Serum-free culture medium for suspension culture of McCoy cells and application of serum-free culture medium

Abstract

The invention relates to a serum-free culture medium for suspension culture of McCoy cells and application of the serum-free culture medium. The serum-free culture medium for suspension culture of the McCoy cells comprises amino acid, vitamins, inorganic salt, glucose, sodium hyaluronate, PF68 and water. Wherein the serum-free culture medium meets at least one of the following characteristics (t1) and (t2): (t1) amino acid contains one or more of asparagine, cysteine, glutamic acid and proline; and (t2) the inorganic salt contains anhydrous disodium chlorine phosphate. The serum-free culture medium provided by the invention can realize high-density suspension culture of McCoy adherent cells without the help of any adsorption carrier, and can realize continuous and stable passage.

Description

Technical Field

[0001] The present invention relates to the technical field of cell culture, in particular to a serum-free culture medium for suspending and culturing McCoy cells and an application thereof, and further to a serum-free culture medium for suspending and culturing McCoy cells, a McCoy cell suspension culture method and an application thereof. Background Art

[0002] McCoy cells are mouse fibroblasts, adherent cells isolated from mouse skin tissue. Fibroblasts are the main cell component of loose connective tissue and are differentiated from mesenchymal cells (MSCs) during the embryonic period. Functionally active cells are called fibroblasts. Fibroblasts are large, with clear outlines, and are mostly protruding spindle-shaped or star-shaped flat structures. Their nuclei are regular oval, with large and obvious nucleoli. Fibrocytes, the most numerous cells in connective tissue, are relatively static cells called fibroblasts. Because of their ease of culture and sensitivity to certain strains, McCoy cells are often used for strain isolation, proliferation and culture. They are also suitable for drug screening, toxicology research and basic cell biology research.

[0003] Porcine proliferative ileitis (sometimes referred to as porcine proliferative enteritis (PPE)) is a common intestinal disease of pigs caused by infection with Lawsonia intracellularis. The disease has a high incidence in the breeding stock industry and often affects weaned pigs between 6 and 20 weeks of age. Clinical signs of pigs with proliferative ileitis include intermittent diarrhea, loss of appetite, marked dullness and emotional apathy, and wasting syndrome. An effective means of preventing the disease is vaccination with an inactivated Lawsonia intracellularis vaccine. Current methods for culturing Lawsonia intracellularis do not allow for high-density cultivation thereof. Summary of the invention

[0004] Based on this, the purpose of the present invention includes providing a serum-free culture medium for suspending and culturing McCoy cells and its application.

[0005] In some embodiments, a serum-free medium for culturing McCoy cells in suspension is provided, wherein the serum-free medium comprises amino acids, vitamins, inorganic salts, glucose, sodium hyaluronate, PF68 and water;

[0006] Wherein, the serum-free medium satisfies at least one of the following characteristics (t1) and (t2):

[0007] (t1) the amino acids include one or more of asparagine, cysteine, glutamic acid and proline;

[0008] (t2) The inorganic salt comprises anhydrous disodium hydrogen phosphate.

[0009] In some embodiments, the serum-free medium provided comprises, in terms of concentration in the serum-free medium, 33.8 mg / L to 77.8 mg / L glycine, 74.1 mg / L to 133.4 mg / L arginine, 55.6 mg / L to 100.0 mg / L L-cystine, 515.1mg / L~927.2mg / Lglutamine, 37.0mg / L~66.7mg / Lhistidine, 92.6mg / L~166.7mg / Lisoleucine, 101.4mg / L~182.6mg / Lleucine, 128.8mg / L~231.8mg / Llysine, 26.5mg / L~47.6mg / Lmethionine, 58.2mg / L~104.8mg / Lphenylalanine, 37.0mg / L~66.7mg / Lserine, 83.8mg / L~150.8mg / Lthreonine, 14.1mg / L~25.4mg / Ltryptophan, 91.7mg / L~165.1mg / L L-tyrosine, 82.9mg / L~149.2mg / L valine, 165.8mg / L~298.4mg / L asparagine, 49.7mg / L~89.5mg / L cysteine, 331.0mg / L~441.4mg / L glutamic acid and 66.3mg / L~119.4mg / L proline.

[0010] In some embodiments, a serum-free medium is provided, wherein the vitamins comprise, in terms of concentration in the serum-free medium, 3.6 mg / L to 6.5 mg / L choline chloride, 4.0 mg / L to 7.7 mg / L nicotinamide, 0.3 mg / L to 0.6 mg / L pyridoxine, 3.4 mg / L to 10 mg / L riboflavin, 6.1 mg / L to 11 mg / L thiamine hydrochloride, and 3.6 mg / L to 6.5 mg / L inositol.

[0011] In some embodiments, the serum-free medium provided, in which the inorganic salts comprise, based on the concentration in the serum-free medium, 0.1 mg / L to 0.2 mg / L ferric nitrate nonahydrate, 68.4 mg / L to 123.1 mg / L magnesium sulfate, 280 mg / L to 504 mg / L potassium chloride, 2590 mg / L to 6622 mg / L sodium bicarbonate, 4480 mg / L to 8064 mg / L sodium chloride, 87.5 mg / L to 157.5 mg / L sodium dihydrogen phosphate monohydrate, and 192.5 mg / L to 346.5 mg / L anhydrous disodium hydrogen phosphate.

[0012] In some embodiments, a serum-free medium is provided that is free of calcium chloride, calcium nitrate, calcium pantothenate, and calciferol.

