A SIEC-S cell suitable for serum-free full suspension culture, a domestication method and an application thereof

Abstract

The present invention discloses a SIEC-S cell suitable for serum-free full suspension and a culture domestication method, belonging to the technical field of SIEC-S cell culture. The SIEC-S cell was deposited with the General Microbiology Center of the China Microbial Culture Collection Management Committee on December 25, 2022, with the deposit number CGMCC No. 45338, and the deposit address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. It is classified and named as porcine small intestinal mucosal epithelial suspension cell. The SIEC-S cell directly grows in a serum-free CD medium, reducing the usage amount of serum and lowering the cost. The SIEC-S can be suspended and cultured on a large scale in a bioreactor, improving the yield of downstream vaccine antigen content.

Description

Technical Field

[0001] The present invention relates to the technical field of whole suspension SIEC-S cell domestication, and more specifically to a SIEC-S cell suitable for serum-free whole suspension culture, a domestication method and an application thereof. Background Art

[0002] Since most animal cells have the characteristics of adherent culture, in the process of large-scale industrial production, the widely used bioreactor cascade amplification process requires cells to have the characteristics of suspension culture to facilitate the realization of the amplification process. Therefore, the suspension domestication of cells becomes indispensable.

[0003] Porcine epidemic diarrhea virus (PEDV) is a highly contagious intestinal disease. After being infected through the oral cavity or nasal cavity and then swallowed into the digestive tract, a large number of intestinal epithelial cells are infected, the microenvironment in the intestine is disordered, a large amount of harmful acids are produced by fermentation in the large intestine, resulting in an increase in intestinal osmotic pressure, and then a large amount of tissue fluid enters the intestine, causing diarrhea. And SIEC suspension cells are exactly separated from porcine small intestinal mucosal epithelium. PEDV virus is an intestinal disease. Therefore, PEDV virus can multiply in large numbers on SIEC adherent cells. After suspension domestication, the obtained SIEC suspension cells can realize the large-scale culture of PEDV virus on a reactor.

[0004] SIEC suspension cells are porcine small intestinal mucosal epithelial cells, and PEDV is an infectious intestinal disease, and intestinal epithelial cells are easily infected. Therefore, SIEC, porcine small intestinal epithelial mucosal cells, was selected to conduct a virus sensitivity verification experiment on PEDV virus. The results showed that SIEC cells were sensitive to PEDV virus. However, the growth mode of SIEC adherent cells restricted the industrial production of PEDV virus. Therefore, the SIEC adherent cells were subjected to suspension domestication, and the SIEC suspension cells after suspension domestication were highly sensitive to PEDV virus. Therefore, SIEC suspension cells were selected for the propagation of PEDV virus. There is no precedent for the domestication of SIEC suspension cells and the culture of PEDV virus reported in domestic literature patents.

[0005] Therefore, how to provide a SIEC-S cell suitable for serum-free whole suspension is an urgent problem to be solved by those skilled in the art. Summary of the Invention

[0006] In view of this, the present invention provides a SIEC-S cell suitable for serum-free whole suspension, a domestication method and an application thereof.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] A SIEC-S cell suitable for serum-free full suspension. The SIEC-S cell was deposited with the General Microbiology Center of the China Microbial Culture Collection Management Committee on December 25, 2022, with the deposit number CGMCC No. 45338 and the deposit address at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. Its scientific description is porcine small intestinal mucosal epithelial suspension cells.

[0009] As an invention concept identical to the above technical solution, the present invention also claims a domestication and culture method for a SIEC-S cell suitable for serum-free full suspension. The process includes: selecting SIEC cells and successively performing low-serum adherent culture, serum-free suspension culture in CD PK259 medium, and passage domestication to obtain SIEC-S cells suitable for serum-free full suspension culture.

[0010] Preferably, the process of the low-serum adherent culture is as follows: Take adherent SIEC cells that have grown to a confluent monolayer, place them in an incubator at 37 °C until the cells reach 100% confluence, digest and disperse them with 0.25% (V / V) trypsin, add them to DMEM medium containing 10% (V / V) newborn bovine serum, and perform adherent serum reduction domestication culture. The serum content is reduced according to the ratio of 10%, 7%, 5%, and 2%. When the serum content of the adherent cells is reduced to 2%, after the cells are fully adapted to the low-serum medium, the adherent cells can be stably passaged in the 2% low-serum culture.

