Papier Trypsninisation 2013

Appl Biochem Biotechnol (2013) 170:1724–1737 DOI 10.1007/s12010-013-0307-y Development of an In Situ Detachment Protocol of Vero Cells Grown on Cytodex1 Microcarriers Under Animal Component-Free Conditions in Stirred Bioreactor Samia Rourou & Nesrine Riahi & Samy Majoul & Khaled Trabelsi & Héla Kallel Received: 1 February 2013 / Accepted: 20 May 2013 / Published online: 5 June 2013 # Springer Science+Business Media New York 2013 Abstract Subcultivation of Vero cells grown in a proprietary animal component-free medium named IPT-AFM, on microcarriers, was studied. TrypLE Select, a non-animal-derived protease, was used as an alternative to trypsin for cell passaging. We first studied the effect of increasing concentrations of TrypLE Select toward cell growth and then studied the inactivation of the protease using either soybean trypsin inhibitor (STI) or the soy hydrolysate Hypep 1510, in sixwell plates. Data showed that cell growth was impaired by residual level of TrypLE Select; STI was identified as an efficient agent to neutralize this effect. To restore cell growth and inactivate TrypLE Select, STI should be added to the medium at least at 0.2 g L−1. Cells were also grown in spinner flask on 2 g L−1 Cytodex1 in IPT-AFM. In these conditions, the cell detachment yield was equal to 78±8 %. Furthermore, cells exhibited a typical growth profile when using the dislodged cells to seed a new culture. A cell detachment yield of 70±19 % was also achieved when the cells were grown in a 2-L stirred bioreactor in IPT-AFM, on 3 g L−1 Cytodex1. This protocol can be of great interest to scale-up the process of Vero cells cultivation in IPT-AFM on Cytodex1 from one stirred bioreactor culture to another. Keywords Vero cells . Stirred bioreactor . Cytodex1 microcarriers . Animal component-free conditions . Subcultivation Introduction Advances in mammalian cell technology have led to its increasing application in the production of vaccines and other biopharmaceuticals. Mammalian cells are generally classified by their requirement for anchorage-dependent growth or by their ability to grow in suspension culture (anchorage-independent cells). S. Rourou : N. Riahi : S. Majoul : K. Trabelsi : H. Kallel (*) LR11IPT01 Laboratory of Molecular Microbiology, Vaccinology and Biotechnology Development, Viral Vaccines Research and Development Unit, Institut Pasteur de Tunis, 13, place Pasteur. BP 74, 1002 Tunis Belvédère, Tunisia e-mail: [email protected] Appl Biochem Biotechnol (2013) 170:1724–1737 1725 Anchorage-dependent cells are used extensively in industrial processes designed for large-scale vaccine production [1, 2]. Among this family, Vero cells are considered as the most widely accepted continuous cell line by the regulatory authorities for the manufacture of viral vaccines [3]. They can be used as a host for the production of different types of viruses. The standard Vero cells culturing process based on growing cells on microcarriers was first described by Van Wezel in 1967 [4]. Starting from that date, microcarrier technology has been applied successfully for the cultivation of anchorage-dependent cells to produce many important biological materials, such as vaccines, tissue plasminogen activator, human interferon, erythropoietin, etc. [5]. Cell detachment is an essential step for the process of subcultivation of adherently growing cells which have to be dissociated and prepared for the subsequent culture. It is a key operation in any cell culture laboratory [6–8]. This is classically achieved by the use of trypsin. Nevertheless, residual trypsin activity in cell cultures damages many cell types and may completely inhibit cell growth in serum-free media [9, 10]. Subpassaging of anchorage-dependent cells from one stirred culture to the next is one of the most crucial steps for successful development of microcarrier technology-based production processes [11–13]. Hence, various methods were developed for cell detachment. The most popular cell-harvesting techniques are enzymatic ones which include the use of proteolytic enzymes, e.g., trypsin [6, 14, 15], collagenase [7, 14], dispase [7, 16], dextranase [17], and accutase [7]. In addition, chemicals such as ethylenediaminetetraacetic acid (EDTA) can be used for cell dislodging [9, 18, 19]. Nevertheless, Cruz et