[0013] In some embodiments, the serum-free medium provided comprises 33.8 mg / L to 77.8 mg / L glycine, 74.1 mg / L to 133.4 mg / L arginine, 55.6 mg / L to 100.0 mg / L L-cystine, 515.1mg / L~927.2mg / Lglutamine, 37.0mg / L~66.7mg / Lhistidine, 92.6mg / L~166.7mg / Lisoleucine, 101.4mg / L~182.6mg / Lleucine, 128.8mg / L~231.8mg / Llysine, 26.5mg / L~47.6mg / Lmethionine, 58.2mg / L~104.8mg / Lphenylalanine, 37.0mg / L~66.7mg / Lserine, 83.8mg / L~150.8mg / Lthreonine, 14.1mg / L~25.4mg / Ltryptophan, 91.7mg / L~165.1mg / L L-tyrosine, 82.9mg / L~149.2mg / L valine, 165.8mg / L~298.4mg / L asparagine, 49.7mg / L~89.5mg / L cysteine, 331.0mg / L~441.4mg / L glutamic acid and 66.3mg / L~119.4mg / L proline, 3.6mg / L~6.5mg / L choline chloride, 4.0mg / L~7.7mg / L nicotinamide, 0.3mg / L~0.6mg / L pyridoxine, 3.4mg / L~10mg / L riboflavin, 6.1mg / L~11mg / L thiamine hydrochloride and 3.6mg / L~6.5mg / L g / L inositol, 0.1mg / L~0.2mg / L ferric nitrate nonahydrate, 68.4mg / L~123.1mg / L magnesium sulfate, 280mg / L~504mg / L potassium chloride, 2590mg / L~6622mg / L sodium bicarbonate, 4480mg / L~8064mg / L sodium chloride, 87.5mg / L~157.5mg / L sodium dihydrogen phosphate monohydrate and 192.5mg / L~346.5mg / L disodium hydrogen phosphate anhydrous, 3150mg / L~5670mg / L glucose, 3.8mg / L~6.9mg / L sodium hyaluronate, 1000mg / L~2000mg / LPF68.

[0014] In some embodiments, a method for culturing McCoy cells in suspension is provided, comprising the following steps: placing McCoy cells in the serum-free culture medium for culturing.

[0015] In some embodiments, the provided McCoy cell suspension culture method comprises the following steps: placing the McCoy cells in the serum-free culture medium for suspension culture for 3 to 5 days, starting perfusion culture, and the perfusion rate is 0.5VVD to 3VVD;

[0016] Optionally, in the step of placing the McCoy cells in the serum-free medium, the initial viable cell density of the McCoy cells is greater than or equal to 1.0×10 6 Pieces / mL.

[0017] In some embodiments, the use of the serum-free culture medium or the McCoy cell suspension culture method in producing Lawsonia intracellularis is provided.

[0018] In some embodiments, a method for producing intracellular Lawsonia is provided, wherein the McCoy cells are placed in the serum-free medium for suspension culture, and intracellular Lawsonia are inoculated, cultured, and harvested;

[0019] Optionally, the McCoy cells are placed in the serum-free medium for suspension culture for 4 to 5 days, perfusion is started, the perfusion rate is 0.5VVD~3VVD, intracellular Lawsoniae are inoculated 40h~60h after perfusion is started, and the intracellular Lawsoniae are cultured for 6 to 8 days after inoculation, and the intracellular Lawsoniae are harvested.

[0020] The serum-free culture medium provided above is protein-free, serum-free, and serum-free, and can achieve high-density suspension culture of McCoy adherent cells without the aid of any adsorption carrier, and can be continuously and stably passaged. The provided McCoy cell suspension culture method uses perfusion technology to culture McCoy suspension cells, and by adjusting the culture parameters, the density of McCoy suspension cells is 10 times higher than that of traditional batch culture. In the aforementioned serum-free culture medium environment, intracellular Lawsonia can efficiently infect host cells. In this culture medium, with the help of perfusion process technology, host cells can grow in high-density suspension and efficiently produce intracellular Lawsonia. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the technical solutions in the implementation methods and examples of the present application and to more completely understand the present application and its beneficial effects, the following is a brief introduction to the drawings required for use in the description of the implementation methods or examples. Obviously, the drawings described below are only some embodiments of the present application, and those skilled in the art can obtain other drawings based on these drawings without creative work.

[0022] Figure 1 This is a graph showing the results of the stability test of experimental formula 3 in Example 2. DETAILED DESCRIPTION

[0023] The present invention will be further described in detail below in conjunction with the embodiments and examples. It should be understood that these embodiments and examples are only used to illustrate the present invention and are not used to limit the scope of the present invention. The purpose of providing these embodiments and examples is to make the understanding of the disclosure of the present invention more thorough and comprehensive. It should also be understood that the present invention can be implemented in many different forms and is not limited to the embodiments and examples described herein. Those skilled in the art can make various changes or modifications without violating the connotation of the present invention, and the equivalent form obtained also falls within the protection scope of the present application. In addition, in the description below, a large number of specific details are given in order to provide a more comprehensive understanding of the present invention. It should be understood that the present invention can be implemented without one or more of these details.

[0024] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing implementation modes and embodiments and are not intended to limit the present invention.

[0025] Unless otherwise specified or incompatible herewith, the terms and phrases used herein shall have the following meanings:

[0026] In the present invention, "plurality", "multiple", "multiple times", "multiple" and the like, unless otherwise specified, refer to a number greater than or equal to 2. For example, "one or more" means one or greater than or equal to two.

