CN118325837B

CN118325837B - Serum-free and matrix-free cell culture method of neutrophils

Info

Publication number: CN118325837B
Application number: CN202410748773.1A
Authority: CN
Inventors: 刘佳
Original assignee: Shenzhen Zhongjia Biomedical Technology Co ltd
Current assignee: Shenzhen Zhongjia Biomedical Technology Co ltd
Priority date: 2024-06-12
Filing date: 2024-06-12
Publication date: 2024-09-03
Anticipated expiration: 2044-06-12
Also published as: CN118325837A

Abstract

The application discloses a serum-free and matrix-free cell culture method of neutrophils. In a first aspect of the present application, there is provided a serum-free and stromal cell-free culture method of neutrophils, comprising the steps of: providing a cell culture vessel having an inner wall coated with Thiazovivin; neutrophils are placed in a cell culture vessel and cultured in a serum-free medium comprising a basal medium and additives comprising CXCR2 and L-selectin to obtain neutrophils. The proposal selects the Thiazovivin in an adhesion form to replace matrix cells and serum, thereby providing better culture density and proliferation effect for neutrophils. In addition, CXCR2 and L-selectin are additionally added during the culture process, so that the use of serum is further avoided, and the establishment and long-term maintenance of a neutrophil line and clone can be supported.

Description

Serum-free and matrix-free cell culture method of neutrophils

Technical Field

The application relates to the technical field of cell culture, in particular to a serum-free and matrix-free cell culture method of neutrophils.

Background

Neutrophils (Neutrophils) are derived from bone marrow stem cells and proliferate and differentiate into mature neutrophils. The ratio of neutrophils as the most abundant white blood cells in blood is 60-70%. Neutrophils have strong phagocytic, digestive and pathogenic microorganism eliminating abilities and chemotactic effects, and can rapidly pass through vascular endothelial cells to reach an infection site under the stimulation of inflammation, thereby playing the role of phagocytic killing and clearing. In addition, neutrophils play an important role not only in early anti-infective immunity of the body, but also in ADCC action under the participation of antibodies, and participate in adaptive immunity.

Taking leukemia patients as an example, there is a severe neutropenic risk period of about 8 to 12 days after receiving chemotherapy and bone marrow transplantation, requiring rapid onset of action by infusion of neutrophils into the patient, thereby avoiding severe bacterial and fungal infections. And patients with bone marrow loss and patients with fungal or bacterial infections that are unresponsive to antimicrobial treatment also require treatment by external infusion of neutrophils. However, neutrophils are updated rapidly and have a short life span, and the difficulty of achieving efficient expansion in an ex vivo environment is great.

Although solutions exist in the related art, the addition of serum is relied upon to maintain cell growth. Although serum provides growth factors required for proliferation, it contains heterologous animal components, and has undefined components, poor reproducibility of culture, risk of contamination with exogenous viruses and pathogenic factors, and is unsuitable for neutrophil infusion therapy. In addition, stromal cell lines such as OP9 can significantly increase the number of differentiated mature neutrophils, but additional introduced cells also result in subsequent need for further isolation prior to neutrophil use. Therefore, it is necessary to provide a serum-free and stromal-free cell culture method for neutrophils.

Disclosure of Invention

The present application aims to solve at least one of the technical problems existing in the prior art. Therefore, the application provides a serum-free and matrix-free cell culture method of neutrophils.

In a first aspect of the present application, there is provided a serum-free and stromal cell-free culture method of neutrophils, comprising the steps of:

Providing a cell culture vessel having an inner wall coated with Thiazovivin;

Neutrophils are placed in a cell culture vessel and cultured in a serum-free medium comprising a basal medium and additives comprising CXCR2 and L-selectin to obtain neutrophils.

The serum-free and matrix-free cell culture method provided by the embodiment of the application has at least the following beneficial effects:

The proposal selects the Thiazovivin in an adhesion form to replace matrix cells and serum, thereby providing better culture density and proliferation effect for neutrophils. In addition, CXCR2 and L-selectin are additionally added during the culture process, so that the use of serum is further avoided, and the establishment and long-term maintenance of a neutrophil line and clone can be supported.

Unlike undifferentiated totipotent stem cells such as embryonic stem cells, neutrophils are a terminal cell with a higher degree of differentiation, but it is speculated from experimental results that thiazoviin (CAS: 1226056-71-8, chemical formula C ₁₅H₁₃N₅ OS, chinese name N-benzyl 2- (pyrimidin-4-ylamino) thiazole-4-carboxamide or N-benzyl-2- (pyrimidin-4-ylamino) thiazole-4-carboxamide) may be protected from death in an ECM-free environment by E-cadherin-mediated intercellular interactions during the culture of neutrophils by increasing the activity of beta 1 integrin mediated by cell-ECM adhesion, thus promoting survival and proliferation of the neutrophils.

In some embodiments of the application, the concentration of CXCR2 (C-X-C motif chemokine receptor, C-X-C motif chemokine receptor 2) in serum-free medium is 1 to 100 μg/mL and may be, for example 1 μg/mL、2 μg/mL、3 μg/mL、4 μg/mL、5 μg/mL、6 μg/mL、7 μg/mL、8 μg/mL、9 μg/mL、10 μg/mL、20 μg/mL、30 μg/mL、40 μg/mL、50 μg/mL、60 μg/mL、70 μg/mL、80 μg/mL、90 μg/mL、100 μg/mL.

