CN114891718B

CN114891718B - Culture medium for suspension culture of bone marrow cells, preparation method, application and method for inducing differentiation of bone marrow-derived cells into macrophages

Info

Publication number: CN114891718B
Application number: CN202210642646.4A
Authority: CN
Inventors: 贺笋; 毛丽萍; 潘晓梅; 候凤; 周涛; 侯红娟
Original assignee: Tiankang Pharmaceutical Co ltd
Current assignee: Tiankang Pharmaceutical Co ltd
Priority date: 2022-06-08
Filing date: 2022-06-08
Publication date: 2024-01-30
Anticipated expiration: 2042-06-08
Also published as: CN114891718A

Abstract

The invention provides a culture medium for suspension culture of bone marrow cells, a preparation method, application and a method for inducing differentiation of bone marrow cells into macrophages, which relate to the technical field of cell culture. In addition, the culture medium has the advantages of definite components, controllable cost and convenient use by independently packaging components with different functions.

Description

Culture medium for suspension culture of bone marrow cells, preparation method, application and method for inducing differentiation of bone marrow-derived cells into macrophages

Technical Field

The invention relates to the technical field of cell culture, in particular to a culture medium for suspension culture of bone marrow cells, a preparation method, application and a method for inducing differentiation of bone marrow-derived cells into macrophages.

Background

Macrophages are important defensive cells of organisms, play a wide biological role in phagocytizing, removing foreign matters, regulating hematopoiesis, participating in immune response and the like, and have important values for virus separation, seed virus propagation and vaccine research and development. The research shows that macrophages are extremely susceptible to various viruses such as Porcine Reproductive Respiratory Syndrome Virus (PRRSV), african Swine Fever Virus (ASFV), porcine Circovirus (PCV) and the like, and the macrophages are reported to be applied to virus isolation, in vitro culture and related vaccine production.

Porcine Reproductive and Respiratory Syndrome (PRRS) is a highly contagious disease that can cause swine morbidity, an acute infectious disease that affects the global pig industry most severely, causing serious economic loss. In PRRSV isolation, PAM cells are the primary target cells for PRRSV infection, and most strains are adaptable due to the higher sensitivity of PRRSV to the virus, and are often used as primary cell isolation and culture of PRRSV. Primary Alveolar Macrophages (PAM) and bone marrow cells are the main hosts for separating and culturing the virus at present, and in the preparation process of the alveolar macrophages, it is found that even SPF pigs, the lung of the pigs is seriously polluted by unknown microorganisms due to the open state of the respiratory system, and the qualified PAM cells are difficult to prepare. In contrast, the marrow is in a physiological closed state, and the external pathogen pollution is slight, so that the method is an ideal choice for PRRSV virus isolation and ASFV vaccine culture at present. However, the virus titer of the undifferentiated marrow cell culture virus is low, and the differentiated marrow-derived mononuclear-macrophages are induced, which is beneficial to the improvement of the virus titer.

There are many ways to separate and culture macrophages from tissues and organs, but they are not easy. For example, in the prior art, freshly prepared bone marrow cells are utilized for static induction culture, the induced bone marrow cells are partially adherent and partially float for growth, and the operation is tedious, time and labor are consumed, the efficiency is low, and the production cost is high in the culture process. How to improve the production efficiency and the product quality, reduce the production cost, and provide a method for inducing differentiation of high-speed and effective bone marrow cells to obtain highly directional differentiated and proliferated macrophages is important.

In view of this, the present invention has been made.

Disclosure of Invention

It is an object of the present invention to provide a culture medium for suspension culture of bone marrow cells, which solves at least one of the technical problems of the prior art.

The second object of the present invention is to provide a method for preparing the culture medium for suspension culture of bone marrow cells.

The third object of the present invention is to provide the use of the culture medium for suspension culture of bone marrow cells in full suspension culture of bone marrow cells and/or inducing differentiation of bone marrow cells into macrophages.

The fourth object of the present invention is to provide a method for inducing differentiation of bone marrow-derived cells into macrophages.

The invention provides a culture medium for suspension culture of bone marrow cells, which comprises an independently packaged cell culture solution and M-CSF;

the cell culture broth comprises basal medium, fetal bovine serum, shear force protectant, anti-cell-clumping agent, and optionally L929 cell supernatant.

Further, the shear force protecting agent is Pluronic F-68;

preferably, the anti-cell-clumping agent is dextran sulfate;

preferably, the basal medium is at least one of DME/F-12 or IMDM.