[0027] As used herein, "combination thereof", "any combination thereof", "any combination thereof" etc. include all suitable combinations of any two or more of the listed items.

[0028] Herein, the “suitable” mentioned in “suitable combination”, “suitable method”, “any suitable method”, etc., shall be based on the ability to implement the technical solution of the present invention, solve the technical problem of the present invention, and achieve the expected technical effect of the present invention.

[0029] Herein, “preferred”, “better”, “more preferred” and “suitable” are merely used to describe implementation methods or examples with better effects, and it should be understood that they do not constitute limitations on the scope of protection of the present invention.

[0030] In the present invention, “further”, “furthermore”, “particularly”, etc. are used for descriptive purposes to indicate differences in content, but should not be construed as limiting the scope of protection of the present invention.

[0031] In the present invention, "optionally", "optional", and "optional" mean optional, that is, any one of the two parallel solutions of "yes" or "no". If multiple "options" appear in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction, each "optional" is independent.

[0032] In the present invention, in the "first aspect", "second aspect", "third aspect", "fourth aspect", etc., the terms "first", "second", "third", "fourth", etc. are used only for descriptive purposes and cannot be understood as indicating or implying relative importance or quantity, nor can they be understood as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first", "second", "third", "fourth", etc. only serve the purpose of non-exhaustive enumeration and description, and it should be understood that they do not constitute a closed limitation on quantity.

[0033] In the present invention, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0034] In the present invention, when it comes to numerical intervals (i.e., numerical ranges), unless otherwise specified, the optional numerical distribution is considered continuous within the above numerical interval, and includes the two numerical endpoints (i.e., the minimum value and the maximum value) of the numerical range, and each numerical value between the two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to the integers within the numerical interval, it includes the two endpoint integers of the numerical range, and each integer between the two endpoints. In this article, it is equivalent to directly listing each integer, such as t is an integer selected from 1 to 10, indicating that t is any integer selected from the integer group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. In addition, when multiple ranges are provided to describe features or characteristics, these ranges can be combined. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all sub-ranges included therein.

[0035] The temperature parameters in the present invention, if not specifically limited, are allowed to be either constant temperature treatment or to vary within a certain temperature range. It should be understood that the constant temperature treatment allows the temperature to fluctuate within the precision range controlled by the instrument. Fluctuations within the range of ±5°C, ±4°C, ±3°C, ±2°C, and ±1°C are allowed.

[0036] In the present invention, %(w / w) and wt% both represent weight percentage, %(v / v) refers to volume percentage, and %(w / v) refers to mass volume percentage.

[0037] In the present invention, "VVD" refers to the tank volume per day in the perfusion process, that is, the volume of culture fluid discharged from the culture tank every day. It is an important parameter in the perfusion process, which is used to measure the frequency of culture fluid replacement and the changes in the cell growth environment during the culture process. For example, 3VVD means that the volume of culture fluid discharged from the culture tank every day is 3 times the tank volume, and 3 times the tank volume of culture medium is added at the same time to maintain the total volume of the culture fluid unchanged. It can be understood by those skilled in the art that when the culture fluid is discharged, the cells in the culture fluid are retained to avoid the discharge of cells.

[0038] In the present invention, "perfusion" refers to the process of continuously adding fresh culture medium to the bioreactor during the actual production process while continuously removing products and metabolic waste through the cell retention device (hollow fiber column). The perfusion process provides an optimized and stable cell physiological environment to bring production capacity and cost advantages to pharmaceutical companies, achieving cost reduction, efficiency improvement and process intensification.

[0039] In the present invention, PF68 refers to polyoxyethylene polyoxypropylene block copolymer F68, CAS No. 9003-11-6, molecular formula , molecular weight about 8350.

[0040] In the present invention, "Day" in the context refers to day, for example, Day 3 refers to the 3rd day.

[0041] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as a reference separately. Unless they conflict with the invention purpose and / or technical solution of the present application, the cited documents involved in the present invention are cited with all contents and all purposes. When the present invention involves cited documents, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited documents are also cited. When the present invention involves cited documents, the examples and preferred embodiments of the cited relevant technical features may also be incorporated into this application as references, but are limited to the ability to implement the present invention. It should be understood that when the content of the citation conflicts with the description in this application, the present application shall prevail or be modified adaptively according to the description of this application.

[0042] McCoy cells are difficult to grow in high-density suspension. The traditional process of culturing McCoy cells adopts an adherent culture method, which relies on serum during the culture process. Large-scale culture of McCoy cells uses microcarriers to attach cells to microcarriers for suspension culture, and still requires a serum-containing culture medium. These characteristics lead to high cell culture costs and relatively complex operations, which restrict McCoy cells in the process of amplification. In response to these defects, the present invention adopts a method of taming and suspending adherent cells to carry out suspension culture. In the adherent culture medium, only changing the culture method cannot make the cells grow normally or even survive. The original adherent cell culture process is replaced by a suspension culture process to develop a full suspension production process for the cell and a culture medium with corresponding requirements. The present invention adjusts and optimizes the culture medium formula so that McCoy adherent cells can be suspended and cultured at a high density in a serum-free culture medium without the need for carrier attachment or the addition of serum.

[0043] In some embodiments, a serum-free medium for culturing McCoy cells in suspension is provided, comprising amino acids, vitamins, inorganic salts, glucose, sodium hyaluronate, PF68 and water;

[0044] The serum-free medium satisfies at least one of the following characteristics (t1) and (t2):

[0045] (t1) the amino acid comprises one or more of asparagine, cysteine, glutamic acid and proline;

[0046] (t2) The inorganic salt comprises anhydrous disodium chloride phosphate.