al. [20] showed that this method was not efficient in comparison to the enzymatic detachment and can be toxic to certain cells such as BHK-21 cells. We also demonstrated in a previous work that EDTA did not allow the detachment of Vero cells grown in monolayer culture under animal component-free conditions [21]. Furthermore, Junge et al. [22] analyzed the detachment of adherent HeLa cells from a substrate after the interaction with a shockwave. They found that the regions of cell detachment are strongly correlated with spatial presence of cavitation bubbles. They had also showed that the cavitation bubble collapse generates a transient high-speed flow along the substrate surface leading to rapid detachment of the cells. Lately, a new non-enzymatic detachment method was developed. Thermoresponsive (or stimuli-responsive) polymers and their hydrogels can be used for enzyme-free cell detachment. Cell monolayers were harvested as single-cell sheets by temperature decrease from 37 to 20 °C. This technique was first developed for monolayer cultures and then for microcarriers [23–26]. Bead-to-bead cell transfer also provides an alternative solution for the scale-up of microcarrier cultures [5, 7]. Although this method is simple to operate, it results in uneven cell distribution on microcarriers and, therefore, in a low growth rate in the subsequent culture [27]. Luo et al. [28] improved this method; they showed that it was feasible to scale-up microcarrier culture of Vero cells through a two-stage bead-to-bead cell transfer process. The process was initiated by mixing confluent microcarriers with fresh microcarriers. In the first stage, the mixture was stirred intermittently for 8 h, and then the second stage started with a continuous agitation at 35 rpm. It was found that with this method a higher percentage of bridged fresh microcarriers was obtained, compared with the classic bead-to-bead cell transfer process. To improve the quality and increase the safety of manufactured products, it is now recommended to remove serum and other components of animal origin, from the manufacturing process. For this purpose, we developed in a previous work an animal component-free process to grow Vero cell culture in agitated bioreactor to produce a human rabies vaccine [21, 29, 30]. However, the scale-up of this process is cumbersome if the seed cells would be prepared in stationary culture mode. This culture technique suffers from several drawbacks such as 1726 Appl Biochem Biotechnol (2013) 170:1724–1737 increased consumption of culture medium, high risk of microbial contamination, and a longer overall production cycle. Thus, the preparation of the inoculum in a bioreactor is the alternative to circumvent these limitations. This work focuses on the development of a protocol for the detachment of Vero cells grown in a 2-L bioreactor in IPT-AFM, using the recombinant trypsin (TrypLE Select). For this purpose, we optimized various parameters to reach the highest cell detachment yield; Vero cells’ behavior grown under these conditions was also assessed. Materials and Methods Cell Line Vero cells at passage 131, provided by the National Laboratory for Control of Biologicals (Tunis, Tunisia) and originally obtained from American Type Culture Collection, ATCC (CCL-81), were used in this study. Culture Media and Chemicals Hypep 1510 (cat. no. 5X99023) was provided by Sheffield-Bioscience (Norwich, NY, USA); it is an ultrafiltered, animal component-free, medium supplement produced by the enzymatic hydrolysis of soy. Soy trypsin inhibitor (cat. no. T6522) was supplied by Sigma Aldrich (St. Louis, USA). IPT-AF medium is an in-house-developed animal component-free medium prepared as described in Rourou et al. [21]. Cell Dissociation Cells were detached using an animal origin free recombinant trypsin called TrypLE Select (Invitrogen, cat. no. 12563-029). Cell subcultivation was performed as described by the manufacturer (for details, see Rourou et al. [29]). Briefly, cells were washed twice with phosphate buffer saline solution (PBS) then 6–7 mL of enzyme solution was added to a T-225 flask, and cells were incubated for 2 min at 37 °C. Dislodged cells were then centrifuged at 1,000 rpm for 10 min. Growth in Static Culture Vero cells cryopreserved in IPT-AF medium were revitalized and grown in static culture