Further, the cell culture broth further comprises one or more of GM-CSF, an antibiotic, a basal nutrient substance, and a buffer substance;

preferably, the antibiotic is penicillin and/or streptomycin;

preferably, the basic nutrient is L-glutamine;

preferably, the buffer substance is HEPES buffer.

Further, the cell culture solution contains 10 to 20 percent v/v of fetal bovine serum, 0.5 to 3g/L of shear force protective agent, 20 to 40mg/L of anti-cell-agglomerating agent, and optionally, 5 to 50ng/mL of GM-CSF, 50 to 150U/mL of penicillin, 50 to 150 mu g/mL of streptomycin, 2 to 2.50mM of L-glutamine, 10 to 20mM HEPES buffer solution and 10 to 30 percent v/v of L929 cell supernatant;

preferably, the cell culture broth comprises 10% v/v fetal bovine serum, 1g/L shear force protectant, 30mg/L anti-cell clumping agent, and optionally 20% v/v GM-CSF, 100U/mL penicillin, 100. Mu.g/mL streptomycin, 2.50mM L-glutamine, 15mM HEPES buffer, and 20% v/v L929 cell supernatant.

The invention also provides a preparation method of the culture medium for suspension culture of bone marrow cells, which comprises the steps of adding fetal bovine serum, a shearing force protective agent and an anti-cell-agglomerating agent into a basic culture medium, and uniformly mixing to obtain the cell culture solution.

Further, adding the formula amount of fetal bovine serum, the shearing force protective agent, the anti-cell-agglomerating agent, GM-CSF, antibiotics, basic nutrients, buffer substances and optional L929 cell supernatant into the basic culture medium, and uniformly mixing to obtain the cell culture solution.

The invention also provides application of the culture medium for suspension culture of bone marrow cells in full suspension culture of bone marrow cells and/or induction of differentiation of bone marrow cells into macrophages.

In addition, the invention also provides a method for inducing the differentiation of bone marrow-derived cells into macrophages, which comprises the steps of adopting the cell culture solution to culture the bone marrow-derived cells in a suspension manner, then adding M-CSF for continuous culture, and inducing the differentiation of the bone marrow-derived cells into macrophages.

Further, the cell culture solution is subjected to half-liquid exchange in 3 to 5 days of culture, and M-CSF is added after the half-liquid exchange;

alternatively, half-changing the liquid and adding M-CSF on day 3 and day 5 of culture, respectively;

alternatively, half-changing the liquid and adding M-CSF on day 4 of culture;

preferably, the addition amount of the M-CSF is 5-50 ng/mL, preferably 20ng/mL;

preferably, the bone marrow-derived cells have an initial cell density of 1.0 to 2.0X10 ⁶ individual/mL;

further, a bioreactor is adopted for cell culture;

preferably, the parameters of the bioreactor comprise at least one of rotating speed 30-50 r/min, temperature 37 ℃, dissolved oxygen value 40-60%, pH value 7.0-7.2 and ventilation rate 50-300 mL/min.

Further, the culture method comprises the following steps:

(a) Fresh preparing bone marrow source cells from femur and tibia of pig, and re-suspending cells with cell culture liquid according to cell density of 1.0-2.0X10 ⁶ Shake flask culture is carried out per ml;

(b) Cells were placed in a carbon dioxide incubator for 7 days, and culture parameters included: temperature 37 ℃,5% CO ₂ ，>60％RH，100～120rpm@50mm orbital throw shaker；

(c) On the fourth day of culture, half-cell exchange was performed, and then 5-50 ng/mL of M-CSF was added thereto, followed by further culture.

Compared with the prior art, the invention has at least the following beneficial effects:

the culture medium for suspension culture of bone marrow cells provided by the invention can realize full suspension culture of bone marrow cells through mutual coordination of specific components, obtain high-quality full suspension cells, and simultaneously promote proliferation and differentiation of bone marrow cells into macrophages. In addition, the culture medium has the advantages of definite components, controllable cost and convenient use by independently packaging components with different functions.

The preparation method of the culture medium provided by the invention has the advantages of simple process, convenience in operation and convenience in mass production.

The method for inducing the differentiation of the bone marrow-derived cells into macrophages, which is provided by the invention, is applied to the culture medium for suspension culture of the bone marrow cells, and can effectively induce the directional differentiation and/or proliferation of the bone marrow-derived cells, and the proliferation amount can be up to 3 times.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.