[0047] Through the screening and optimization of amino acids and inorganic salts, a protein-free, serum-free and serum-replacement-free culture medium is provided, and the culture medium can be used for high-density suspension culture of McCoy cells.

[0048] In some embodiments, in the serum-free medium provided, the amino acids include one or more of 165.8 mg / L to 298.4 mg / L asparagine, 49.7 mg / L to 89.5 mg / L cysteine, 331.0 mg / L to 441.4 mg / L glutamate, and 66.3 mg / L to 119.4 mg / L proline, based on the concentration in the serum-free medium.

[0049] In some embodiments, the serum-free medium provided contains amino acids including glycine, arginine, L-cystine, glutamine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, serine, threonine, tryptophan, L-tyrosine, valine, asparagine, cysteine, glutamic acid and proline. In some embodiments, the serum-free medium provided contains amino acids including 33.8 mg / L to 77.8 mg / L glycine, 74.1 mg / L to 133.4 mg / L arginine, 55.6 mg / L to 100.0 mg / L arginine, and 100.0 mg / L arginine. L-cystine, 515.1mg / L~927.2mg / Lglutamine, 37.0mg / L~66.7mg / Lhistidine, 92.6mg / L~166.7mg / Lisoleucine, 101.4mg / L~182.6mg / Lleucine, 128.8mg / L~231.8mg / Llysine, 26.5mg / L~47.6mg / Lmethionine, 58.2mg / L~104.8mg / Lphenylalanine, 37.0mg / L~66.7mg / Lserine, 83.8mg / L~150.8mg / Lthreonine, 14.1mg / L~25.4mg / Ltryptophan, 91.7mg / L~165.1mg / L L-tyrosine, 82.9mg / L~149.2mg / L valine, 165.8mg / L~298.4mg / L asparagine, 49.7mg / L~89.5mg / L cysteine, 331.0mg / L~441.4mg / L glutamic acid and 66.3mg / L~119.4mg / L proline.

[0050] In some embodiments, in the serum-free medium provided, the vitamins include 3.6 mg / L to 6.5 mg / L choline chloride, 4.0 mg / L to 7.7 mg / L nicotinamide, 0.3 mg / L to 0.6 mg / L pyridoxine, 3.4 mg / L to 10 mg / L riboflavin, 6.1 mg / L to 11 mg / L thiamine hydrochloride and 3.6 mg / L to 6.5 mg / L inositol, based on the concentration in the serum-free medium.

[0051] In some embodiments, in the serum-free culture medium provided, the inorganic salts include, by concentration in the serum-free culture medium, 0.1 mg / L to 0.2 mg / L ferric nitrate nonahydrate, 68.4 mg / L to 123.1 mg / L magnesium sulfate, 280 mg / L to 504 mg / L potassium chloride, 2590 mg / L to 6622 mg / L sodium bicarbonate, 4480 mg / L to 8064 mg / L sodium chloride, 87.5 mg / L to 157.5 mg / L sodium dihydrogen phosphate monohydrate, and 192.5 mg / L to 346.5 mg / L anhydrous disodium hydrogen phosphate.

[0052] In some embodiments, a serum-free medium is provided that is free of calcium chloride, calcium nitrate, calcium pantothenate, and calciferol.

[0053] In some embodiments, the serum-free medium provided comprises, in terms of concentration in the serum-free medium, 33.8 mg / L to 77.8 mg / L glycine, 74.1 mg / L to 133.4 mg / L arginine, 55.6 mg / L to 100.0 mg / L L-cystine, 515.1mg / L~927.2mg / Lglutamine, 37.0mg / L~66.7mg / Lhistidine, 92.6mg / L~166.7mg / Lisoleucine, 101.4mg / L~182.6mg / Lleucine, 128.8mg / L~231.8mg / Llysine, 26.5mg / L~47.6mg / Lmethionine, 58.2mg / L~104.8mg / Lphenylalanine, 37.0mg / L~66.7mg / Lserine, 83.8mg / L~150.8mg / Lthreonine, 14.1mg / L~25.4mg / Ltryptophan, 91.7mg / L~165.1mg / L L-tyrosine, 82.9mg / L~149.2mg / L valine, 165.8mg / L~298.4mg / L asparagine, 49.7mg / L~89.5mg / L cysteine, 331.0mg / L~441.4mg / L glutamic acid and 66.3mg / L~119.4mg / L proline, 3.6mg / L~6.5mg / L choline chloride, 4.0mg / L~7.7mg / L nicotinamide, 0.3mg / L~0.6mg / L pyridoxine, 3.4mg / L~10mg / L riboflavin, 6.1mg / L~11mg / L thiamine hydrochloride and 3.6mg / L~6.5mg / L g / L inositol, 0.1mg / L~0.2mg / L ferric nitrate nonahydrate, 68.4mg / L~123.1mg / L magnesium sulfate, 280mg / L~504mg / L potassium chloride, 2590mg / L~6622mg / L sodium bicarbonate, 4480mg / L~8064mg / L sodium chloride, 87.5mg / L~157.5mg / L sodium dihydrogen phosphate monohydrate and 192.5mg / L~346.5mg / L disodium hydrogen phosphate anhydrous, 3150mg / L~5670mg / L glucose, 3.8mg / L~6.9mg / L sodium hyaluronate, 1000mg / L~2000mg / LPF68.