at 37 °C and 5 % CO2. Cell culture flasks were coated with teleostean (Sigma) as detailed in Rourou et al. [30] only during the first passage after defrosting. Static cultures were carried out under the following conditions: a seeding density of 8×104 cells/cm2, 37 °C, and 5 % CO2. Six-Well Plate Experiments “Nunclon Δ” plates (Nunc, Denmark) were used for monolayer cultures; microcarrier cultures were performed in “Costar” low-binding plates (Costar, NY, USA). Cultures were seeded at 2×105 cells mL−1; Cytodex1 concentration was 0.5 g L−1. The plates were incubated for 3 days at 37 °C in 5 % CO2 incubator. Appl Biochem Biotechnol (2013) 170:1724–1737 1727 During subcultivation, cell suspensions were not centrifuged to avoid the removal of the protease. All the experiments were performed in duplicate. To inactivate the residual activity of TrypLE Select, we studied the effect of two products: Hypep1510 peptone which is an ultrafiltrated enzymatic hydrolysate of soy grits and the commercial soybean trypsin inhibitor. Each inhibitor was tested at different concentrations; Hypep1510 was tested at 0, 2, 5, 10, 25, and 50 g L−1. For soybean trypsin inhibitor (STI), the following concentrations 0.01, 0.05, 0.1, and 0.2 g L−1 were used. For monolayer culture, the medium was removed, and about 0.5 mL/well of TrypLE Select was added. After incubation for 2 min at room temperature, the content of each well was recovered and counted. For Cytodex1 cultures, the content of each well was recovered in a 15-mL sterile tube. After settling, the pellet was washed twice with PBS. The microcarriers were then covered by a 0.5 mL of TrypLE Select and left at room temperature for 5 min. After detachment, cells were recovered in the presence of the inhibitor to be tested and counted. Microcarrier Preparation Cytodex1 microcarriers from GE Healthcare (Uppsala, Sweden) were prepared and sterilized according to the manufacturer’s instructions. Cell Growth in Spinner Flasks Cultures were carried out in 250-mL spinner flasks (Techne, UK) containing 200 mL of cultured cells, at 37 °C in a 5 % CO2 incubator as described by Trabelsi et al. [31]. Cells were cultivated in IPT-AF medium. The stirring speed was maintained at 30 rpm. The spinners were seeded with 2×105 cells mL−1 using 2 g L−1 Cytodex1 microcarriers. Bioreactor Cultures The cultures were performed in a 2-L bioreactor (Inceltech, France) containing 1.2 L as a working volume equipped with a pitched-blade impeller and a spin filter (pore size, 20 μm) fixed on the axis. To inoculate the bioreactor, cells were detached from T-flasks using TrypLE Select as described above, washed twice with PBS, and incubated in the reactor in the presence of microcarriers. The culture was seeded with 2.5×105 cells mL−1 using 3 g L−1 Cytodex1 microcarriers and was continuously agitated at 30 rpm. pH was maintained at 7.2 by CO2 injection or addition of NaHCO3 at 88 g L−1; pO2 was regulated at 50 % air saturation by either air injection through a sparger at a maximum flow rate of 15 mL min−1 or by headspace aeration. Temperature was regulated at 37 °C and agitation speed at 30 rpm. Samples were taken daily for cell counting and to monitor microcarriers load. Bioreactor culture was repeated twice. Trypsinization Protocol in Spinner Once the maximum cell density was reached, the cells were settled and the medium was discarded. After two washing steps with PBS, 10 mL of TrypLE Select was added and the cells were detached under agitation at 30 rpm. The detachment process was followed by regular microscopic observations. After detachment, an equal amount of STI (i.e., 10 mL) was added, the efficiency of detachment was determined, and cells were used to re-inoculate a new culture. 