In some embodiments of the application, the concentration of L-selectin in serum-free medium is 1-100 ng/mL, for example, it may be 1 ng/mL、2 ng/mL、3 ng/mL、4 ng/mL、5 ng/mL、6 ng/mL、7 ng/mL、8 ng/mL、9 ng/mL、10 ng/mL、20 ng/mL、30 ng/mL、40 ng/mL、50 ng/mL、60 ng/mL、70 ng/mL、80 ng/mL、90 ng/mL、100 ng/mL.

In some embodiments of the application, the concentration of CXCR2 in serum-free medium is 1 to 100 μg/mL and the concentration of L-selectin is 1 to 100 ng/mL, for example CXCR2 may be present at a concentration of 1 μg/mL、2 μg/mL、3 μg/mL、4 μg/mL、5 μg/mL、6 μg/mL、7 μg/mL、8 μg/mL、9 μg/mL、10 μg/mL、20 μg/mL、30 μg/mL、40 μg/mL、50 μg/mL、60 μg/mL、70 μg/mL、80 μg/mL、90 μg/mL、100 μg/mL,L- selectin 1 ng/mL、2 ng/mL、3 ng/mL、4 ng/mL、5 ng/mL、6 ng/mL、7 ng/mL、8 ng/mL、9 ng/mL、10 ng/mL、20 ng/mL、30 ng/mL、40 ng/mL、50 ng/mL、60 ng/mL、70 ng/mL、80 ng/mL、90 ng/mL、100 ng/mL.

In some embodiments of the application, the basal medium comprises any one of DMEM medium, RPMI 1640 medium, IMDM medium, MEM medium.

In some embodiments of the application, the additive further comprises at least one of a polyamine, an antioxidant, insulin, siderophore, B-27 supplement, a stem cell factor, a granulocyte colony stimulating factor, IL-8, an fms-related tyrosine kinase 3 ligand, a granulocyte-macrophage colony stimulating factor, a thrombopoietin.

In some embodiments of the application, the additive further comprises 1 to 500. Mu.M of a polyamine, such as may be 1 μM、2 μM、3 μM、4 μM、5 μM、6 μM、7 μM、8 μM、9 μM、10 μM、20 μM、30 μM、40 μM、50 μM、60 μM、70 μM、80 μM、90 μM、100 μM、200 μM、300 μM、400 μM、500 μM.

In some embodiments of the present application, the polyamine comprises at least one of putrescine, spermine, spermidine.

In some embodiments of the application, the additive further comprises 1-10 ng/mL of an antioxidant, for example, 1 ng/mL, 2 ng/mL, 3 ng/mL, 4 ng/mL, 5 ng/mL, 6 ng/mL, 7 ng/mL, 8 ng/mL, 9 ng/mL of an antioxidant.

In some embodiments of the application, the antioxidant comprises selenium, which may be provided and metered in selenite form.

In some embodiments of the application, the additive further comprises 1-100 μg/mL insulin, such as 1 μg/mL、2 μg/mL、3 μg/mL、4 μg/mL、5 μg/mL、6 μg/mL、7 μg/mL、8 μg/mL、9 μg/mL、10 μg/mL、20 μg/mL、30 μg/mL、40 μg/mL、50 μg/mL、60 μg/mL、70 μg/mL、80 μg/mL、90 μg/mL、100 μg/mL insulin.

In some embodiments of the application, the additive further comprises a siderophore of 1 to 100 μg/mL, such as a siderophore that may be 1 μg/mL、2 μg/mL、3 μg/mL、4 μg/mL、5 μg/mL、6 μg/mL、7 μg/mL、8 μg/mL、9 μg/mL、10 μg/mL、20 μg/mL、30 μg/mL、40 μg/mL、50 μg/mL、60 μg/mL、70 μg/mL、80 μg/mL、90 μg/mL、100 μg/mL.

In some embodiments of the application, the siderophore comprises transferrin.

In some embodiments of the application, the additive further comprises 1-10 ng/mL selenite, 1-100 μg/mL insulin, and 1-100 μg/mL transferrin.

In some embodiments of the application, the additive further comprises 1-5 v/v% of a B-27 supplement, which may be, for example, 1 v/v%, 2 v/v%, 3 v/v%, 4 v/v%, 5 v/v% of a B-27 supplement.

In some embodiments of the application, the additive further comprises Stem Cell Factor (SCF) of 1-500 ng/mL, such as 1 ng/mL、2 ng/mL、3 ng/mL、4 ng/mL、5 ng/mL、6 ng/mL、7 ng/mL、8 ng/mL、9 ng/mL、10 ng/mL、20 ng/mL、30 ng/mL、40 ng/mL、50 ng/mL、60 ng/mL、70 ng/mL、80 ng/mL、90 ng/mL、100 ng/mL、200 ng/mL、300 ng/mL、400 ng/mL、500 ng/mL.

In some embodiments of the application, the additive further comprises a granulocyte colony stimulating factor (Granulocyte colony stimulating factor, G-CSF) of 1-100 ng/mL, which may be 1 ng/mL、2 ng/mL、3 ng/mL、4 ng/mL、5 ng/mL、6 ng/mL、7 ng/mL、8 ng/mL、9 ng/mL、10 ng/mL、20 ng/mL、30 ng/mL、40 ng/mL、50 ng/mL、60 ng/mL、70 ng/mL、80 ng/mL、90 ng/mL、100 ng/mL, for example.