[0054] The culture medium provided by the present invention may not contain phenol red or may contain phenol red as required.

[0055] McCoy cells were cultured in suspension using the serum-free medium provided. The density of McCoy cells can reach 2.5×107 cells / mL. When McCoy cells in suspension culture were inoculated with intracellular Lawsonia, the yield of intracellular Lawsonia could reach 10 8.3 TCID 50 / mL.

[0056] By optimizing and adjusting the types and concentrations of amino acids, vitamins, and inorganic salts in the culture medium, high-density suspension growth of McCoy cells is achieved. The amino acids in the serum-free culture medium for suspending and culturing McCoy cells provided play an important role in protein synthesis and other physiological processes in the body. And the formula contains anhydrous disodium hydrogen phosphate, does not contain calcium chloride, calcium pantothenate, calcium nitrate, and calciferol, so that McCoy adherent cells can be suspended and cultured at high density without the aid of any adsorption carrier, and can be continuously and stably passaged. In addition, the accumulation of lactic acid and ammonium is unfavorable to the growth of cells. The culture medium provided by the present invention includes four amino acids, asparagine, cysteine, glutamic acid, and proline. According to the detection of the metabolic efficiency of lactic acid and ammonium, it is found that lactic acid is converted from accumulation to consumption, and the accumulation of ammonium is also less. The culture medium provided allows McCoy cells to be dispersed and non-agglomerated during suspension growth, which is beneficial to cell proliferation and helps to efficiently produce intracellular Lawsonia. At the same time, the growth rate of cells and the yield of Lawsonia are significantly improved.

[0057] In some embodiments, a method for culturing McCoy cells in suspension is provided, comprising the following steps: culturing McCoy cells in a serum-free medium.

[0058] In some embodiments, the provided McCoy cell suspension culture method comprises the following steps: placing the McCoy cells in a serum-free medium for suspension culture for 3 to 5 days, starting perfusion culture, and the perfusion rate is 0.5VVD to 3VVD; for example, the perfusion rate can be 0.5VVD, 1VVD, 1.5VVD, 2VVD, 2.5VVD, 3VVD, etc., and can also be a range consisting of any two of the aforementioned values.

[0059] In some embodiments, in the provided McCoy cell suspension culture method, in the step of placing the McCoy cells in the serum-free medium, the initial viable cell density of the McCoy cells is greater than or equal to 1.0×10 6 Pieces / mL.

[0060] In some embodiments, use of serum-free culture medium or McCoy cell suspension culture method for producing Lawsonia intracellularis is provided.

[0061] In some embodiments, a method for producing intracellular Lawsonia is provided, comprising the steps of placing McCoy cells in a serum-free medium for suspension culture, inoculating intracellular Lawsonia, culturing, and harvesting intracellular Lawsonia.

[0062] In some embodiments, the provided method for producing intracellular Lawsonia comprises the following steps: placing the McCoy cells in the serum-free medium for suspension culture for 4 to 5 days, starting perfusion with a perfusion rate of 0.5VVD to 3VVD, inoculating intracellular Lawsonia after starting perfusion for 40h to 60h, culturing for 6 to 8 days after inoculating intracellular Lawsonia, and harvesting the intracellular Lawsonia.

[0063] In order to achieve a higher cell density, perfusion technology was used to culture McCoy suspension cells. By adjusting the culture parameters, the density of McCoy suspension cells reached a new peak value, which was 10 times higher than that of traditional batch culture. In this culture medium environment, intracellular Lawsonia can efficiently infect host cells. The provided culture medium can be used for high-density suspension culture of McCoy cells and can also be used for intracellular Lawsonia production, which is suitable for large-scale vaccine production.

[0064] The embodiments of the present invention will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples that do not specify specific conditions are preferably referred to the guidance provided in the present invention, and can also be based on the experimental manual or normal conditions in this area, can also be based on the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0065] In the following specific embodiments, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.

[0066] In the embodiment: PF68 was purchased from Sigma, item number K4894; FITC-labeled goat anti-mouse IgG was purchased from Bio-Tech; McCoy adherent cells were purchased from ATCC; 2L bioreactor was purchased from Applikon; ATF was purchased from Repligen; counter was purchased from Beckman; bioprocess biochemical analyzer was purchased from Shenzhen Silman Technology Co., Ltd.

[0067] Example 1 Adherent cell acclimation suspension

[0068] After digestion of McCoy adherent cells, the cells were transferred to a centrifuge tube and centrifuged at 1000 rpm for 5 min. The supernatant was discarded and the precipitate was diluted to 1.0 × 10 6The cells were inoculated at a density of 100 cells / mL into serum-free medium for suspension growth of different formulations shown in Tables 1 to 3, and suspension culture was carried out under the same culture conditions (37°C, 5% CO2) (shaking culture was carried out at a speed of 135 rpm in a shaker with a 25 mm orbital diameter), and the cells were subcultured every 3 days. The cell density and viability were tested. The results are shown in Tables 4 and 5.