1728 Appl Biochem Biotechnol (2013) 170:1724–1737 In Situ Trypsinization of Microcarrier Bioreactor Culture TrypLE Select solution was pre-warmed at 37 °C prior to use. The culture medium was drained out; PBS was pumped into the reactor to wash the microcarriers and then removed. The washing procedure was repeated twice. After the second PBS wash, TrypLE Select solution was aseptically connected to the vessel, and 200 mL was pumped into the reactor. Then, around 30 mL of enzyme was kept, and the excess was discarded. This point is considered as the start of the trypsinization incubation period under agitation. The culture was incubated for 10 min. Samples were taken at 2-min intervals to monitor cell detachment by microscopic observation. When the vast majority of cells were detached, the action of TrypLE Select was stopped by the addition of an equal amount of 1 g L−1 STI. The resulting suspension of detached cells and microcarriers was then sampled for cell count and used to seed the next bioreactor culture. Cell Counting For viable cell count after cell detachment, cells were stained with Trypan Blue (0.2 % (w/v) in PBS) and counted using a hemacytometer. For nucleus count before detachment, 5 mL of Vero cell culture was washed three times with PBS then treated in 5 mL of 0.1 M citric acid (Sigma) containing 0.1 % Crystal Violet (Sigma) and 0.1 % Triton X-100 (USB, Cleveland, OH, USA) and incubated at least for 1 h at 37 °C. The released nuclei were counted using a hemacytometer. Calculations The following calculations were performed: The specific growth rate, μ (per hour), was estimated by the following equation μ¼ lnX n −lnX n−1 tn −tn−1 Where X represents the viable cell density per milliliter, t represents the time points of sampling expressed in hour, the subscripts n and n−1 stand for two succeeding sampling points. The detachment yield (Y) was calculated as follows: Y ¼ Total cell density level after detachment  100 Total cell density level before detachment Results Effect of TrypLE Select Concentration on Vero Cell Growth To study the effect of TrypLE Select concentration on cell attachment and growth, Vero cells were cultivated in IPT-AFM in stationary mode and in static culture on 0.5 g L−1 Cytodex1 microcarriers in six-well plates. Vero cells were cultivated in IPT-AFM containing 0.2, 0.4, 0.5, 0.8, 1, 2.5, 5, and 10 % (v/v) of TrypLE Select and in IPT-AFM without the enzyme (control). Cell density was determined after 3 days of culture for all the conditions, corresponding to the highest cell density level. Appl Biochem Biotechnol (2013) 170:1724–1737 1729 Data displayed in Fig. 1 indicate that cell attachment and growth were affected by the protease level, particularly when its concentration was higher that 0.5 %. Below this level, the drop of cell density was less than 20 % when compared with the control (culture devoid of TrypLE Select). The effect of TrypLE Select on Vero cell growth was more pronounced in static microcarrier culture. At a protease level equal to 10 %, a dramatic decrease of cell density was observed; 65 and 23 % of cell density was achieved as compared with the control, for monolayer and static microcarrier six-well plate cultures, respectively. Based on these results, it appears that the inactivation of TrypLE Select is needed. TrypLE Select Inactivation To inactivate the activity of the enzyme, we investigated the use of soy-derived products, which are Hypep 1510 and the commercial STI. Vero cells were cultivated in IPT-AFM in static microcarrier six-well plate cultures using 0.5 g L−1 Cytodex1. Cell density was determined after 3 days of culture. Three subsequent subcultivations using the same seeding cell density for all the passages were performed. As a control, we used Vero cells grown in IPT-AFM with total removal of TrypLE Select by centrifugation during subcultivation. Independently of the concentration of Hypep 1510 used to neutralize the residual activity of TrypLE Select, few cells were attached to Cytodex1 beads after detachment (Fig. 2). The majority of the cells were weakly attached, and cell growth decreased over passages. At the second passage, cell density level was around 40 % as compared with the control when Hypep 1510 was used at 2, 5, and 10 g L−1, whereas for higher Hypep level (25 and 50 g L−1), lower cell density level was obtained. In addition, no cell growth was observed at the third passage. Therefore, it appears that Hypep1510 is not efficient to inactivate the residual concentration of TrypLE Select. Results obtained with the soybean trypsin inhibitor were much better. At low concentrations (0.01 and 0.05 g L−1), a cell density level of 60–70 % of the control was maintained through the passages. At 0.1 g L–1 of STI, cell density level was improved; cell density level through passages was close to 75 % as compared with the control. For 0.2 g L–1 STI, normal cell growth and attachment were