIBs in the capsules; , PIBs in the medium; 䊉, cell density in the capsules and line: AcMNPV infection).
J.H. Son et al. / Biochemical Engineering Journal 29 (2006) 55–61 Table 1 Production of polyhedral inclusion bodies (PIBs) in suspension and immobilised Sf21 insect cell cultivation Value
Suspension cultivation
Immobilised cultivation
TOI (106 cells/ml)a PIBs/ml and production time (day)
1 2.75 × 107 (4)b
30 3.9 × 108 (1)c 1.76 × 1010 (15)d
Relative PIBs productivity Specific PIB productivity in 4 dpi (PIBs/cell) Relative yield of PIBs (PIBs/cell)
1e 27.5
58 52
1f
1.9
a
Time of infection (TOI: cell density when virus infection). The density of baculoviruses (PIBs/ml) produced for 4 days. c The density of baculoviruses (PIBs/ml) produced for 1 day. d The density of baculoviruses (PIBs/ml) produced for 15 days. e Relative PIBs productivity (2.75 × 107 ) in the suspension condition as set to 1 to compared with the value in the immobilised condition. f Relative yield of PIBs (27.5) in the suspension condition as set to 1 to compared with the value in the immobilised condition. b
production term in the suspension culture was just 5 dpi (Fig. 3A), which was terminated due to the death of infected cells. Therefore, the relative PIBs productivity in the immobilised cell culture was 58 times higher as compared to that in the suspension cultivation. 3.3. GFP analysis by SDS-PAGE Suspended and immobilised Sf21 cells were infected with Ac-omega-GFP baculovirus. Then, GFP light from the cultures could be detected from 24 hpi under a fluorescence microscope (492 nm). Fig. 4 shows SDS-PAGE results of the suspension culture (Fig. 4A) and the immobilised culture (Fig. 4B) infected by Ac-omega-GFP baculovirus. In Fig. 4A, GFP was expressed with the molecular weight of 27 kD at 96 hpi (lane no. 3) and 144 hpi (lane no. 4), but not 24 hpi (lane no. 2) because GFP was expressed under the polyhedrin promoter control at very late in the virus infection cycle. The protein content of one lane on the gel was corresponded to the extract of 6 × 104 cells.
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In the immobilised culture (Fig. 4B), GFP expressed could be observed from 72 hpi (lane no. 3). The protein content of one lane was corresponded to the extract of about 2.4 × 104 cells. In addition, the GFP from the cultured medium in the immobilised culture was not detected (lane no. 4). 3.4. Quantification of GFP in the suspended and immobilised culture Fig. 5A shows Sf21 insect cell culture in a 500 ml spinner flask in a perfusion batch system. The highest noninfected Sf21 insect cell density obtained was 1.2 × 106 cells/ml. After the infection of Ac-omega-GFP baculovirus, the medium was changed every 2 days, however, the infected cell density decreased significantly. The concentration of the GFP produced in the suspension culture was 0.0012 mg/ml at 48 hpi, and the highest GFP production reached was 0.0048 mg/ml at 144 hpi (Fig. 5A). The suspension cultivation could not be performed after 168 hpi in spite of fresh culture condition by medium exchange every 2 days after virus infection. In the immobilised cell cultivation, the highest cell density achieved was 1 × 108 cells/ml in the microspheres before the virus infection as shown in Fig. 5B. After the virus infection, the immobilised cell density was maintained about 5 × 107 cells/ml in the microspheres throughout the cultivation period (510 hpi). After 510 hpi, the immobilised culture was kept to without medium exchange, hence decreasing of the immobilised cell density after 4 days of culture. The concentration of GFP produced was 0.057 mg/ml in the microspheres at 120 hpi, and the highest GFP concentration achieved was 0.159 mg/ml at 300 hpi in the microspheres (Fig. 5B). This concentration was about 33 times higher than that in the suspension culture. In addition, 0.2–0.43 g/ml of GFP in the spent medium was constantly detected every 2 days. In the immobilised culture system, trypan blue exclusion is not valid for the measurement of the viable cells because the cells are entrapped by capsule polymer, thus it is impossible to distinguish the dead and living cells. Another approach for measurement of viable cell density was performed after rup-
Fig. 4. Expression of green fluorescent protein from suspended (A) and immobilised culture (B) by SDS-PAGE.