[0069] Table 1. Serum-free culture medium, unit: mg / L

[0070]

[0071] Table 2. Serum-free culture medium for control formulations 1 to 4, unit: mg / L

[0072]

[0073] Table 3. Serum-free culture medium for control formula 5 to 8, unit: mg / L

[0074]

[0075] Table 4. Summary of the culture effects of serum-free medium shown in Table 1

[0076]

[0077] Summary of the culture effects of serum-free culture media of control formulations 1 to 8 shown in Table 5, Table 2 and Table 3

[0078]

[0079] According to the experimental results in Tables 4 and 5, the digested McCoy adherent cells were subjected to suspension acclimation. In the culture medium of Experimental Formulas 1 to 4, the McCoy cells were able to be cultured in suspension. After digestion, the McCoy adherent cells were inoculated in the culture medium of Experimental Formulas 1 to 4 for suspension culture. After 3 days of culture, as shown in Table 4, the average cell density on Day 3 and the average cell viability on Day 3, the average cell density reached 3.03×10 6 / mL to 8.63×10 6 / mL, and the average cell viability reached 97.6%~99.7%. Especially in the culture medium of experimental formula 2 and experimental formula 3, after 3 days of culture, the average cell density of suspended cells reached 6.12×10 6 / mL and 8.63×10 6 In the control formulations, especially the culture medium of control formulations 1, 3 to 8, after 3 days of culture, as shown in Table 5, the average cell density of suspension cells was low, which was not suitable for suspension culture of McCoy cells.

[0080] Example 2 Suspension culture medium screening

[0081] The McCoy suspension cells obtained by acclimating the experimental formula 3 in Example 1 were cultured at 1.0×10 6 The cells were inoculated into the culture medium formula described in Table 1 at a density of 1.0×10 / mL. Specifically, the cells were transferred to a centrifuge tube, centrifuged at 1000 rpm for 5 min, the supernatant was discarded, and the cell density was adjusted to 1.0×10 / mL using the 4 culture media listed in Table 1 and the control formula 2 culture medium. 6 / mL, placed in a 37°C, 5% CO2, 125rpm shaking incubator for culture, and tested cell density and viability every 24 hours. The culture was continued for 6 days to compare parameters such as cell growth rate, maximum cell density, and the length of cell maintenance period in the culture medium. The results are shown in Table 6. From the results in Table 6, it can be seen that experimental formulas 2 and 3 are preferred, and experimental formula 3 is more preferred. The cells in the experimental group formula 3 grew fastest, and the maximum cell density could reach 9.98×10 6 / mL, and the cells were maintained for the longest period in this culture medium.

[0082] Experimental formulation 3 was used for stability subculture, with a seeding density of 1.0 × 10 6 / mL, placed in 37 ℃, 5% CO2, 125rpm shaking incubator for culture, every 3 days for subculture, the results are shown in Figure 1 The density and viability of the medium passage were higher than 8.0×10 on the third day. 6 / mL, and the viability is above 95%.

[0083] Table 6, Example 2 Results Summary

[0084]

[0085] Example 3 Cellular metabolism

[0086] The McCoy suspension cells obtained by acclimating the experimental formula 3 in Example 1 were cultured at 1.0×10 6 The cells were inoculated into experimental formula 3 and control formula 2 at a density of 1.0×10 / mL for culture. Specifically, the cells were transferred to a centrifuge tube, centrifuged at 1000 rpm for 5 min, the supernatant was discarded, and the cell density was adjusted to 1.0×10 / mL using the experimental formula 3 and control formula 2 culture medium. 6 The cells were cultured in a shaking incubator at 37°C, 5% CO2, and 125 rpm. The cell supernatants on the first, third, and fifth days were collected for biochemical testing using a biochemical analyzer. The metabolism of lactate and ammonium by the cells was specifically measured. The results are shown in Table 7.

[0087] The accumulation of lactic acid and ammonium is not conducive to cell growth. According to the experimental results in Table 7, the experimental formula 3 medium has a better ability to metabolize lactic acid, which can convert lactic acid from accumulation to consumption, and the accumulation of ammonium is also less. When McCoy cells were cultured in the control formula 2 medium, lactic acid continued to accumulate and the accumulation of ammonium was also higher.

[0088] Table 7. Comparison of cell metabolism in preferred culture medium

[0089]

[0090] Example 4 Perfusion culture process-perfusion time optimization

[0091] The McCoy suspension cells obtained by acclimating the experimental formula 3 in Example 1 were cultured at 1.0×10 6 The cells were inoculated into experimental formula 3 in Table 1 at a density of 1.0×10 / mL. After culturing for 3 days, perfusion was started. Specifically, the cells were transferred to a centrifuge tube and centrifuged at 1000 rpm for 5 min. The supernatant was discarded and the cell density was adjusted to 1.0×10 / mL using the experimental formula 3 medium and the control formula 2 medium in Table 1. 6 / mL, cultured in the reactor, and perfusion started after 3 days. Perfusion started on the 3rd, 4th and 5th days, respectively, with a perfusion rate of 1VVD. After perfusion was started, the cell density and viability were tested every 24 hours from Day 0. The comparison of the highest viable cell density between the control formula 2 and the experimental group formula 3 is shown in Table 8.

[0092] According to the experimental results in Table 8, the McCoy cells were suspended and cultured using experimental formula 3. The perfusion was started on the third day, and the highest cell density reached 18.3×10 6 / mL, the cell viability was 99.7%; especially on the fourth day of perfusion, the highest cell density reached 25.1×10 6 / mL, the cell viability reached 99.1%; perfusion began on the 5th day, and the highest cell density reached 21.2×10 6 / mL, the cell viability reached 99.2%. When McCoy cells were cultured in suspension using control formula 2 and perfusion was started on the 4th day, the highest cell density that could be achieved was 6.52×10 6 Pieces / mL.