FIG. 1A is a photograph of cells of example 10 of experimental example 1 of the present invention after induction of 7 d;

FIG. 1B is a photograph of cells of example 6 of the experimental example 1 of the present invention after induction of 7 d;

FIG. 2 shows a cell photograph (a) and a fluorescent photograph (b) of bone marrow cells induced into mononuclear-macrophages according to experimental example 2 of the present invention;

FIG. 3 is a graph of PAM virus content measurement cells (a with lesions, b without lesions) provided in Experimental example 3 of the present invention;

fig. 4 is a picture of PAM immunofluorescence assay fluorescent cells (a with lesions, b without lesions) provided in experimental example 3 of the present invention.

Detailed Description

Unless defined otherwise herein, scientific and technical terms used in connection with the present invention shall have the meanings commonly understood by one of ordinary skill in the art. The meaning and scope of terms should be clear, however, in the event of any potential ambiguity, the definitions provided herein take precedence over any dictionary or extraneous definition. In this application, the use of "or" means "and/or" unless stated otherwise. Furthermore, the use of the term "include" and other forms is not limiting.

Generally, the nomenclature used in connection with the cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein and the techniques thereof are those well known and commonly employed in the art. Unless otherwise indicated, the methods and techniques of the present invention are generally well known in the art and are performed according to conventional methods as described in various general and more specific references cited and discussed throughout the present specification. Enzymatic reactions and purification techniques are performed according to manufacturer's instructions, as commonly accomplished in the art, or as described herein. Nomenclature used in connection with the analytical chemistry, synthetic organic chemistry, and medical and pharmaceutical chemistry described herein, and the laboratory procedures and techniques therefor, are those well known and commonly employed in the art.

The technical solutions of the present invention will be clearly and completely described in connection with the embodiments, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

According to one aspect of the present invention there is provided a medium for suspension culture of bone marrow cells comprising an independently packaged cell culture broth and M-CSF;

The cell culture solution and the M-CSF in the culture medium for suspension culture of the bone marrow cells are respectively and independently packaged, the adding time of the M-CSF can be flexibly selected according to the actual culture condition, the full suspension culture efficiency of the bone marrow cells is effectively improved, and the use is convenient.

The cell culture medium of the present invention may or may not contain L929 cell supernatant, and differentiation of bone marrow cells can be achieved regardless of whether the L929 cell supernatant is contained. When the culture target is biased toward cell proliferation, the cell culture medium mixed with the L929 cell supernatant is more effective.

Wherein, the L929 cells are mouse fibroblast strains, and the supernatant fluid of the cultured L929 contains high concentration of macrophage colony stimulating factor (M-CSF), which can induce the in vitro undifferentiated bone marrow cells to differentiate into macrophages and promote the proliferation of the macrophages.

The cell culture solution can realize the full suspension culture of bone marrow-derived cells by the mutual coordination of specific components, and obtain high-quality full suspension cells. The culture medium has definite components and controllable cost.

Optionally, the shear force protectant comprises Pluronic F-68;

optionally, the anti-cell-clumping agent comprises dextran sulfate;

optionally, the basal medium comprises at least one of DME/F-12 or IMDM.

In some preferred embodiments, the cell culture broth further comprises one or more of GM-CSF, an antibiotic, a basal nutrient substance, and a buffer substance.

The GM-CSF alone, the antibiotic, the basic nutrient, the buffer substance, or any two of the above three substances may be added to the cell culture solution, or all of the above three substances may be added to the cell culture solution. The effect is optimal when an embodiment is selected in which all of the above three substances are added to the cell culture liquid.

Wherein the antibiotic comprises penicillin and/or streptomycin; the base nutrients include glutamine; the buffer substance comprises HEPES buffer.

The specific components are preferably added in specific amounts to maximize the effect of the combination, so that the cell culture broth preferably contains 10 to 20% v/v of fetal bovine serum, 0.5 to 3g/L of shear force protectant, 20 to 40mg/L of anti-cell-agglomerating agent, and optionally 5 to 50ng/mL of GM-CSF, 50 to 150U/mL of penicillin, 50 to 150. Mu.g/mL of streptomycin, 2 to 2.50mM of L-glutamine, 10 to 20mM of HEPES buffer, and 10 to 30% v/v of L929 cell supernatant.

