Culture method and composition for synergistically prolonging in-vitro survival time of neutrophil

Abstract

The invention provides a culture method and a composition for synergistically prolonging the in-vitro survival time of neutrophil. The composition provided by the invention comprises prednisolone and beta-mercaptoethanol, and the prednisolone and the beta-mercaptoethanol can synergistically prolong the survival time of neutrophile granulocytes. The method provided by the invention is low in cost, simple and convenient to operate and small in interference on cell functions, can ensure an experimental window of 3-4 days, and has a good application value.

Description

Technical Field

[0001] The present invention relates to the field of in vitro cell culture, and in particular to a culture method and composition for synergistically prolonging the survival time of neutrophils in vitro. Background Art

[0002] The development and maturation of neutrophils is a delicate and complex process. In the bone marrow, a hematopoietic organ, granulocyte precursor cells undergo a series of differentiation stages and eventually transform into mature neutrophils. This maturation process can be divided into five key stages: first, the primitive granulocyte stage, which is characterized by a larger nucleus; followed by the promyelocyte stage, when the nucleus begins to lobate; then the myelocyte stage, with the nucleus taking a horseshoe shape; then the metamyelocyte stage, with the degree of lobation of the nucleus further increased; and finally the band nucleus and polymorphonuclear granulocyte stage, where the nuclear morphology transforms into a rod or polymorphonuclear nucleus to lay the foundation for the formation of mature polymorphonuclear neutrophils. During these consecutive stages, the morphology and function of neutrophils gradually evolve to adapt to their final physiological function. As an active hematopoietic site, the bone marrow can produce up to 2×10 11 neutrophils, which then undergo a maturation process.

[0003] Neutrophils are a classic type of immune cell, generally considered to have a short lifespan. They circulate in the blood for approximately 18.5 hours and survive for only a few hours outside the body. Their inability to be stored outside the body significantly limits in vitro research on neutrophils. In theory, infusion of neutrophils into the body could help eliminate bacteria and mitigate organ damage caused by infection. Improving or prolonging the survival time of neutrophils in vitro holds great promise for clinical and scientific applications.

[0004] Existing methods for extending neutrophil lifespan have limitations. Currently, commonly used methods include the addition of cytokines (such as G-CSF and GM-CSF) or the use of glucocorticoids (such as dexamethasone) to extend neutrophil lifespan in vitro. However, these methods have the following disadvantages: ① High cost: Cytokines are expensive, increasing experimental costs; ② Potential effects on cell function: Some methods may alter the functional state of neutrophils, affecting the accuracy of experimental results. For example, slightly higher doses of dexamethasone can inhibit chemotaxis and NET formation; ③ Limited efficacy: The lifespan extension effect is limited, making it difficult to meet the requirements of long-term experiments; ④ Complex operation: The recently proposed CLON-G (caspase + lysosome + oxidation + necrosis + G-CSF) method requires the addition of ≥5 reagents at a time, which has a high implementation threshold. Furthermore, it affects multiple metabolic pathways, hindering subsequent experiments. Currently, there is no simple, low-cost method that can significantly extend neutrophil lifespan in vitro, which limits the development of related research.

[0005] Therefore, if a method that is simple to operate, low-cost, and efficient can be found to prolong the survival time of neutrophils in vitro, it will be of great significance for the biological research of neutrophils and their clinical application. Summary of the Invention

[0006] In view of this, in order to make up for the deficiencies of the prior art, the present invention is proposed.

[0007] A first aspect of the present invention provides a composition for preserving neutrophils / prolonging the survival time of neutrophils, wherein the composition comprises prednisolone and β-mercaptoethanol.

[0008] In the present invention, the neutrophils may be purified neutrophils, or may be a cell population containing other cells and neutrophils.

[0009] In the present invention, the prolonging of neutrophil survival time includes increasing the survival time of neutrophils by at least 1%, 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%.

[0010] In some embodiments, the extended neutrophil survival time comprises increasing the viability or function of the treated neutrophils by at least 1%, 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100% compared to untreated neutrophils over the same time period.

[0011] In some embodiments, the molar ratio of prednisolone to β-mercaptoethanol is (0.1-10):(20-100).

[0012] In a specific embodiment, the molar ratio of prednisolone to β-mercaptoethanol is 1:5.

[0013] In some embodiments, the molar concentration of prednisolone is 0.1-10 μM.

[0014] In a specific embodiment, the molar concentration of prednisolone is 10 μM.

[0015] In some embodiments, the molar concentration of β-mercaptoethanol is 20–100 μM.

[0016] In a specific embodiment, the molar concentration of β-mercaptoethanol is 50 μM.

[0017] In some embodiments, the composition further comprises a pharmaceutically acceptable carrier and / or excipient.