[0093] Table 8. Optimization of perfusion time

[0094]

[0095] Example 5 Perfusion culture process-VVD optimization

[0096] The McCoy suspension cells obtained by acclimating the experimental formula 3 in Example 1 were cultured at 1.0×10 6The cells were inoculated into the experimental formula 3 described in Table 1 at a density of 1.0×10 / mL. After culturing for 4 days, perfusion was started. Specifically, the cells were transferred to a centrifuge tube, centrifuged at 1000 rpm for 5 min, the supernatant was discarded, and the cell density was adjusted to 1.0×10 / mL using the medium of formula 3 in Table 1 and the medium of control formula 2. 6 / mL, cultured in a reactor, and after 4 days, perfusion was started, with the perfusion rates set at 0.5VVD, 1.0VVD, 1.5VVD, 2VVD, and 3VVD, respectively, and the cell density and viability were tested every 24 hours. The results are shown in Table 9.

[0097] According to the experimental results in Table 9, under the perfusion process, in the experimental formula 3 culture medium, the cell density can reach 16.3×10 6 / mL to 25.7×10 6 / mL, especially when the perfusion rate was 1.0VVD~1.5VVD, the cell density reached 24.3×10 6 / mL to 25.7×10 6 In the culture medium of control formula 2, the cell density was 5.05×10 6 / mL to 6.52×10 6 Pieces / mL.

[0098] Table 9. VVD optimization

[0099]

[0100] Example 6 Production of Lawsonia intracellularis by perfusion process

[0101] The McCoy suspension cells obtained by acclimating the experimental formula 3 in Example 1 were cultured at 1.0×10 6 The cells were inoculated into the experimental formula 3 described in Table 1 at a density of 1.0×10 / mL. After 4 days of culture, perfusion and inoculation of intracellular Lawsonia were started. Specifically, the cells were transferred to a centrifuge tube, centrifuged at 1000 rpm for 5 min, the supernatant was discarded, and the cell density was adjusted to 1.0×10 / mL using the experimental formula 3 culture medium and the control formula 2 culture medium in Table 1. 6 / mL, cultured in a reactor, and after 4 days, perfusion was started at a rate of 1.0VVD. After 48 hours of perfusion, intracellular Lawsoniae were inoculated. After 7 days of culture, the content of intracellular Lawsoniae was detected.

[0102] The intracellular Lawsonia solani content was measured by using the tissue culture infectious dose 50 per milliliter. ) was determined by the following method: 2.0 ml of the culture to be tested was removed and the cells were lysed by passing through a 25 gauge needle 4 times. The sample was diluted in a series of 1:10 in DMEM / 5% FBS containing vancomycin (100 μg / ml) and amphotericin B (2.0 μg / ml). The dilution was added to a 96-well microtiter plate at 0.1 ml per well. McCoy cells were seeded in microtiter plates at 1250 cells / well and grown for 18-24 hours before infection. 3-6 wells were used for each dilution concentration. The microtiter plate was incubated in 8.0% oxygen, 8.8% carbon dioxide, and 83.2% hydrogen for 6 days. The cells were fixed with cold 50% acetone and 50% methanol for 2 minutes. 0.03 ml / well of homemade Omp2 protein (Xiao Ning 2022) was added to each well. The antibody was diluted 1:2000 in PBS. The plate was incubated at 37°C for 30 minutes and then washed three times with PBS. FITC-labeled goat anti-mouse IgG diluted 1:30 was added at 0.03 ml / well and incubated at 37°C for 30 minutes. The plate was washed three times with double distilled water, dried, and the samples were observed under a fluorescence microscope to determine According to the experimental results, the content of intracellular Lawsonia solani in the experimental formula 3 culture medium can reach .

[0103] The technical features of the above-mentioned implementation modes and examples can be combined in any appropriate manner. To make the description concise, not all possible combinations of the technical features in the above-mentioned implementation modes and examples are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0104] The above-described embodiments only express several implementation methods of the present invention, which is convenient for understanding the technical solutions of the present invention in detail, but cannot be understood as limiting the scope of protection of the invention patent. It should be pointed out that, for those of ordinary skill in the art, without departing from the concept of the present invention, several variations and improvements can be made, which all belong to the protection scope of the present invention. In addition, it should be understood that after reading the above-mentioned teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and the equivalent forms obtained also fall within the protection scope of this application. It should also be understood that the technical solutions obtained by those skilled in the art on the basis of the technical solutions provided by the present invention through logical analysis, reasoning or limited experiments are all within the protection scope of the claims attached to the present invention. Therefore, the protection scope of the patent of the present invention shall be based on the contents of the attached claims, and the description and drawings can be used to explain the contents of the claims.

Claims

1. A serum-free medium for culturing McCoy cells in suspension, characterized in that: The serum-free medium contains amino acids, vitamins, inorganic salts, glucose, sodium hyaluronate, PF68 and water; Wherein, the serum-free medium satisfies at least one of the following characteristics (t1) and (t2): (t1) the amino acids include one or more of asparagine, cysteine, glutamic acid and proline; (t2) The inorganic salt comprises anhydrous disodium hydrogen phosphate.

2. The serum-free medium according to claim 1, characterized in that In terms of concentration in the serum-free medium, the amino acids include 33.8 mg / L to 77.8 mg / L glycine, 74.1 mg / L to 133.4 mg / L arginine, 55.6 mg / L to 100.0 mg / L L-cystine, 515.1mg / L~927.2mg / Lglutamine, 37.0mg / L~66.7mg / Lhistidine, 92.6mg / L~166.7mg / Lisoleucine, 101.4mg / L~182.6mg / Lleucine, 128.8mg / L~231.8mg / Llysine, 26.5mg / L~47.6mg / Lmethionine, 58.2mg / L~104.8mg / Lphenylalanine, 37.0mg / L~66.7mg / Lserine, 83.8mg / L~150.8mg / Lthreonine, 14.1mg / L~25.4mg / Ltryptophan, 91.7mg / L~165.1mg / L L-tyrosine, 82.9mg / L~149.2mg / L valine, 165.8mg / L~298.4mg / L asparagine, 49.7mg / L~89.5mg / L cysteine, 331.0mg / L~441.4mg / L glutamic acid and 66.3mg / L~119.4mg / L proline.