In some embodiments of the application, the additive further comprises 0.1 to 10 ng/mL IL-8, which may be 0.1 ng/mL、0.2 ng/mL、0.3 ng/mL、0.4 ng/mL、0.5 ng/mL、0.6 ng/mL、0.7 ng/mL、0.8 ng/mL、0.9 ng/mL、1 ng/mL、2 ng/mL、3 ng/mL、4 ng/mL、5 ng/mL、6 ng/mL、7 ng/mL、8 ng/mL、9 ng/mL、10 ng/mL, for example.

In some embodiments of the application, the additive further comprises 1-500 ng/mL of an fms-related tyrosine kinase 3 ligand (Flt-3L), which may be 1 ng/mL、2 ng/mL、3 ng/mL、4 ng/mL、5 ng/mL、6 ng/mL、7 ng/mL、8 ng/mL、9 ng/mL、10 ng/mL、20 ng/mL、30 ng/mL、40 ng/mL、50 ng/mL、60 ng/mL、70 ng/mL、80 ng/mL、90 ng/mL、100 ng/mL、200 ng/mL、300 ng/mL、400 ng/mL、500 ng/mL, for example, an fms-related tyrosine kinase 3 ligand.

In some embodiments of the application, the additive further comprises 1-50 ng/mL granulocyte-macrophage colony-stimulating factor (Granulocyte-macrophage colony stimulating factor, GM-CSF), which may be 1 ng/mL、2 ng/mL、3 ng/mL、4 ng/mL、5 ng/mL、6 ng/mL、7 ng/mL、8 ng/mL、9 ng/mL、10 ng/mL、20 ng/mL、30 ng/mL、40 ng/mL、50 ng/mL, for example.

In some embodiments of the application, the additive further comprises 1-100 ng/mL thrombopoietin (Thrombopoietin, TPO), which may be 1 ng/mL、2 ng/mL、3 ng/mL、4 ng/mL、5 ng/mL、6 ng/mL、7 ng/mL、8 ng/mL、9 ng/mL、10 ng/mL、20 ng/mL、30 ng/mL、40 ng/mL、50 ng/mL、60 ng/mL、70 ng/mL、80 ng/mL、90 ng/mL、100 ng/mL, for example.

In some embodiments of the application, the additive further comprises 1-500. Mu.M polyamine, 1-10 ng/mL antioxidant, 1-100. Mu.g/mL insulin, 1-100. Mu.g/mL siderophore, 1-5 v/v% B-27 supplement, 1-500 ng/mL stem cell factor, 1-100 ng/mL granulocyte colony stimulating factor, 0.1-10 ng/mL IL-8, 1-500 ng/mL fms-associated tyrosine kinase 3 ligand, 1-50 ng/mL granulocyte-macrophage colony stimulating factor, 1-100 ng/mL thrombopoietin.

In some embodiments of the application, the additive further comprises 1-500. Mu.M putrescine, 1-10 ng/mL selenium, 1-100. Mu.g/mL insulin, 1-100. Mu.g/mL transferrin, 1-5 v/v% B-27 supplement, 1-500 ng/mL stem cell factor, 1-100 ng/mL granulocyte colony stimulating factor, 0.1-10 ng/mL IL-8, 1-500 ng/mL fms-associated tyrosine kinase 3 ligand, 1-50 ng/mL granulocyte-macrophage colony stimulating factor, 1-100 ng/mL thrombopoietin.

In some embodiments of the application, the method of coating Thiazovivin comprises the steps of:

Adding a solution of laminin (laminin) into a cell culture container, incubating, washing and drying to obtain a pre-coated cell culture container;

Adding the thiazovin working solution into the pre-coated cell culture container, incubating, washing and drying to obtain the cell culture container with the inner wall coated with the thiazovin.

In some embodiments of the application, the concentration of the laminin solution is 1-100 mg/mL, for example, it may be 1 mg/mL、2 mg/mL、3 mg/mL、4 mg/mL、5 mg/mL、6 mg/mL、7 mg/mL、8 mg/mL、9 mg/mL、10 mg/mL、20 mg/mL、30 mg/mL、40 mg/mL、50 mg/mL、60 mg/mL、70 mg/mL、80 mg/mL、90 mg/mL、100 mg/mL.

In some embodiments of the application, the laminin is coated on the inner wall of the cell culture vessel in an amount of 0.1-10 μg/cm ², for example, it may be 0.1 μg/cm²、0.2 μg/cm²、0.3 μg/cm²、0.4 μg/cm²、0.5 μg/cm²、0.6 μg/cm²、0.7 μg/cm²、0.8 μg/cm²、0.9 μg/cm²、1 μg/cm²、2 μg/cm²、3 μg/cm²、4 μg/cm²、5 μg/cm²、6 μg/cm²、7 μg/cm²、8 μg/cm²、9 μg/cm²、10 μg/cm².

In some embodiments of the application, the solution of laminin is a buffer of laminin, such as any of PBS, HBSS, DPBS, EBSS, etc.

In some embodiments of the application, the incubation time of laminin is 6-24 hours, e.g., 6 h, 8 h, 12 h, 16 h, 20 h, 24 h.