[0018] In the present invention, the pharmaceutically acceptable carriers and / or excipients include but are not limited to diluents, binders, surfactants, wetting agents, adsorption carriers, lubricants and / or disintegrants. Among them, diluents include but are not limited to lactose, sodium chloride, glucose, urea, starch, and water; binders include but are not limited to starch, pregelatinized starch, dextrin, maltodextrin, sucrose, gum arabic, gelatin, methylcellulose, carboxymethylcellulose, ethylcellulose, polyvinyl alcohol, polyethylene glycol, polyvinyl pyrrolidone, alginic acid and alginates, xanthan gum, hydroxypropyl cellulose and hydroxypropyl methylcellulose; surfactants include but are not limited to polyoxyethylene sorbitan fatty acid esters, sodium lauryl sulfate, monoglyceride of stearic acid, and cetyl alcohol; wetting agents include but are not limited to glycerol and starch; adsorption carriers include but are not limited to starch, lactose, bentonite, silica gel, kaolin, and bentonite; lubricants include but are not limited to zinc stearate, glyceride monostearate, polyethylene glycol, talc, calcium and magnesium stearate, polyethylene glycol, boric acid powder, hydrogenated vegetable oil, sodium stearyl fumarate, polyoxyethylene monostearate, monolauric sucrose ester, sodium lauryl sulfate, magnesium lauryl sulfate, and magnesium lauryl sulfate.

[0019] A second aspect of the present invention provides a culture medium for preserving neutrophils / prolonging the survival time of neutrophils, wherein the culture medium comprises a basal culture medium, prednisolone and β-mercaptoethanol.

[0020] In some embodiments, the molar ratio of prednisolone to β-mercaptoethanol is (0.1-10):(20-100).

[0021] In some embodiments, the molar ratio of prednisolone to β-mercaptoethanol is 1:5.

[0022] In some embodiments, the molar concentration of prednisolone is 0.1-10 μM.

[0023] In a specific embodiment, the molar concentration of prednisolone is 10 μM.

[0024] In some embodiments, the molar concentration of β-mercaptoethanol is 20–100 μM.

[0025] In a specific embodiment, the molar concentration of β-mercaptoethanol is 50 μM.

[0026] In some embodiments, the basal culture medium includes but is not limited to RPMI-1640, IMDM, or DMEM.

[0027] In a specific embodiment, the basal culture medium is selected from RPMI-1640.

[0028] In some embodiments, the culture medium further comprises serum, which can be of human or non-human origin.

[0029] Serum from non-human sources includes, but is not limited to, fetal bovine serum, calf serum, adult bovine serum, horse serum, sheep serum, pig serum, rabbit serum, chicken serum, mouse serum, and rat serum.

[0030] In a specific embodiment, the serum is preferably human serum.

[0031] In some embodiments, the mass concentration of the serum is 5-20%.

[0032] In a specific embodiment, the mass concentration of the serum is 10%.

[0033] In some embodiments, the culture medium further comprises growth factors, hormones, adhesion factors, carrier proteins, cytokines, antibiotics, and buffers.

[0034] In some embodiments, the cytokines include but are not limited to granulocyte colony stimulating factor, granulocyte-macrophage colony stimulating factor.

[0035] In some embodiments, the antibiotics include but are not limited to penicillin and streptomycin.

[0036] In some embodiments, the buffer includes but is not limited to phosphate buffered saline, Hank's balanced salt solution.

[0037] The third aspect of the present invention provides a method for preserving neutrophils / prolonging the survival time of neutrophils, which comprises administering the composition of the first aspect of the present invention or the culture medium of the second aspect of the present invention to culture neutrophils.

[0038] In some embodiments, the density of the neutrophils is 5×10 5 -20×10 5 cells / mL.

[0039] In a specific embodiment, the density of the neutrophils is 11×10 5 cells / mL.

[0040] In some embodiments, the culture condition is 30-40°C.

[0041] In a specific embodiment, the culture condition is 37°C.

[0042] In some embodiments, the culture conditions are 1-30% CO2.

[0043] In a specific embodiment, the culture condition is 5-21% CO2.

[0044] In some embodiments, the culture condition is 21% O2.

[0045] In some embodiments, the method further comprises maintaining the molar concentration of β-mercaptoethanol at 50 μM.

[0046] In a specific embodiment, the method for maintaining the molar concentration of β-mercaptoethanol at 50 μM comprises adding β-mercaptoethanol once every 12-36 hours.

[0047] In a specific embodiment, the method for maintaining the molar concentration of β-mercaptoethanol at 50 μM comprises adding β-mercaptoethanol once every 24 hours.

[0048] In some embodiments, the method further comprises a step of isolating and purifying neutrophils.

[0049] In some embodiments, the separation and purification step comprises density gradient centrifugation, lysis of red blood cells, and washing.

[0050] In some embodiments, the density gradient centrifugation is performed using Ficoll-Paque™ PLUS / Lymphoprep™ or polysucrose-diatrizoate dedicated layering solution.

[0051] In some embodiments, the polysucrose-diatrizoate dedicated layering fluid includes but is not limited to Polymorphprep™, Histopaque®-1077 / 1119 combination, and Percoll®.

[0052] In some embodiments, the density gradient centrifugation is selected from Ficoll-Paque™ PLUS / Lymphoprep™.

[0053] In some embodiments, the lysing of red blood cells is performed using a red blood cell lysing solution.

[0054] In some embodiments, the red blood cell lysis solution includes but is not limited to sodium chloride and ammonium chloride.