Wherein the content of said fetal bovine serum may be, for example, but not limited to, 10% v/v, 12% v/v, 15% v/v, 18% v/v or 20% v/v, preferably 10% v/v; the shear force protectant may be present, for example, but not limited to, at 0.5g/L, 1g/L, 1.5g/L, 2g/L, 2.5g/L, or 3g/L, preferably 1g/L; the content of the anti-cell-agglomerating agent may be, for example, but not limited to, 20mg/L, 25mg/L, 30mg/L, 35mg/L or 40mg/L, preferably 30mg/L. And, when any one of GM-CSF, penicillin, streptomycin, L-glutamine or HEPES buffer is contained in the cell culture broth, the addition amount of GM-CSF may be, for example, but not limited to, 5ng/mL, 10ng/mL, 20ng/mL, 30ng/mL, 40ng/mL or 50ng/mL, preferably 20ng/mL; the penicillin content may be, for example, but not limited to, 50U/ml, 80U/ml, 100U/ml, 120U/ml or 150U/ml, preferably 100U/ml; the streptomycin content may be, for example, but not limited to, 50 μg/ml, 80 μg/ml, 100 μg/ml, 120 μg/ml or 150 μg/ml, preferably 100 μg/ml; the L-glutamine content can be, for example, but not limited to, 1.50mM, 2mM, 2.50mM, 3.0mM, 3.5mM, preferably 2.50mM; the HEPES buffer may be present in an amount of, for example, but not limited to, 10mM, 12mM, 15mM, 18mM or 20mM, preferably 15mM; the content of the L929 cell supernatant may be, for example, but not limited to, 10% v/v, 15% v/v, 20% v/v, 25% v/v or 30% v/v, preferably 20% v/v.

According to a second aspect of the present invention, the present invention also provides a method for preparing the culture medium for suspension culture of bone marrow cells, comprising adding fetal bovine serum, a shear force protecting agent and an anti-cell-agglomerating agent to a basal medium, and uniformly mixing to obtain the cell culture solution.

In some preferred embodiments, the cell culture broth is obtained by adding to the basal medium a formulated amount of fetal bovine serum, a shear force protectant, an anti-cell-clumping agent, GM-CSF, an antibiotic, a basal nutrient, a buffer substance, and optionally L929 cell supernatant, and mixing uniformly.

Specifically, the method may include the steps of:

a. bone marrow cell culture DME/F-12 was selected as the basal medium with the addition of 2.5mM L-glutamine and 15mM HEPES buffer.

b. Adding 100U/ml penicillin and 100 μg/ml streptomycin to step (a) under aseptic conditions;

c. adding total 15% Fetal Bovine Serum (FBS) to the step (b) under aseptic condition, and uniformly mixing;

d. under aseptic conditions, adding a shear force protecting agent of Pluronic F-68 (1 g/L) into the step (c), and uniformly mixing;

e. and (3) adding dextran sulfate (30 mg/L) serving as an anti-cell-aggregation agent into the step (d) under the aseptic condition, and uniformly mixing to obtain the cell culture solution.

Based on the beneficial effects of the culture medium for suspension culture of bone marrow cells provided by the invention, the invention also provides the application of the culture medium for suspension culture of bone marrow cells in full suspension culture of bone marrow cells and/or induction of differentiation of bone marrow cells into macrophages.

It should be noted that "and/or" means that the cell culture solution in the medium may be used alone to culture bone marrow-derived cells in full suspension, or that the M-CSF in the medium may be used alone to induce differentiation of bone marrow-derived cells into macrophages, or that the cell culture solution in the medium and the M-CSF are used in combination to induce differentiation of bone marrow-derived cells into macrophages in full suspension culture of bone marrow-derived cells.

According to a fourth aspect of the present invention, there is also provided a method for inducing differentiation of bone marrow-derived cells into macrophages, comprising culturing bone marrow-derived cells in suspension using the above-described cell culture broth, and then continuing the culture by adding M-CSF to induce differentiation of bone marrow-derived cells into macrophages.

The method for inducing the differentiation of the bone marrow-derived cells into macrophages, which is provided by the invention, is applied to the culture medium for suspension culture of the bone marrow cells, and can effectively induce the directional differentiation and proliferation of the bone marrow-derived cells, and the proliferation amount can be up to 3 times.

In some preferred embodiments, the cell culture broth is half-changed on days 3-5 of culture, and M-CSF is added after half-change;

alternatively, half-changing and adding M-CSF on days 3 and 5 of culture, respectively, or half-changing and adding M-CSF on day 4 of culture may be selected. The two effects are equivalent, and in order to save time and operation cost, the addition is preferable at the 4 th day of cultivation.

The amount of M-CSF added is 5 to 50ng/mL, and may be, for example, but not limited to, 5ng/mL, 10ng/mL, 15ng/mL, 20ng/mL, 25ng/mL, 30ng/mL, 35ng/mL, 40ng/mL, 45ng/mL or 50ng/mL.