In some embodiments of the application, the incubation temperature of laminin is 4-40 ℃, for example, 4 ℃,10 ℃, 15 ℃, 20 ℃,25 ℃, 30 ℃, 35 ℃, 37 ℃, 40 ℃.

In some embodiments of the application, the wash solution of laminin is any one of buffers, such as PBS, HBSS, DPBS, EBSS, and the like.

In some embodiments of the application, the solvent of the working fluid of Thiazovivin comprises PEG and water.

In some embodiments of the application, PEG comprises PEG400.

In some embodiments of the application, the solvent further comprises a surfactant.

In some embodiments of the application, the surfactant comprises Tween, which may be Tween80, for example.

In some embodiments of the present application, the volume ratio of PEG to water in the working fluid is 1:0.1-10, for example, 1:0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.8, 1,2, 3,4,5, 6,7,8, 9, 10.

In some embodiments of the present application, the concentration of the working fluid of Thiazovivin is 1-100 mg/mL, for example, it may be 1 mg/mL、2 mg/mL、3 mg/mL、4 mg/mL、5 mg/mL、6 mg/mL、7 mg/mL、8 mg/mL、9 mg/mL、10 mg/mL、20 mg/mL、30 mg/mL、40 mg/mL、50 mg/mL、60 mg/mL、70 mg/mL、80 mg/mL、90 mg/mL、100 mg/mL.

In some embodiments of the application, the incubation time of Thiazovivin is 1-10 h, for example 1h, 2h, 3h, 4h, 5 h, 6h, 7 h, 8 h, 9 h, 10 h.

In some embodiments of the present application, the incubation temperature of Thiazovivin is 4-40 ℃, for example, 4 ℃,10 ℃, 15 ℃, 20 ℃,25 ℃,30 ℃, 35 ℃, 37 ℃, 40 ℃.

In some embodiments of the application, the wash solution of Thiazovivin is any one of buffers, such as PBS, HBSS, DPBS, EBSS, and the like.

In some embodiments of the application, the neutrophils are bone marrow-derived or peripheral blood-derived neutrophils.

In some embodiments of the application, the neutrophils are those of mammalian (e.g., monocular, marsupials, food worm, jumping shrew, beast, winged, primates, lean, scaly, lagomorpha, rodentia, carnivorous, beef, hoof-and-rabbit, tube tooth, fanciful, whale, etc.) origin, including specifically those of at least one of rodentia (e.g., mice, rats, mice, guinea pigs), lagomorpha (e.g., rabbits), fanciful (e.g., horses), artiodactyla (e.g., sheep, pigs), primates (e.g., monkeys, gorillas, chimpanzees, humans), carnivores (e.g., dogs), etc.

In some embodiments of the application, the cell culture vessel comprises a cell culture plate (e.g., a 4-well, 6-well, 8-well, 12-well, 24-well, 48-well, 96-well, 384-well, 1536-well plate), a cell culture dish (e.g., a 35 mm, 60 mm, 100 mm, 150 mm dish), a cell culture flask (e.g., a T25, T75, T175, T225 flask), a cell factory (e.g., a 1-layer, 2-layer, 3-layer, 4-layer, 5-layer, 10-layer, 40-layer cell factory), or the like.

In some embodiments of the application, the cell culture vessel is prepared from a material comprising a polymer, such as polystyrene.

In some embodiments of the application, neutrophils are cultured in a serum-free medium in a cell culture vessel for 1 hour or more, 2 hours or more, 4 hours or more, 6 hours or more, 12 hours or more, 16 hours or more, 20 hours or more, 1 day or more, 2 days or more, 3 days or more, 4 days or more, 5 days or more, 6 days or more, 8 days or more, 10 days or more, 12 days or more, 14 days or more, 16 days or more, 18 days or more, 20 days or more, 22 days or more, 24 days or more, 26 days or more, 28 days or more, 30 days or more.

In some embodiments of the application, the neutrophils are cultured in a serum-free medium in a cell culture vessel for 1-30 days, e.g., 1,2,3,4, 5,6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30 days.

In some embodiments of the application, the neutrophils are subcultured in the cell culture vessel with serum-free medium at intervals of 1 to 5 days, for example, at intervals of 1,2, 3,4, 5 days.

In a second aspect of the application, there is provided a kit for serum-free and stromal cell-free culture of neutrophils, the kit comprising:

a cell culture vessel having an inner wall coated with Thiazovivin;

Serum-free medium, including basal medium and additives, including CXCR2 and L-selectin.

In some embodiments of the application, the concentration of CXCR2 in the serum-free medium is 1 to 100 μg/mL.

In some embodiments of the application, the concentration of L-selectin in serum-free medium is 1-100 ng/mL.

In some embodiments of the application, the concentration of CXCR2 in serum-free medium is 1-100 μg/mL and the concentration of L-selectin is 1-100 ng/mL.

In some embodiments of the application, the additive further comprises 1 to 500. Mu.M of a polyamine.

In some embodiments of the application, the additive further comprises 1-10 ng/mL of an antioxidant.

In some embodiments of the application, the additive further comprises 1-100 μg/mL insulin.

In some embodiments of the application, the additive further comprises 1-100 μg/mL siderophores.

In some embodiments of the application, the siderophore comprises transferrin.

In some embodiments of the application, the additive further comprises 1-500 ng/mL stem cell factor.