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Fig. 5. Production of green fluorescent protein in suspended (A) and immobilised (B) culture. The medium was changed every 2 days after virus infection (A: 䊉, cell density; , GFP produced; ♦, specific GFP production; ↓, the time of medium perfusion, B: 䊉, the immobilised Sf21 cell density; , GFP produced; ♦, specific GFP production).
turing capsules gently and the cell suspension washed with fresh medium was measured using trypan blue exclusion. Then, the cell concentration was compared to the values of MTT-test. The deviation between trypan blue exclusion and MTT-test of immobilised cell density was about 30%. However, many viable cells in capsules might die in the step of capsule rupturing for measurement of trypan blue exclusion. Practically, the calibration results of MTT-test between suspension and immobilised culture were about the same in the previous work [13]. This indicated that the reaction of MTTchemical in a capsule was performed was just viable cells in the microspheres. Thus the MTT-test in immobilised cultures was used as a simple method for the measurement of immobilised insect cell density in this study. The volumetric PIBs (PIBs/ml) production in the microspheres was 58 times higher as compared to that in the suspension culture. The specific PIBs production in the microspheres compared to in the suspension culture is shown in Table 1. In the suspension culture, one infected cell produced 27.5 PIBs, whereas, one immobilised cell produced 52 PIBs. When the PIB productivity per cell (PIBs/cell) in the suspension culture was set to 1, the relative PIB productivity
(PIBs/cell) in the microspheres was a little higher of 1.9 times. In the suspension culture, the PIB productivity per cell can be considered as an optimal production value in this study with high cell viability (over 90%) and replacement of the medium just before virus infection. However, the specific productivity in the suspension culture was lower than that in the microspheres, because in the suspension culture it is very likely that some cells died before they were able to accumulate the maximum PIB number inside the cell. In the previous works, high insect cell densities and production of wild type baculoviruses were obtained. Sf9 cells in NaCSPDADMAC microspheres were successfully cultured for a long time (13 weeks) maintaining high cell density [5] and Choristoneura fumiferana (Cf-2C1) cells were immobilised using NaCS-PDADMAC polymer and the immobilised cells after CfMNP virus infection produced 83 times higher CfMNPV PIBs as compared to that in the suspension condition [13]. In this study, the recombinant protein, GFP, was produced in immobilised Sf21 insect cell culture. The specific GFP production was little higher (11 ng/106 cells h) in the suspension culture rather than that (10 ng/106 cells h) in the immobilised culture as shown in Fig. 5. The total GFP produced amount in the immobilised culture was, however, 1.193 mg in 7.5 ml microspheres, which value was about two times higher than that (0.6 mg in 125 ml) in the suspension culture. In the immobilised condition, the high cell density could be maintained for a long time, thus the virus infection was performed at a high TOI (time of infection). Therefore, a higher cell density and production yields of wild type PIBs as well as recombinant proteins in the immobilised condition with the relative high TOI could be achieved as compared to that in the suspension culture. This indicates that a high TOI does not necessarily lead to a decrease of the productivity. It seems to be more important to maintain the optimal culture conditions with minimal biological and physical stresses to the cells during producing the desired products [15,16].
Acknowledgments This research was supported by a grant (P-2004-02) from Marine Bioprocess Research Center of the Marine Bio 21 center funded by the Ministry of Maritime Affairs & Fisheries, Republic of Korea. Dr. Jeong Hwa Son is financially supported by a fund from the Brain Korea 21 project of the Ministry of Education, Korea.
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