In order to promote the proliferation of bone marrow-derived cells in suspension culture, it is preferable to set the initial cell density of bone marrow-derived cells to 1.0 to 2.0X10 ⁶ The volume per mL may be, for example, but not limited to, 1.0X10 ⁶ individual/mL, 1.5X10) ⁶ individual/mL or 2.0X10 ⁶ And each mL.

In order to promote differentiation of bone marrow-derived cells in suspension culture, it is preferable to add 5 to 50ng/mL of GM-CSF to the cell culture medium and then to culture the bone marrow-derived cells in suspension, and the amount added may be, for example, but not limited to, 5ng/mL, 10ng/mL, 20ng/mL, 30ng/mL, 40ng/mL or 50ng/mL.

Cascading bioreactors is the dominant mode of current bioproduct production, so to increase culture efficiency, bioreactors are used in some preferred embodiments for cell culture.

The parameters of the bioreactor comprise the rotating speed of 30-50 r/min, such as, but not limited to, 30r/min, 35r/min, 40r/min, 45r/min or 50r/min; the temperature is 37 ℃; the dissolved oxygen value is 40% -60%, for example, but not limited to 40%, 45%, 50%, 55% or 60%; the pH value is 7.0-7.2, for example, but not limited to 7.0, 7.1 or 7.2; the ventilation is 50 to 300mL/min, and may be, for example, but not limited to, 50mL/min, 100mL/min, 150mL/min, 200mL/min, 250mL/min, or 300mL/min.

The invention is further illustrated by the following examples. The materials in the examples were prepared according to the existing methods or were directly commercially available unless otherwise specified.

Example 1

The present example provides a medium for suspension culture of bone marrow cells comprising an independently packaged cell culture broth and M-CSF;

the cell culture broth included DME/F-12, GM-CSF 20ng/mL, fetal bovine serum 10% v/v, pluronic F-68 1g/L, dextran sulfate 30mg/L, penicillin 100U/mL, streptomycin 100 μg/mL, 2.50mM L-glutamine, 15mM HEPES buffer, and L929 cell supernatant 20% v/v.

Example 2

This example provides a medium for suspension culture of bone marrow cells, which differs from example 1 in that the cell culture broth comprises DME/F-12, GM-CSF 50ng/mL, fetal bovine serum 15% v/v, pluronic F-68 3g/L, dextran sulfate 20mg/L, penicillin 150U/mL, streptomycin 50 μg/mL, 2mM L-glutamine, 20mM HEPES buffer, and L929 cell supernatant 30% v/v.

Example 3

This example provides a medium for suspension culture of bone marrow cells, which differs from example 1 in that the cell culture broth comprises DME/F-12, GM-CSF 5ng/mL, fetal bovine serum 20% v/v, pluronic F-68.5 g/L, dextran sulfate 40mg/L, penicillin 50U/mL, streptomycin 150. Mu.g/mL, 2.50mM L-glutamine, 10mM HEPES buffer, and L929 cell supernatant 10% v/v.

Example 4

This example provides a medium for suspension culture of bone marrow cells, which differs from example 1 in that it does not contain penicillin, streptomycin, L-glutamine and HEPES buffer.

Example 5

This example provides a medium for suspension culture of bone marrow cells, which differs from example 1 in that it does not contain L929 cell supernatant.

Examples 6 to 10

The embodiment provides a shake flask suspension culture method for inducing bone marrow-derived cells to become macrophages, which comprises the following steps:

A. fresh preparation of bone marrow-derived cells from femur and tibia of pig using the cells provided in examples 1 to 5, respectivelyThe cells were resuspended in a cell density of 1.0 to 2.0X10 ⁶ Shake flask culture is carried out per ml;

B. cells were placed in a carbon dioxide incubator for 7d, culture parameters: the temperature is 37 ℃,5 percent CO2, >60 percent RH, 100-120rpm@50mm orbital throw shaker;

C. cell half-changing was performed on the fourth day of culture, followed by the addition of 20ng/mL M-CSF;

D. and continuing to culture to obtain the macrophage.

Example 11

The present example provides a shake flask suspension culture method for inducing bone marrow-derived cells to become macrophages, which is different from example 6 in that: the amount of M-CSF added was 5ng/mL.

Example 12

The present example provides a shake flask suspension culture method for inducing bone marrow-derived cells to become macrophages, which is different from example 6 in that: the amount of M-CSF added was 50ng/mL.

Example 13

The present example provides a shake flask suspension culture method for inducing bone marrow-derived cells to become macrophages, which is different from example 6 in that: GM-CSF was not added.