In some embodiments of the application, the additive further comprises a granulocyte colony stimulating factor of 1-100 ng/mL.

In some embodiments of the application, the additive further comprises 0.1 to 10 ng/mL IL-8.

In some embodiments of the application, the additive further comprises 1-500 ng/mL of fms-related tyrosine kinase 3 ligand.

In some embodiments of the application, the additive further comprises granulocyte-macrophage colony stimulating factor in the range of 1-50 ng/mL.

In some embodiments of the application, the additive further comprises 1-100 ng/mL thrombopoietin.

In some embodiments of the application, thiazovivin is coated on the inner wall of a cell culture vessel by:

In some embodiments of the application, the concentration of the solution of laminin is 1-100 mg/mL.

In some embodiments of the application, the amount of laminin coated on the inner wall of the cell culture vessel is 0.1-10 μg/cm ².

In some embodiments of the application, the incubation time of laminin is 6-24 hours.

In some embodiments of the application, the incubation temperature of laminin is 4-40 ℃.

In some embodiments of the application, PEG comprises PEG400.

In some embodiments of the application, the volume ratio of PEG to water in the working fluid is 1:0.1-10.

In some embodiments of the application, the concentration of the working fluid of Thiazovivin is 1-100 mg/mL.

In some embodiments of the application, the incubation time of Thiazovivin is 1-10 hours.

In some embodiments of the application, the incubation temperature of Thiazovivin is 4-40 ℃.

In a third aspect of the application, there is provided the use of the serum-free and stromal cell-free culture method or kit described above for promoting neutrophil proliferation or for the preparation of a product for promoting neutrophil proliferation.

In some embodiments of the application, the use of the aforementioned serum-free stromal cell-free culture method to promote neutrophil proliferation comprises culturing neutrophils according to the serum-free stromal cell-free culture method.

In some embodiments of the application, the use of the aforementioned kit to promote neutrophil proliferation comprises culturing neutrophils using the kit.

In some embodiments of the application, the use of the aforementioned kit in the preparation of a product that promotes neutrophil proliferation comprises providing the kit in a product that promotes neutrophil proliferation.

In a fourth aspect of the application, there is provided the use of the serum-free and stromal cell-free culture method or kit described above for promoting the differentiation of neutrophils into mature neutrophils or for preparing a product that promotes the differentiation of neutrophils into mature neutrophils.

In some embodiments of the application, the use of the aforementioned serum-free and stromal cell-free culture method to promote differentiation of neutrophils to mature neutrophils comprises culturing neutrophils according to the serum-free and stromal cell-free culture method and then isolating mature neutrophils.

In some embodiments of the application, the use of the aforementioned kit to promote differentiation of neutrophils to mature neutrophils comprises culturing neutrophils using the kit and then isolating mature neutrophils.

In some embodiments of the application, the use of the aforementioned kit in the preparation of a product that promotes differentiation of neutrophils to mature neutrophils comprises providing the kit in a product that promotes differentiation of neutrophils to mature neutrophils.

In a fifth aspect of the application, there is provided a method of differentiating neutrophils into mature neutrophils, the method comprising the steps of:

Providing a cell culture vessel having an inner wall coated with Thiazovivin;

Neutrophils are placed in a cell culture vessel and cultured in a serum-free medium comprising a basal medium and additives comprising CXCR2 and L-selectin, and mature neutrophils are isolated.

In some embodiments of the application, the siderophore comprises transferrin.

In some embodiments of the application, PEG comprises PEG400.

It will be appreciated that the neutrophils differentiated towards mature neutrophils are immature neutrophils, for example, in some embodiments, the neutrophils differentiated towards mature neutrophils may be precursor neutrophils, and in particular may be bone marrow or peripheral blood derived precursor neutrophils.

Additional aspects and advantages of the application will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the application.

Drawings

FIG. 1 is a graph showing the proliferation of neutrophils under different culture conditions, including culture with OP9 stromal cells and serum-containing medium, in accordance with an embodiment of the application; culturing the microplate coating Thiazovivin (Thi) with a serum-containing medium; culturing the microplate coating Thiazovivin (Thi) with a serum-free medium; culturing with Thiazovivin (Thi) -dissolved serum-containing medium.

FIG. 2 is a microscopic examination of neutrophils cultured in different forms according to an embodiment of the application. Wherein, the left side is the result of culturing by coating the Thiazovivin on the culture plate in an adhering form, and the right side is the result of culturing by dissolving the Thiazovivin in a culture medium in a soluble form.

FIG. 3 shows the cell proliferation of Thiazovivin coated in culture flasks at optimal concentration and using serum-free medium supplemented with CXCR2 and L-selectin, and other control groups, in the case of continuous culture of peripheral blood-derived neutrophils for 30 days, in accordance with one embodiment of the present application.

FIG. 4 is a graph showing the effect of bone marrow-derived neutrophils on cell proliferation by adding L-selectin or CXCR2 to serum-free medium under Thiazovivin coating conditions in accordance with one embodiment of the application.

FIG. 5 shows the proliferation of cells in a continuous culture of bone marrow-derived neutrophils in a serum-free medium supplemented with CXCR2 and L-selectin under Thiazovivin-coated conditions, and in a further control group for 30 days, in accordance with an embodiment of the application.

FIG. 6 shows the results of testing neutrophil cloning efficiency using serum-free medium supplemented with CXCR2 and L-selectin and limiting dilution with other control groups under Thiazovivin coating conditions in an embodiment of the application.