[0011] Preferably, the process of serum-free suspension culture and passage domestication in CD PK 259 medium is as follows: Suspend 2% low-serum adherent cells in CD PK 259 medium for suspension culture and passage to obtain preliminarily suspended SIEC cells, and then continuously centrifuge and passage the SIEC cells. The precipitate is directly added to serum-free CD PK 259 suspension medium for further domestication. After 5 generations of domestication, SIEC-S cells that can be stably passaged and suspended are obtained.

[0012] Preferably, the centrifugation is at 1000 rpm for 5 min.

[0013] As an invention concept identical to the above technical solution, the present invention also claims the application of the SIEC-S cells or the SIEC-S cells obtained by the above domestication and culture method in culturing porcine epidemic diarrhea virus PEDV.

[0014] As an invention concept identical to the above technical solution, the present invention also claims the application of the SIEC-S cells or the SIEC-S cells obtained by the above method in the preparation of PEDV vaccines.

[0015] As can be seen from the above technical solutions, compared with the prior art, the SIEC-S cells prepared by the present invention have a stable growth state, high sensitivity to porcine epidemic diarrhea virus (PEDV), good cell dispersion, uniform size, and the viable cell density can reach 1.12×10 7 cells / ml, which is suitable for large-scale production of PEDV virus and provides a basis for the research and development of (PEDV) vaccines.

[0016] At the same time, in the present invention, the SIEC adherent cells are domesticated by autonomous suspension, and SIEC-S can be passaged in CD PK 259 medium. After the 3rd passage, the final density of the cells can reach 3.00×10 6 cells / mL; inoculating porcine epidemic diarrhea virus (PEDV), the virus titer can reach 10 7.75 TCID50 / mL, which lays a technical foundation for obtaining high-density SIEC-S cells and improving the proliferation efficiency of PEDV.

[0017] In summary, the domesticated SIEC-S cells of the present invention grow directly in a serum-free CD medium, reducing the amount of serum used and lowering the cost. SIEC-S can be cultured in suspension on a bioreactor on a large scale, improving the yield of downstream vaccine antigen content.

[0018] The adherent cells are first domesticated by reducing the serum content while adherent, and the cells can be passaged and grown normally in a medium with 2% serum, enabling the adherent cells to gradually reduce their dependence on serum, which is beneficial to the success of serum-free suspension domestication; then the low-serum adherent cells are screened through a suspension solution screening experiment to select a suspension medium suitable for the growth of this type of cell; finally, domestication screening is carried out in the optimal suspension medium to obtain a continuously passaged fully suspended cell line. Compared with the existing domestication methods, this domestication method is more effective, doubling the cell density during the 24h - 72h of culture.

[0019] Using this domestication method (first reducing the serum content of adherent cells to get rid of the dependence on serum; then screening the suspension medium to select a medium suitable for SIEC suspension culture; finally, quickly completing the suspension domestication in this medium). This method omits the intermediate culture transition period of the cells in the mixed medium of adherent medium + suspension medium compared with the conventional suspension domestication method, and instead screens no less than 4 kinds of suspension media to complete one-step suspension domestication. Under such conditions, it is possible to quickly enrich the cell line adapted to suspension growth, saving more time and having a higher success rate.

[0020] This method is relatively fast, simple, and has good repeatability; the vitality of the suspension cell line after successful domestication is high, and its sensitivity to the virus is good; the suspension cell line prepared by this method can be continuously produced on a conventional reactor, greatly reducing the production cost and increasing the vaccine yield. Description of the Drawings

[0021] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the drawings in the following description are only the embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on the provided drawings.

[0022] Figure 1 The drawing is a microscope image of adherent SIEC cells;

[0023] Figure 2 The drawing is a suspension curve of SIEC cells in different suspension media;

[0024] Figure 3 The drawing is a microscope image of domesticated SIEC-S cells (1:100X);

[0025] Figure 4 The drawing is a growth curve of SIEC-S in serum-free suspension medium;

[0026] Figure 5 The drawing is a nutrient consumption curve of glutamine, glucose, and lactate during the growth of SIEC-S CGMCC NO.45338;

[0027] Figure 6 The drawing is a cell density experiment diagram of SIEC-S inoculated with PEDV;

[0028] Figure 7 The drawing is a MOI experiment diagram of SIEC-S inoculated with PEDV. Detailed Embodiments

[0029] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.