Example 14

The present example provides a shake flask suspension culture method for inducing bone marrow-derived cells to become macrophages, which is different from example 6 in that: and (C) performing no half liquid exchange.

Example 15

The present example provides a shake flask suspension culture method for inducing bone marrow-derived cells to become macrophages, which is different from example 6 in that: cell half-exchanges were performed on day 3 and day 5 of culture, respectively, and 20ng/mL M-CSF was added after the exchanges.

Example 16

The present example provides a shake flask suspension culture method for inducing bone marrow-derived cells to become macrophages, which is different from example 6 in that: 20ng/mL M-CSF was added on day 1 of culture and no further addition was repeated on day 4.

Comparative example 1

This comparative example provides a shake flask suspension culture method for inducing bone marrow-derived cells into macrophages, which differs from example 6 in that the cell culture broth does not contain fetal bovine serum.

Comparative example 2

This comparative example provides a shake flask suspension culture method for inducing bone marrow-derived cells into macrophages, which is different from example 6 in that the cell culture broth does not contain Pluronic F-68.

Comparative example 3

This comparative example provides a shake flask suspension culture method for inducing bone marrow-derived cells into macrophages, which is different from example 6 in that the cell culture broth does not contain dextran sulfate.

Experimental example 1

In order to verify the culture medium for suspension culture of bone marrow cells and the shake flask suspension culture method for inducing bone marrow-derived cells to become macrophages provided by the present invention, after culturing for 7 days using the culture method step D provided in examples 6 to 16 and comparative examples 1 to 3, examination of proliferation and differentiation of bone marrow cells was performed, and the results are shown in Table 1 below:

wherein, the pig bone marrow-derived cells of example 10 were cultured for 7d, the cell morphology became enlarged and rounded, the floating cells exhibited dendritic morphology, and the stimulation and differentiation effects were remarkable. Cell counts after induction for 7d, and no cell proliferation of the induced BM cells occurred, see fig. 1A.

The cells of example 6 were counted after 7d induction of the bone marrow-derived cells of swine, and the induced BM cells showed 3-fold cell proliferation, as shown in FIG. 1B.

Experimental example 2 immunofluorescence assay of bone marrow cells induced to differentiate

1. Taking out BM cells obtained by induction in example 6 and non-induced BM cells, spreading the BM cells on 96 wells, centrifuging at 2000rpm for 5min, and discarding the liquid;

2. fixing with 4% paraformaldehyde at room temperature for 10min, air drying, and washing with PBST;

3. cells were penetrated with 3% Triton, room temperature for 10min, pbst wash;

4. blocking with 1% PBA, cleaning with PBST at 37deg.C for 20 min;

5. adding primary anti-murine MAC387, incubating for 2h at 37 ℃, and cleaning by PBST;

6. secondary antibody (anti-mouse) was added in the dark, incubated at 37℃for 1h and PBST was washed.

7. The results are shown in FIG. 2 when observed in a fluorescence inverted microscope.

From the figure, it can be seen that more than 95% of the cells produced specific fluorescence, indicating that bone marrow cells induced a large number of macrophages.

Experimental example 3 passage domestication of porcine reproductive and respiratory syndrome Virus NADC30 Strain on shake flask suspension cell line and Virus content determination

The NADC30 strains prepared by the adherent cells are continuously domesticated for 3 generations on the suspension cells obtained by culturing in examples 6-16 and comparative examples 1-3, the toxin receiving doses are 0.01, 0.02 and 0.05MOI, and the samples are respectively taken at toxin receiving time of 12 hours, 24 hours, 36 hours, 48 hours, 60 hours, 72 hours, 84 hours and 96 hours, wherein the culture parameters are as follows: the virus content of the culture at 37℃for 72 hours, 5% CO2, >60% RH, 100-120rpm@50mm orbital throw shaker, is shown in Table 2 below:

group of	Virus content
		Example 6	Not less than 10 ^6.5 TCID ₅₀ /ml
Example 7	Not less than 10 ^6.5 TCID ₅₀ /ml
		Example 8	Not less than 10 ^6.0 TCID ₅₀ /ml
Example 9	Not higher than 10 ^5.5 TCID ₅₀ /ml
		Example 10	Not less than 10 ^6.0 TCID ₅₀ /ml
Example 11	Not less than 10 ^6.0 TCID ₅₀ /ml
		Example 12	Not less than 10 ^6.5 TCID ₅₀ /ml
Example 13	Not higher than 10 ^5.5 TCID ₅₀ /ml
		Example 14	Not less than 10 ^6.0 TCID ₅₀ /ml
Example 15	Not less than 10 ^6.5 TCID ₅₀ /ml
		Example 16	Not higher than 10 ^5.8 TCID ₅₀ /ml
Comparative example 1	Not higher than 10 ^4.5 TCID ₅₀ /ml
		Comparative example 2	Not higher than 10 ^5.0 TCID ₅₀ /ml
Comparative example 3	Not higher than 10 ^5.0 TCID ₅₀ /ml