recovered, and a cell density level around 90 % of the control was reached (Fig. 2). At this concentration, STI inactivated the residual activity of TrypLE Select without affecting cell growth. Toxicity Assays The toxicity of STI toward cell attachment and growth was investigated in monolayer culture and in static microcarrier six-well plate experiments. Cells were grown in IPT-AFM supplemented with 0.01, 0.05, 0.1, and 0.2 g L−1 STI. As shown in Fig. 3 independently of the level of STI used, Vero cells exhibited normal cell growth and attachment in both monolayer and static microcarrier cultures. All wells showed a cell aspect similar to the negative control (absence of inhibitor). In addition, Fig. 3c indicates that the viable cell density achieved was not affected by STI concentration. A viable cell density around 2×106 and 2.5×106 cells/well was reached under monolayer culture and static microcarrier cultures, respectively. Hence, STI has no effect on cell growth for all concentrations tested. STI was then not toxic to Vero cells grown in IPT-AFM. Therefore, it can be used to inactivate the residual activity of TrypLE Select without having any negative effect on cell attachment and growth. 1730 Appl Biochem Biotechnol (2013) 170:1724–1737 (A) 0% (Control without TrypLE select) 0.2% 0.5% 0.8% 1% 2.5% 5% 10% 0% (Control without TrypLE select) 0.2% 0.5% 0.8% 1% 2.5% 5% 10% (B) 55 65 69 77 67 77 77 75 80 70 0.4 85 84 0.2 85 100 93 120 92 98 60 40 23 Viable cell density relative to the control (%) (C) 20 0 0.5 0.8 1 2.5 TrypleSelect level (%) Monolayer culture 5 10 Static microcarrier culture Fig. 1 Vero cells growth in IPT-AFM containing different concentrations of TrypLE Select. Microscopic observation of a monolayer culture, b static microcarrier six-well plates culture on 0.5 g L−1 Cytodex1. c Cell density level obtained at different concentrations of TrypLE Select. Cells were observed with an inverted light microscope without staining at ×100 Appl Biochem Biotechnol (2013) 170:1724–1737 1731 (A) 2 g L-1 Hypep 1510 Control 10 g L-1 Hypep 1510 0.05 g L-1 STI 0.2 g L-1 STI 100 100 90 90 Cell density/control (%) Cell density/control (%) (B) 80 70 60 50 40 30 20 80 70 60 50 40 30 20 10 10 0 0 0 First passage 2 5 10 25 Hypep 1510 level (g L-1) Second passage 50 Third passage 0 0.01 0.05 0.1 0.2 STI level (g L-1) First passage Second passage Third passage Fig. 2 Microscopic observation (a) and Vero cell density (b) after inactivation of TrypLE Select. Cells were grown in IPT-AFM in static microcarrier six-well plates on 0.5 g L−1 Cytodex1. To neutralize the protease, Hypep 1510 and soybean trypsin inhibitor were tested at different concentrations during three subsequent subcultivations Spinner Experiments The preliminary results obtained during the six-well plate experiments showed that STI can be used to inactivate the residual TrypLE Select activity without impairing cell attachment and growth. To optimize STI concentration, spinner experiments were carried out. Vero cells were grown in IPT-AFM on 2 g L−1 Cytodex1. Once the maximum cell density was reached, cells were detached from the microcarriers according to the protocol detailed in “Materials and Methods.” Three concentrations of STI were tested to inactivate TrypLE Select—0.2, 0.5, and 1 g L−1. The detachment time was in the range of 10 to 15 min depending on the cell density. The detachment yield was 69, 78, and 86.7 % when STI was used at 0.2, 0.5, and 1 g L−1, respectively. Dislodged cells were used to start new cultures. As depicted in Fig. 4, the highest cell density achieved after subcultivation varied from 1.35×106 to 2×106 cells mL−1. The average specific growth rate (μ) was 0.011, 0.018, and 0.022 h−1 for 0.2, 0.5, and 1 g L−1 STI, respectively. Figure 4 also shows that cell density level obtained when 1 g L−1 STI was used to neutralize TrypLE Select activity was comparable to the level obtained for the control where TrypLE Select was removed by centrifugation. Cell growth after subcultivation was improved when using 1 g L−1 STI compared with 0.2 and 0.5 g L−1 (Fig. 4). Therefore, STI at 1 g L−1 appears to be a sufficient amount for efficient inactivation of the protease. Hence, STI at 1 g L−1 was selected as the optimal level to neutralize the residual activity of TrypLE Select during in situ detachment of Vero cells grown in stirred conditions. 