[0030] Example 1

[0031] SIEC cells were used in the experiment. They were isolated from porcine intestinal epithelial cells and obtained by JinYu Baoling Biological Products Co., Ltd. A commercially available DMEM medium (purchased from: GIBCO) was used, supplemented with 10% (volume percentage, V / V) newborn bovine serum (purchased from: Jinyuankang Bioengineering Co., Ltd.). The cells were cultured statically at 37°C. After the cells grew to confluence, they were digested and dispersed with 0.25% (V / V) trypsin and subcultured at a dilution ratio of 1:3 to 1:4 to expand the culture, obtaining adherent SIEC cells. The microscope images are as shown in Figure 1 shown;

[0032] Suspension acclimation of adherent SIEC cells was carried out as follows:

[0033] (1) Low-serum adherent culture

[0034] Adherent SIEC cells that had grown to confluence were digested and dispersed with 0.25% (V / V) trypsin and then added to DMEM medium containing 10% (V / V) newborn bovine serum. The serum was gradually reduced for adherent culture. The serum content was reduced according to the ratios of 10%, 7%, 5%, and 2%. When the serum content of the adherent cells was reduced to 2%, after the cells had fully adapted to the low-serum medium, the cells grew stably in the 2% low-serum medium. During the process of reducing the serum, bovine insulin was added to the cell nutrient solution to promote cell proliferation. The concentration of added bovine insulin was 10 μg / ml, and the cell morphology became more regular and the cell edges became clearer.

[0035] (2) Screening of serum-free suspension media

[0036] SIEC adherent cells with 2% low serum were digested with 0.25% (V / V) trypsin and then directly suspended in 4 different suspension media and cultured in a shaker at 37°C. The suspension cells were counted daily. Trypan blue was used to stain the suspension cells, and a Countstar cell counter was used to measure the cell density and viability of SIEC-S CGMCC NO.45338 until the cell viability began to drop to 30%. According to the growth curves of SIEC-S CGMCC NO.45338 in different suspension media, 1 to 2 optimal suspension media were initially explored.

[0037] The 4 different suspension media include:

[0038] Medium No. 5: CD ST 258 (Gansu Jianshun Biotech), product number 10604-258

[0039] Medium No. 9: CD Vero 693 (Gansu Jianshun Biotech), product number 10904-693

[0040] 29# Medium Name: CD PK15259 (Gansu Jianshun Biotech), Catalog Number 10701 - 259

[0041] 30# Medium Name: CD MDBK 286 (Gansu Jianshun Biotech), Catalog Number 10501 - 286

[0042] The culture results in each medium are shown in Figure 2 , from Figure 2 It can be seen that the suspension culture effects of CD PK15259 and CD MDBK 286 are better.

[0043] (3) SIEC - S CGMCC NO.45338 is adapted to serum - free culture: After screening out the optimal 2 suspension media, 2% low - serum adherent cells are suspended and passaged in CD PK15259 and CD MDBK 286 suspension media to test the effect of the suspension media on cell growth during cell passage. After 5 generations of cell domestication, 1 suspension medium is most suitable for the proliferation of SIEC - S, which is CD PK 259 suspension medium. The microscopic image of the obtained SIEC - S cells is as shown in Figure 3 Shown. SIEC - S cells were deposited in the China General Microbiological Culture Collection Center on December 25, 2022, with the deposit number CGMCC No.45338, the deposit address being No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, and the scientific description being porcine small intestinal mucosal epithelial suspension cells.

[0044] Example 2

[0045] The SIEC - S suspension cells obtained in Example 1 were cultured using a bioreactor, and batch production was carried out using serum - free CD PK259 medium. The density of SIEC - S CGMCC NO.45338 can be greater than 5.0×10 6 cells / mL. SIEC - S CGMCC NO.45338 grows rapidly in a 3.5L reactor. The initial seeding density of the cells is 0.93×10 6 cells / ml. After 96h of culture, the cell density can reach 9.05×10 6 cells / ml (see Table 1).