The data show that the porcine reproductive and respiratory syndrome virus has good sensibility to the macrophage line obtained by the culture of the method, and the suspension cell line obtained by the culture process provided by the invention can be used as a candidate cell line for the subsequent process amplification production, thereby completing the adaptive passage domestication of the porcine reproductive and respiratory syndrome virus on the suspension cell line.

The virus content of the sample after virus inoculation by using alveolar macrophages (PAM) is determined as follows:

(1) the RPMI-1640+4% FBS culture solution is used according to the ratio of 1.0 to 1.5X10 ⁶ Spread to 96-well cell culture plate at 100 μl/well;

(2) for 24h, 10-fold serial dilutions (dilution from 10) were performed on virus samples using cell culture broth ^-1 To 10 ^-7 ) Each dilution was inoculated into 96-well cell culture plates, 8 wells per dilution, 100. Mu.l/well.

(3) Virus controls and normal cell controls were established simultaneously. The 96-well cell culture plate was placed in a constant temperature incubator at 37℃for 5 days, cytopathic effect was observed, and virus half-cell infection (TCID 50) was calculated by the Reed-Muench method.

(4) The results showed that the virus control group showed all cytopathic effects characterized by cell disruption and shedding, and that the cell control group showed normal cytopathic effects, as shown in FIG. 3.

Immunofluorescence detection is carried out on the pore plate with the measured virus content, and the steps are as follows:

(1) after fixation with 80% pre-chilled acetone for 10min, PBST was washed 3 times;

(2) porcine reproductive and respiratory syndrome virus mab (1:1000) was added and incubated for 1 hour at 37℃and PBST was washed 3 times.

(3) Anti-mouse FITC secondary antibody (1:200) was added and incubated for 1 hour at 37℃and PBST was washed 3 times. Finally 50 μl of PBST was added per well.

(4) When the virus and the positive serum react with each other by using an inverted fluorescence microscope, after the fluorescent secondary antibody is added, obvious specific fluorescence appears in the visual field, and a blank control hole is not fluorescent, so that the result is shown in figure 4.

TABLE 3 determination of the viral content at different time of harvest (unit: log10TCID ₅₀ /ml)

From the above results, it can be seen that PRRSV virus can be propagated using macrophages obtained after induction.

The virus inoculation dose will affect the final virus content, with the virus inoculation dose increasing from 0.01MOI to 0.05MOI, the final virus content being from 10 ^6.0 TCID ₅₀ The volume per ml is increased to 10 ^6.7 TCID ₅₀ Per ml, the inoculum size in the invention is selected to be 0.02-0.05 MOI;

the virus collection time of different viruses can directly influence the final virus content, and the trend of increasing and decreasing is presented along with the increase of the culture time, and the optimal virus collection time of the invention is 72 hours.

Experimental example 4 bioreactor process development-2.5L, 5L torrent bioreactor experimental design and experimental method

1. Cell suspension culture in 2.5L and 5L reaction bags

Fresh preparation of bone marrow-derived cells from femur and tibia of pig at 1.0X10 ⁶ Inoculating the culture medium to a 2.5L (5L) reaction bag for culture, wherein the culture volume is 1.0L (2.0L), and adjusting the control parameters of the reactor (the rotating speed is 40r/min, the temperature is 37 ℃, the dissolved oxygen value is 40-60%, the pH value is 7.0-7.2 and the ventilation rate is 50-300 ml/min). On the first day of culture, 10ng/mL of GM-CSF was added as an inducer, and on the fourth day of culture, 10ng/mL of M-CSF was added to reactThe control parameters are unchanged, and the culture is continued until the seventh day.

2. NADC30 virus inoculation, culture and harvest

The cell density in the reaction bag was adjusted to 1.0X10 ⁶ Virus inoculation is carried out within the cell density range of each mL, the inoculation dose is inoculated according to 0.02MOI, the parameters of the reactor are unchanged, and samples are taken 72 hours and 96 hours after inoculation, and virus content detection is carried out.