1732 Appl Biochem Biotechnol (2013) 170:1724–1737 (A) 0.01 g L-1 STI Without STI 0.05 g L-1 STI 0.1 g L-1 STI 0.2 g L-1 STI (B) 0.01 g L-1 STI Without STI 0.05 g L-1 STI 0.1 g L-1 STI 0.2 g L-1 STI Viable cell density (cells/well) (C) 3.0E+06 2.5E+06 2.0E+06 1.5E+06 1.0E+06 5.0E+05 0.0E+00 Control 0.01 0.05 0.1 STI level (g L-1 ) Monolayer culture 0.2 Static microcarrier culture Fig. 3 Toxicity studies in six-well plates. Microscopic observation of Vero cells grown in IPT-AFM containing different concentrations of soybean trypsin inhibitor in a monolayer, and b static microcarrier six-well plate cultures, and c viable cell density in the presence of different concentrations of STI 1733 2.5E+06 0.025 2.0E+06 0.02 1.5E+06 0.015 1.0E+06 0.01 5.0E+05 0.005 0.0E+00 Average µ (h-1) Fig. 4 Kinetic parameters of Vero cell growth after cell detachment. Cells were grown in spinner flask in IPT-AFM on 2 g L−1 Cytodex1 and detached with TrypLE Select. Protease inactivation was carried out with 0.2, 0.5, and 1 g L−1 of STI. TrypLE Select was removed by centrifugation for the control culture Highest cell density level (cells mL-1) Appl Biochem Biotechnol (2013) 170:1724–1737 0 Control 0.2 0.5 1 STI level (g L-1) Highest cell density level Average µ Bioreactor Experiments Vero cells were cultured in 2-L bioreactor operating in batch mode on 3 g L−1 Cytodex1, in IPT-AF medium (Fig. 5). After 5 days of culture, cell density level reached 2×106 cells mL−1, the average specific cell growth was equal to 0.02 h−1. At this time, cells were dislodged in situ with TrypLE Select as described in the “Materials and methods.” After a contact time of 10 min and using 1 g L−1 STI, the residual activity of the protease was neutralized. Cells were totally detached from the beads as indicated in Fig. 5a. The detachment yield was equal to 70±19 %. The dislodged cells were used as a seed to start another 2-L bioreactor culture. Data shown in Fig. 5b indicated that the cells started to grow after a 24-h lag phase. Four days post-subcultivation cells were also able to adhere correctly to Cytodex1 beads as shown in Fig. 5a. After 144 h, cell density level was around 2×106 cells mL−1. The average specific growth rate was equal to 0.024 h−1. Discussion The manufacture of biotechnology-derived products requires large-scale propagation of mammalian cells. In conventional production processes, the cost of serum and other animal-derived proteins may represent over 50 % of the medium cost. The use of non-animal-derived material offers additional benefits such as simplified downstream purification protocols and a reduced requirement for quality testing. Moreover, the safety aspects of the products will be highly warranted. These advantages are likely to ensure that animal component-free processes for pharmaceuticals production are the outgrowth of choice for future biotechnology production. Subcultivation is a crucial step when adherent cells are used. During this work, we demonstrated that TrypLE Select can effectively detach Vero cells grown in IPT-AFM on microcarriers and developed a protocol that allows the preparation of an inoculum of Vero cells grown on Cytodex1 microcarriers. The recovered cells can be used to start a culture in a larger bioreactor, enabling therefore the scale-up of Vero cell culture process, under fully animal component-free conditions. The study of the influence of TrypLE Select concentration on cell growth showed that the enzyme should be inactivated to obtain a normal cell growth particularly for the agitated culture. On the contrary, we observed a negative effect on cell attachment and growth. This result correlates with data reported by Umegaki et al. [32] who studied the effect of trypsin 1734 Appl Biochem Biotechnol (2013) 170:1724–1737 (A) 5 days of culture before cell detachment During cell detachment 4 days of culture post detachment and reinoculation Viable cell density (ells mL-1) (B) 2.5E+06 2.0E+06 1.5E+06 1.0E+06 5.0E+05 0.0E+00 0 50 100 150 200 250 300 Time (h) Before trypsinisation After trypsinisation Fig. 5 Vero cell subcultivation in a 2-L stirred bioreactor. Cells were grown in IPT-AFM on 3 g L−1 Cytodex1; cells were detached with TrypLE Select then the residual activity of the protease was neutralized with 1 g L−1 STI. a Microscopic