FIG. 7 is a graph showing the results of testing the ability of neutrophils to differentiate into mature neutrophils using serum-free medium supplemented with CXCR2 and L-selectin under Thiazovivin coating conditions in one embodiment of the application, as well as other control groups.

Detailed Description

The conception and the technical effects produced by the present application will be clearly and completely described in conjunction with the embodiments below to fully understand the objects, features and effects of the present application. It is apparent that the described embodiments are only some embodiments of the present application, but not all embodiments, and that other embodiments obtained by those skilled in the art without inventive effort are within the scope of the present application based on the embodiments of the present application.

The following detailed description of embodiments of the application is exemplary and is provided merely to illustrate the application and is not to be construed as limiting the application.

In the description of the present application, the meaning of a number is one or more, the meaning of a number is two or more, and greater than, less than, exceeding, etc. are understood to exclude the present number, and the meaning of a number is understood to include the present number. The description of the first and second is for the purpose of distinguishing between technical features only and should not be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated or implicitly indicating the precedence of the technical features indicated.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing embodiments of the application only and is not intended to be limiting of the application.

In the description of the present application, the descriptions of the terms "one embodiment," "some embodiments," "illustrative embodiments," "examples," "specific examples," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present application. In this specification, schematic representations of the above terms do not necessarily refer to the same embodiments or examples. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

Example 1

A Thiazovivin coated microplate was prepared by the following steps:

1. preparing Thiazovivin working solution

Taking 1mL working fluid as an example, the steps are as follows: taking 300 mu L of 100 mg/mL of clarified PEG400 stock solution of Thiazovivin, adding the stock solution into 5 mu L of Tween 80, and uniformly mixing to clarify the stock solution; continuously adding 50 mu L of propylene glycol into the mixture, and uniformly mixing to clarify the mixture; then 645. Mu.L of ddH ₂ O was added and the volume was set to 1mL to give 30. 30 mg/mL (96.35 mM) of Thiazovivin working solution. Stored in a-20 ℃ refrigerator and diluted to the required concentration with ddH ₂ O before use.

2. Microplate pre-coated laminin

The laminin is diluted by a sterile HBSS balanced salt solution, then a proper amount of the laminin is dripped into a 96-well plate, after overnight incubation at 37 ℃, the excessive solution is sucked away and washed twice by PBS, and the laminin is dried in the air, so that the 96-well plate pre-coated with the laminin is obtained, and the coating amount of the laminin is about 1 mug/cm ².

3. Microwell plate coated Thiazovivin

Thiazovivin working solutions of different concentrations were added dropwise to 96-well plates pre-coated with laminin at 50 μl/well, incubated at room temperature for 2h, washed 3 times with 200 μl PBS/0.05% tween-20, then 100 μl PBS solution was added, and incubated at 4deg.C for 2 h. After washing 5 times with PBS/0.05% tween-20, 100. Mu.L of 1 XTMB solution was added and the absorbance at 450 nm was measured using a microplate reader.

Example 2

1. Preparing culture medium

Serum-containing medium and serum-free medium were prepared separately according to the following compositions.

Serum-containing medium: IMDM (including 4 mM L-glutamine, 3024 mg/L NaHCO ₃, 4500 mg/L D-glucose, 1mM sodium pyruvate) was used as a base solution, and 0.036 mM NaHCO ₃, 50. Mu.M beta-mercaptoethanol, 0.03 wt% peptone, 1X kanamycin, 1X nonessential amino acids, 1 ng/mL IL-8, 2% (v/v) Fetal Bovine Serum (FBS), 10. Mu.g/mL human transferrin, 0.005% fatty acid supplement were supplemented on a final concentration basis.

Serum-free medium: IMDM (including 4mM L-glutamine, 3024 mg/L NaHCO ₃, 4500 mg/L D-glucose, 1mM sodium pyruvate) was used as a base solution, and 100. Mu.M putrescine, 5 ng/mL selenium, 25. Mu.g/mL insulin, 50. Mu.g/mL transferrin, 2% (v/v) B-27 supplement, 100 ng/mL Stem Cell Factor (SCF), 50 ng/mL granulocyte colony-stimulating factor (G-CSF), 1 ng/mL IL-8, 100 ng/mL fms-associated tyrosine kinase 3 ligand (Flt-3L), 15 ng/mL granulocyte-macrophage colony-stimulating factor (GM-CSF), 20 ng/mL Thrombopoietin (TPO) were supplemented at final concentrations.

2. Obtaining neutrophils

Neutrophils were isolated from heparinized venous blood after informed consent was obtained from 5 healthy adults. The specific process comprises the following steps: heparin venous blood 10mL is taken, 2.5 mL of 6% dextrose tincture physiological saline is added, the mixture is uniformly mixed, the mixture is kept stand in a constant temperature cabinet at 37 ℃ for 30min, and the upper layer is taken according to the following formula 1:1 to the top of the Ficoll separating liquid, 500 rpm to centrifuge 30 to min, discarding the supernatant, adding 0.155M NH ₄ C1 solution to precipitate to re-suspend and lyse the red blood cells, and washing the Hanks equilibrium liquid for 3 times to obtain the neutrophils. Neutrophils were then resuspended in the two media described above, respectively, to give the corresponding cell suspensions.