3. The serum-free medium according to claim 1 or 2, characterized in that In terms of concentration in the serum-free medium, the vitamins include 3.6 mg / L to 6.5 mg / L choline chloride, 4.0 mg / L to 7.7 mg / L nicotinamide, 0.3 mg / L to 0.6 mg / L pyridoxine, 3.4 mg / L to 10 mg / L riboflavin, 6.1 mg / L to 11 mg / L thiamine hydrochloride and 3.6 mg / L to 6.5 mg / L inositol.

4. The serum-free medium according to any one of claims 1 to 3, characterized in that In terms of concentration in the serum-free medium, the inorganic salts include 0.1 mg / L to 0.2 mg / L ferric nitrate nonahydrate, 68.4 mg / L to 123.1 mg / L magnesium sulfate, 280 mg / L to 504 mg / L potassium chloride, 2590 mg / L to 6622 mg / L sodium bicarbonate, 4480 mg / L to 8064 mg / L sodium chloride, 87.5 mg / L to 157.5 mg / L sodium dihydrogen phosphate monohydrate and 192.5 mg / L to 346.5 mg / L anhydrous disodium hydrogen phosphate.

5. The serum-free medium according to any one of claims 1 to 4, characterized in that The serum-free medium does not contain calcium chloride, calcium nitrate, calcium pantothenate, and calciferol.

6. The serum-free medium according to any one of claims 1 to 5, characterized in that In terms of concentration in the serum-free medium, the serum-free medium contains 33.8 mg / L to 77.8 mg / L glycine, 74.1 mg / L to 133.4 mg / L arginine, 55.6 mg / L to 100.0 mg / L L-cystine, 515.1mg / L~927.2mg / Lglutamine, 37.0mg / L~66.7mg / Lhistidine, 92.6mg / L~166.7mg / Lisoleucine, 101.4mg / L~182.6mg / Lleucine, 128.8mg / L~231.8mg / Llysine, 26.5mg / L~47.6mg / Lmethionine, 58.2mg / L~104.8mg / Lphenylalanine, 37.0mg / L~66.7mg / Lserine, 83.8mg / L~150.8mg / Lthreonine, 14.1mg / L~25.4mg / Ltryptophan, 91.7mg / L~165.1mg / L L-tyrosine, 82.9mg / L~149.2mg / L valine, 165.8mg / L~298.4mg / L asparagine, 49.7mg / L~89.5mg / L cysteine, 331.0mg / L~441.4mg / L glutamic acid and 66.3mg / L~119.4mg / L proline, 3.6mg / L~6.5mg / L choline chloride, 4.0mg / L~7.7mg / L nicotinamide, 0.3mg / L~0.6mg / L pyridoxine, 3.4mg / L~10mg / L riboflavin, 6.1mg / L~11mg / L thiamine hydrochloride and 3.6mg / L~6.5mg / L 5mg / L inositol, 0.1mg / L~0.2mg / L ferric nitrate nonahydrate, 68.4mg / L~123.1mg / L magnesium sulfate, 280mg / L~504mg / L potassium chloride, 2590mg / L~6622mg / L sodium bicarbonate, 4480mg / L~8064mg / L sodium chloride, 87.5mg / L~157.5mg / L sodium dihydrogen phosphate monohydrate and 192.5mg / L~346.5mg / L disodium hydrogen phosphate anhydrous, 3150mg / L~5670mg / L glucose, 3.8mg / L~6.9mg / L sodium hyaluronate, 1000mg / L~2000mg / L PF68.

7. A McCoy cell suspension culture method, characterized in that: The method comprises the following steps: placing McCoy cells in the serum-free culture medium according to any one of claims 1 to 6 for culturing.

8. The McCoy cell suspension culture method according to claim 7, characterized in that: The method comprises the following steps: placing the McCoy cells in the serum-free culture medium for suspension culture for 3 to 5 days, and starting perfusion culture at a perfusion rate of 0.5 VVD to 3 VVD; Optionally, in the step of placing the McCoy cells in the serum-free medium, the initial viable cell density of the McCoy cells is greater than or equal to 1.0×10 6 Pieces / mL.

9. Use of the serum-free medium according to any one of claims 1 to 6 or the McCoy cell suspension culture method according to claim 7 or 8 in producing Lawsonia intracellularis.

10. A method for producing Lawsonia intracellularis, characterized in that: The method comprises the following steps: placing the McCoy cells in the serum-free medium according to any one of claims 1 to 6 for suspension culture, inoculating intracellular Lawsonia spp., culturing, and harvesting the intracellular Lawsonia spp.; Optionally, the method for producing intracellular Lawsoniae comprises the following steps: placing the McCoy cells in the serum-free medium for suspension culture for 4 to 5 days, starting perfusion, the perfusion rate being 0.5VVD to 3VVD, inoculating intracellular Lawsoniae after starting perfusion for 40h to 60h, culturing for 6 to 8 days after inoculating intracellular Lawsoniae, and harvesting the intracellular Lawsoniae.

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