observation and b cell growth profile on human keratinocyte cells growth; they showed that the enzyme induced a decrease in cell density although it did not affect cell viability. Nevertheless, conflicting data were reported in the literature; another study [20] showed that BHK-21 cells in the presence of trypsin acquired a rounded shape after alteration of their cell membrane. In addition, cells become more sensitive to shear forces generated by the agitation. The cells recovered after detachment can be used to start a new culture when TrypLE Select is removed by centrifugation. However, at a large scale such step would not be feasible. Thus, it is necessary to inactivate residual TrypLE Select to use the recovered cells as a seed. The capacity of the soy peptone “Hypep1510” to inactivate the residual activity of TrypLE Select was very limited for all the concentrations tested. Moreover, the use of amounts higher than 10 g L−1 becomes itself an inhibitor of cell growth. Therefore, the use of Hypep1510 to inactivate the residual TrypLE Select activity was not efficient. Soybean trypsin inhibitor appears as a better agent for the inactivation of TrypLE Select. The concentrations used (0.2–1 g L−1) were able to effectively neutralize TrypLE Select without any harmful effect either on cell attachment or growth. To simulate the conditions of large-scale cultivation, we tested different concentrations of STI in spinner flasks. The yields obtained were comparable and even higher to those obtained during the trypsinization of Vero cells cultivated in serum-containing medium (data not shown). Appl Biochem Biotechnol (2013) 170:1724–1737 1735 In the same context, Sun and Zhang [27] applied a cell-detaching reactor to the anchoragedependent recombinant CHO and Vero cell processes and demonstrated its operating feasibility in inoculation between bioreactors using trypsin and a serum-containing medium. Cruz et al. [20] studied the inactivation of trypsin using STI at 1 g L−1 for the subcultivation of BHK-21 cells in a commercial animal component-free medium (SMIF6). However, the highest cell density reached was low; they obtained 0.28×106 cells mL−1 after 4 days of culture. They studied different (STI/trypsin) ratios to allow a complete inhibition of the enzyme. They showed that the higher the ratio is the better cell growth and viability are. They demonstrated that the optimum volume ratio was 1:1. This result correlates with those obtained in the current study where the same ratio was used. Spinner and bioreactor cultures showed that, after cell detachment according to the protocol that we optimized, Vero cells were elongated and were able to reattach to the microcarriers. The cell density reached 2×106 cells mL−1 after 6 days of culture in the bioreactor. The amount of inhibitor used is equivalent to the TrypLE Select. Inactivation of the residual activity of TrypLE Select using STI at 1 g L−1 was successful. Ozawa and Laskowski [33] explained how soybean trypsin inhibitor effectively inhibits trypsin. They showed that this inhibition was due to the covalent bond between the active site of trypsin and soybean trypsin inhibitor. Knowing that TrypLE Select is a recombinant form of trypsin, its inhibition by STI should occur according to a similar mechanism. Conclusion In this study, a protocol was developed for the detachment of Vero cells grown in IPT-AFM on Cytodex1 microcarriers, to achieve the scale-up of the process. We demonstrated that the residual concentration of TrypLE Select used for cell detachment can be efficiently inactivated by STI at 1 g L−1 when the cells were grown on 3 g L−1 Cytodex1 in stirred bioreactor. The detachment yield was around 80 %; the dislodged cells were healthy and were successfully used to start a new bioreactor culture. Acknowledgments We are grateful for Arno van der Ark and Tiny van der Velden from RIVM (Bilthoven, The Netherlands) for fruitful discussion. We also thank Sheffield-Bioscience for supplying the Hypeps. References 1. Birch, J. R. (1999). Suspension culture of animal cells. In M. C. Flickinger & S. W. Drew (Eds.), Encyclopedia of bioprocess technology: fermentation, biocatalysis, and bioseparation (pp. 2509–2516). New York: Wiley. 2. Shao, M., Lei, J., Wei, C., & Ouyang, F. (2002). 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