3. Results of the virus-receiving culture in the reaction bag

The virus content of the cells of the 5L and 50L reaction bags can reach 10 after 72 hours after virus inoculation ^7.0 TCID50/ml, which is significantly higher than the virus content of shake flask propagated viruses, indicates that the current reactor process can be scaled up for production, and the detailed results are shown in Table 4.

TABLE 4 determination of the content of seed viruses in 5L reactor cultures (unit: log10 TCID) ₅₀ /ml)

From the above results, it can be seen that the virus content obtained by 2.5L reactor culture and 5L reactor culture in the virus amplification culture process was 10 ^7.0 TCID _50/ Above mL, the virus content is obviously higher than that of shake flask propagation virus, which indicates that the reactor process can be used for large-scale production.

Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.

Claims

1. A method for inducing bone marrow-derived cells to differentiate into macrophages, which is characterized by comprising the steps of suspending and culturing bone marrow-derived cells by using a cell culture solution, and then adding M-CSF for continuous culture to induce the bone marrow-derived cells to differentiate into macrophages;

the cell culture solution comprises a basal medium, fetal bovine serum, a shear force protective agent, an anti-cell-agglomerating agent and an L929 cell supernatant;

the cell culture solution contains 10% -20% v/v of fetal bovine serum, 0.5% -3 g/L of shear force protective agent, 20% -40 mg/L of anti-cell-agglomerating agent, 5% -50 ng/mL of GM-CSF, 50% -150U/mL of penicillin, 50% -150 mu g/mL of streptomycin, 2% -2.50 mM of L-glutamine, 10% -20 mM of HEPES buffer solution and 10% -30% v/v of L929 cell supernatant;

the shear force protecting agent is Pluronic F-68;

the anti-cell-caking agent is dextran sulfate;

the basal medium is one of DME/F-12 or IMDM;

the continuous culture of the added M-CSF is as follows: and (3) half-changing the cell culture solution on the 3 rd to 5 th days of culture, and adding M-CSF after half-changing the solution.

2. The method of inducing differentiation of bone marrow-derived cells into macrophages according to claim 1, wherein the cell culture broth comprises 10% v/v fetal bovine serum, 1g/L shear force protectant, 30mg/L anti-cell-agglomerating agent, 20ng/mL GM-CSF, 100U/mL penicillin, 100 μg/mL streptomycin, 2.50mM L-glutamine, 15mM HEPES buffer and 20% v/v L929 cell supernatant.

3. The method of inducing differentiation of bone marrow-derived cells into macrophages according to claim 1, wherein the preparation of the cell culture broth comprises: adding the fetal bovine serum, the shear force protective agent, the anti-cell-agglomerating agent, the GM-CSF, the penicillin, the streptomycin, the L-glutamine, the HEPES buffer solution and the L929 cell supernatant into the basic culture medium according to the formula amount, and uniformly mixing to obtain the cell culture solution.

4. The method of inducing differentiation of bone marrow-derived cells into macrophages according to claim 1, wherein the addition of M-CSF continues to culture as follows: half-changing and M-CSF addition were performed on day 3 and day 5 of culture, respectively.

5. The method of inducing differentiation of bone marrow-derived cells into macrophages according to claim 1, wherein the addition of M-CSF continues to culture as follows: half-changing and M-CSF was added on day 4 of culture.

6. The method of claim 1, wherein the amount of M-CSF added in the continuous culture of M-CSF is 5-50 ng/mL.

7. The method for inducing differentiation of bone marrow-derived cells into macrophages according to claim 1, wherein the bone marrow-derived cells have an initial cell density of 1.0 to 2.0 x 10 ⁶ And each mL.

8. The method of inducing differentiation of bone marrow-derived cells into macrophages according to claim 1, wherein the cell culture is performed using a bioreactor;

the parameters of the bioreactor comprise at least one of rotating speed 30-50 r/min, temperature 37 ℃, dissolved oxygen value 40-60%, pH value 7.0-7.2 and ventilation rate 50-300 mL/min.

9. The method of inducing differentiation of bone marrow-derived cells into macrophages according to claim 1, comprising the steps of:

(a) Fresh preparing bone marrow-derived cells from femur and tibia of pig, and re-suspending the cells with cell culture solution according to cell density of 1.0-2.0X10 ⁶ Shake flask culture is carried out per ml;

(b) Cells were placed in a carbon dioxide incubator for 7 days, and culture parameters included: temperature 37 ℃,5% CO ₂ ，>60% RH，100～120 rpm@50mm orbital throw shaker；

(c) After the fourth day of culture, half cell exchange is performed, and then 5-50 ng/mL of M-CSF is added to continue culture.