3. Inoculating culture

The cell suspension was seeded into 96-well plates at a density of 1X 10 ⁴ cells/mL, 200. Mu.L per well.

Wherein, when the culture medium containing serum is added for culture, OP9 cells are precoated on a microplate according to 1X 10 ⁴ cells/cm ² in advance, and are used as matrix cells for culture, and the total amount of the matrix cells is irradiated with 30Gy of X-rays.

When a serum-free medium is added for culture, the Thiazovivin is coated on a laminin-coated pretreated microplate at different concentrations of 1 ng-10 mug/mL in advance in reference example 1, incubated for 2 hours at room temperature and washed 3 times with PBS. In addition, a control of non-adherent thiazovin was provided, and soluble thiazovin was added directly to the cell suspension in serum-free medium at the same concentration as adherent thiazovin.

4. Passage of

Cells were passaged every 3 or 4 days into another well pre-coated with OP9 cells or Thiazovivin, and cell proliferation was assessed by counting the number of propidium iodide negative living cells by flow cytometry analysis. Data analysis was performed using FlowJo.

In this example, microplates were coated with varying concentrations of thiazovin and laminin to simulate the stromal cell coating normally present on the surface of microplates, and neutrophil line proliferation was tested, and as shown in fig. 1, it can be seen that the adhesion of thiazovin to the laminin-coated plates, together with IL-8, stimulated long-term proliferation of cells in a dose-dependent manner. When Thiazovivin is coated at a concentration of 1 μg/ml and cultured in a serum-containing medium without stromal cells, the proliferation rate of neutrophils is maximized and the proliferation efficiency is almost the same as that of stromal cells cultured in the serum-containing medium; while Thiazovivin has relatively less stimulating effect at lower or higher concentrations. In addition, thiazovivin does not stimulate proliferation of neutrophils when cultured in a soluble form. FIG. 2 is a microscopic observation showing that adherent Thiazovin stimulated neutrophils in growth to adhere to the bottom of the microplate, similar to stromal cells, while cells float in the medium when Thiazovin is cultured in soluble, non-adherent form. These results indicate that adherent Thiazovivin binds to receptors on neutrophils and cross-links these receptors to stimulate neutrophil proliferation. While too low or too high a concentration is detrimental to neutrophil stimulation, cell receptor cross-linking provides the greatest proliferation stimulation in the best mode at the optimal concentration. Thus, adherent Thiazovivin may be employed as a replacement for stromal cells to promote neutrophil survival and proliferation in ECM-free environments.

Example 3

With reference to example 2, neutrophils were cultured at a density of 1X 10 ⁴/mL in a culture flask pre-coated with Thiazovivin at a concentration of 1. Mu.g/mL using a serum-free medium containing IL-8, while L-selectin at a final concentration of 50 ng/mL and CXCR2 at a concentration of 1. Mu.g/mL were additionally added to the medium. And reference was made to a control set without the addition of L-selectin and CXCR2, while serum-containing medium was used.

As a result of the culture, as shown in fig. 3, it was found that, on day 26, the proliferation rate of neutrophils cultured in the serum-free medium was half that of neutrophils cultured in the serum-containing medium under the condition that thiazoviin was coated in an adherent manner, and the proliferation rate was 3 to 4 times that of neutrophils cultured in the serum-containing medium after adding L-selectin and CXCR2 to the serum-free medium.

Example 4

1. Acquisition of bone marrow derived precursor neutrophils

Several C57BL/6 mice of 8 to 12 weeks old were sacrificed by cervical dislocation, femur was isolated, bone marrow cells were collected by repeated rinsing with 5 mL of RPMI 1640 complete medium (containing 1% penicillin-streptomycin, 10% FBS and 2mM EDTA), 10 min was treated with erythrocyte lysate, then filtered through a 70 μm cell filter, the supernatant was centrifuged off, and the RPMI 1640 complete medium was added for resuspension. Flow cytometry was performed to collect bone marrow derived precursor neutrophils (CD 45 ⁺CD11b⁺Ly6G^-CD115^-SSC^{High height}).

2. Culture of bone marrow derived precursor neutrophils

The effect of culturing for 4 days with the serum-free medium containing IL-8 of example 2 additionally added with 50 ng/mL of L-selectin and 1 μg/mL of CXCR2 and pre-coated with Thiazovivin as culture conditions, the collected bone marrow-derived precursor neutrophils were cultured and negative controls of L-selectin and CXCR2 were set, and the control of the manner of culturing with the serum-containing medium of example 2 was set, as shown in fig. 4, it was seen that the proliferation efficiency of the bone marrow-derived precursor neutrophils was not significantly different from that of the cells without addition of either L-selectin or CXCR2, but after the combination of L-selectin and CXCR2 was added, the proliferation efficiency of the cells was significantly improved and not significantly different from that of the cells cultured with the serum-containing medium of example 2.

The results of the long-term culture are shown in FIG. 5, and cell lines employing bone marrow-derived precursor neutrophils can be established and maintained for long periods of time in serum-free medium containing IL-8, CXCR2 and L-selectin, and adherent to Thiazovin, but not in any serum-containing medium.

Example 5

Cloning efficiency of human peripheral blood-derived neutrophils was tested by limiting dilution under adherent Thiazovivin stimulation, serum-free medium containing IL-8, L-selectin, CXCR2, stroma-free culture conditions.