[0046] The culture process of SIEC-S suspension cells in a reactor is as follows: First, revive 1 vial of SIEC suspension cells. Take out a cell cryopreservation tube from liquid nitrogen and quickly thaw it in water at 37°C. Place it in a 50-ml centrifuge tube pre-filled with medium and perform centrifugation at 1000 r for 5 min. After centrifugation, discard the supernatant, gently tap the bottom of the centrifuge tube to loosen the cells, and add 30 ml of CD PK 259. The cell density is 0.5×10 6 ~0.8×10 6 cells / ml, the shaker speed is 100 rpm / min to 120 rpm / min, the culture temperature is 36.0°C to 37.0°C, the CO2 concentration is 5.0%, the cells are cultured for 48 h - 72 h, and the cells are passaged. The initial seeding density of the cells after passage is 0.5×10 6 ~0.8×10 6 cells / ml. Until the cells expand to 200 ml to 500 ml, the cells are inoculated into a 3.5-L reactor. After dilution, the cell density is 0.5×10 6 cells / ml to 0.8×10 6 cells / ml. Continue to culture in a 3.5-L stirred bioreactor (purchased from Sartorius) under the conditions of pH 7.2 and dissolved oxygen concentration of 40% for the whole suspension culture of SIEC suspension cells; when the cell density reaches 2.0×10 6 cells / mL and the viability is above 95.0%, add EDTA-trypsin with a final concentration of 10 μg / ml to 30 μg / ml and 1.0% - 2.0% of PEDV seed virus into the culture medium. After inoculation, control the reactor temperature at 36.0°C to 37.0°C, the dissolved oxygen concentration at 30% - 50%, and the stirring speed at: 40 rpm / min to 60 rpm / min, and culture under the conditions of pH 7.0 - 7.4. After the culture is completed, harvest the virus solution and measure the virulence of porcine epidemic diarrhea virus in the virus solution. As can be seen from the data in Table 2 above for the measurement results after inoculation of PEDV in the bioreactor, when the PEDV strain is inoculated into SIEC suspension cells for proliferation culture using a bioreactor and the virus culture solution is harvested at 72 h after inoculation. Thus, based on the SIEC-S suspension cell line provided by the present invention, a bioreactor can be used to batch suspend culture PEDV. The proliferated PEDV is stable, and when cultured for 72 h after inoculation, a virus culture solution with relatively high virulence can be stably obtained. The titer of the virus culture solution ranges from 10 6.50 TCID50 / mL to 10 7.75 TCID50 / mL. (See Table 2) Table 1 Cell information of the 3.5-L reactor control of SIEC

[0047]

[0048]

[0049] Table 2 Exploration of the amplification process of SIEC-S inoculated with BRV and PEDV

[0050]

[0051] Example 3 Test on the growth characteristics of SIEC-S and its sensitivity to viruses

[0052] During the domestication process of SIEC-S cell culture, the highest density can reach 1.12×10 7 cells / ml (see Figure 4 ). Using a Nova biochemical analyzer to analyze the culture medium of SIEC suspension cells, from the start of inoculation to the sixth day of culture of SIEC cells, the glutamine content, glucose content, and accumulated lactic acid content in the nutrient solution were detected daily, see Figure 5 Nutrient consumption curve. During the growth of SIEC-S, glutamine and glucose are continuously consumed, and the metabolite lactic acid is accumulated. From the consumption of the nutrient solution, it can be seen that the consumption of glutamine is relatively large during 0h - 72h of cell culture, and the glutamine content decreases from 5.17 mmol / L to 0.37 mmol / L. The consumption of lactic acid gradually accumulates, and the accumulated lactic acid amount can reach 1.78 mmol / L - 1.98 mmol / L on the 5th - 6th day of cell culture.

[0053] The initial inoculation density of SIEC-S cells inoculated with PEDV virus is 2.0×10 6 cells / ml, and the virus is harvested at 72h, and the highest titer can reach 10 7.50 TCID50 / mL, see Figure 6 .

[0054] SIEC-S is highly sensitive to the virus. The changes in the virus titer were explored when the cells were inoculated with the virus at different MOIs and different cell densities. The specific process is as follows: When the SIEC suspension cells are cultured to a density greater than 2.0×10 6 cells / ml, PEDV virus is inoculated according to MOI = 0.01 - 1.0. When MOI = 0.01, it is best to harvest the virus at 72h, and the virus titer is 10 7.5 TCID50 / mL, see Figure 7 . The virus titer also varies when the inoculated cell density ranges from 1.0×10 6 to 4.0×10 6 . When the cell density is 2.0×10 6 cells / ml, the virus titer reaches 10 7.50TCID50 / mL.

[0055] The various embodiments in this specification are described in a progressive manner. Each embodiment focuses on the differences from other embodiments. For the same or similar parts among the various embodiments, reference can be made to each other.

[0056] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be obvious to those skilled in the art. The general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather will be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A SIEC-S cell suitable for serum-free full suspension, characterized in that, The SIEC-S cells were deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on December 25, 2022, with the deposit number of CGMCC No. 45338, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The scientific description is porcine small intestinal mucosal epithelial suspension cells.

2. Use of the SIEC-S cells according to claim 1 in culturing porcine epidemic diarrhea virus PEDV.

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