The specific process of limiting dilution method is as follows: serum-free medium containing neutrophils was diluted at a cell density of 200 cells/mL, and after dilution, 0.1 mL of 4 replicates were added to each well of a 96-well plate (pre-coated Thiazovivin), 20 per well was inoculated in a 3-fold ratio dilution, followed by sequential inoculation to obtain 2-row well plates of 6.6, 2.2, 0.7 cells per well. After 7 days of culture, the microscope scores the constituent neutrophil colonies and calculates the frequency of neutrophils from poisson distribution.

As a result, as shown in FIG. 6, the cloning efficiency of neutrophils adhering to Thiazovin was 1/6 in serum-free medium in the presence of CXCR2 and L-selectin, and 1/5 in serum-containing medium of stromal cells, i.e., 1 in 5 cells, both of which were almost the same. Whereas under serum-containing culture conditions without CXCR2 and L-selectin, the efficiency of cloning was reduced by a factor of 2 (1/13), indicating the importance of both additives. Whereas, when Thiazovivin is added to the medium in a non-adherent, soluble form to participate in the culture, the cloning efficiency of neutrophils is much lower, with only 1 out of less than 100 cells.

Example 6

Neutrophils were incubated in serum-free medium pre-coated with Thiazovivin for 5 days using the methods described above, labeled with antibodies, with APC-Cy7 labeled with anti-CD 45 antibody, PE-Cy5 labeled with anti-CD 11b antibody, FITC labeled with anti-CD 115 antibody, mixed on ice, incubated in the dark for 30 min, washed off excess cell surface staining antibodies using DPBS, up-flow cells stained with APC-Cy7 labeled with anti-CD 3 antibody and PE-Cy5 labeled with anti-CD 56 antibody, and subjected to expression analysis on ice for 15 min, and flow cytometry detected differentiated mature neutrophil phenotype cd45+cd11b+cd115-, with the results shown in fig. 7, the added CXCR2 and L-selectin significantly increased the differentiation frequency, as indicated by the culture with OP9 stromal cells in serum-containing medium.

The present application has been described in detail with reference to the embodiments, but the present application is not limited to the embodiments, and various changes can be made within the knowledge of those skilled in the art without departing from the spirit of the present application. Furthermore, embodiments of the application and features of the embodiments may be combined with each other without conflict.

Claims

1. A serum-free and stromal-free cell culture method for neutrophils, comprising the steps of:

Providing a cell culture vessel having an inner wall coated with Thiazovivin;

placing the neutrophils into the cell culture vessel, and culturing in a serum-free medium, wherein the serum-free medium comprises a basal medium and an additive, the additive comprises CXCR2 and L-selectin, and the additive further comprises IL-8.

2. The serum-free and stromal cell-free culture method of claim 1, wherein the concentration of CXCR2 in the serum-free medium is 1-100 μg/mL and the concentration of L-selectin is 1-100 ng/mL.

3. The serum-free and stromal cell-free culture method of claim 1, wherein the basal medium comprises any one of DMEM medium, RPMI 1640 medium, IMDM medium, MEM medium.

4. The serum-free and stromal cell-free culture method of claim 3, wherein the additive further comprises at least one of 1-500. Mu.M polyamine, 1-10 ng/mL antioxidant, 1-100. Mu.g/mL insulin, 1-100. Mu.g/mL siderophore, 1-5 v/v% B-27 supplement, 1-500 ng/mL stem cell factor, 1-100 ng/mL granulocyte colony stimulating factor, 0.1-10 ng/mL IL-8, 1-500 ng/mL fms-associated tyrosine kinase 3 ligand, 1-50 ng/mL granulocyte-macrophage colony stimulating factor, 1-100 ng/mL thrombopoietin.

5. The serum-free and stromal cell-free culture method of claim 1, characterized in that the method of coating Thiazovivin comprises the steps of:

adding a solution of laminin into a cell culture container, incubating, and washing to obtain a pre-coated cell culture container;

and adding the Thiazovivin working solution into the pre-coated cell culture container, incubating and washing to obtain the cell culture container with the inner wall coated with the Thiazovivin.

6. The serum-free stromal cell-free culture method of claim 1, wherein the neutrophils are bone marrow-derived or peripheral blood-derived neutrophils.

7. A kit for serum-free and stromal cell-free culture of neutrophils, comprising:

A cell culture vessel having an inner wall coated with Thiazovivin;

Serum-free medium comprising a basal medium and an additive comprising CXCR2 and L-selectin, said additive further comprising IL-8.

8. Use of the serum-free and stromal cell-free culture method of any one of claims 1-6 or the kit of claim 7 for promoting neutrophil proliferation or for the preparation of a product that promotes neutrophil proliferation.

9. Use of the serum-free and stromal cell-free culture method of any one of claims 1 to 6 or the kit of claim 7 for promoting differentiation of neutrophils into mature neutrophils or for the preparation of a product promoting differentiation of neutrophils into mature neutrophils.

10. A method for differentiating neutrophils into mature neutrophils, comprising the steps of:

Providing a cell culture vessel having an inner wall coated with Thiazovivin;

Placing the neutrophils into the cell culture container, culturing in a serum-free medium, and separating the mature neutrophils, wherein the serum-free medium comprises a basal medium and an additive, the additive comprises CXCR2 and L-selectin, and the additive further comprises IL-8.