[0055] In a specific embodiment, the red blood cell lysis solution is selected from ammonium chloride.

[0056] In some embodiments, the washing is performed using a cell washing solution.

[0057] In some embodiments, the washing includes but is not limited to PBS, HBSS.

[0058] In some embodiments, the wash is selected from PBS.

[0059] In some embodiments, neutrophils are isolated from peripheral blood.

[0060] In some embodiments, the peripheral blood is derived from a human or a non-human mammal.

[0061] In the present invention, non-human mammals refer to all members of the class Mammalia except humans, such as domesticated livestock (e.g., cattle, horses, pigs), pets (e.g., dogs, cats), or rodents. The term "rodent" refers to any and all members of the phylogenetic class Rodentia (e.g., mice, rats, squirrels, beavers, woodchucks, gophers, voles, groundhogs, hamsters, guinea pigs, and agouti), including any descendants derived therefrom.

[0062] In some embodiments, the cell culture container used in the culture of neutrophils is not particularly limited as long as it can culture neutrophils, and includes but is not limited to bottles, tissue culture bottles, dishes, petri dishes, tissue culture dishes, multi-dishes, microplates, microwell plates, multi-plates, multi-well plates, microscope slides, chamber slides, culture dishes, tubes, trays, culture bags, or roller bottles.

[0063] The cell culture vessel may be either cell-adhesive or cell-non-adhesive, and may be selected appropriately depending on the intended purpose. A cell-adhesive cell culture vessel may be coated with any cell-supporting matrix, such as an extracellular matrix (ECM), to enhance the adhesion of the cell culture vessel surface to the cells. The cell-supporting matrix may be any substance intended to promote neutrophil adhesion.

[0064] The fourth aspect of the present invention provides use of the composition described in the first aspect of the present invention or the culture medium described in the second aspect of the present invention in preserving neutrophils / prolonging the survival time of neutrophils.

[0065] The fifth aspect of the present invention provides a neutrophil, wherein the neutrophil is preserved by the method described in the third aspect of the present invention.

[0066] The sixth aspect of the present invention provides a cell population, which includes the neutrophils described in the fifth aspect of the present invention.

[0067] The seventh aspect of the present invention provides a pharmaceutical composition, which comprises the neutrophils described in the fifth aspect of the present invention or the cell population described in the sixth aspect of the present invention.

[0068] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or excipient.

[0069] In the present invention, the pharmaceutically acceptable carriers and / or excipients include but are not limited to diluents, binders, surfactants, wetting agents, adsorption carriers, lubricants and / or disintegrants. Among them, diluents include but are not limited to lactose, sodium chloride, glucose, urea, starch, and water; binders include but are not limited to starch, pregelatinized starch, dextrin, maltodextrin, sucrose, gum arabic, gelatin, methylcellulose, carboxymethylcellulose, ethylcellulose, polyvinyl alcohol, polyethylene glycol, polyvinyl pyrrolidone, alginic acid and alginates, xanthan gum, hydroxypropyl cellulose and hydroxypropyl methylcellulose; surfactants include but are not limited to polyoxyethylene sorbitan fatty acid esters, sodium lauryl sulfate, monoglyceride of stearic acid, and cetyl alcohol; wetting agents include but are not limited to glycerol and starch; adsorption carriers include but are not limited to starch, lactose, bentonite, silica gel, kaolin, and bentonite; lubricants include but are not limited to zinc stearate, glyceride monostearate, polyethylene glycol, talc, calcium and magnesium stearate, polyethylene glycol, boric acid powder, hydrogenated vegetable oil, sodium stearyl fumarate, polyoxyethylene monostearate, monolauric sucrose ester, sodium lauryl sulfate, magnesium lauryl sulfate, and magnesium lauryl sulfate.

[0070] The eighth aspect of the present invention provides the use of the neutrophils described in the fifth aspect of the present invention, the cell population described in the sixth aspect of the present invention, or the pharmaceutical composition described in the seventh aspect of the present invention in the preparation of a pharmaceutical composition for treating, preventing / treating diseases, wherein the diseases include neutropenia-related diseases or conditions and autoimmune diseases.

[0071] In some embodiments, the neutropenia-associated diseases or disorders include, but are not limited to, myeloablative hypoplasia, viral infection, typhoid fever, paratyphoid fever, certain parasitic diseases, hyperthyroidism, and hypersplenism.

[0072] In some embodiments, the autoimmune disease includes but is not limited to systemic lupus erythematosus, rheumatoid arthritis, Sjögren's syndrome, systemic vasculitis, and systemic sclerosis.

[0073] A ninth aspect of the present invention provides a system, device or apparatus for preserving neutrophils / extending neutrophil survival time, wherein the system, device or apparatus performs the method described in the third aspect of the present invention when in operation.

[0074] In some embodiments, the system, device, or apparatus comprises:

[0075] Mixed unit: Neutrophils are mixed with prednisolone and beta-mercaptoethanol.

[0076] Culture unit: Cultivate the mixed cells.

[0077] The tenth aspect of the present invention provides any one of the following applications:

[0078] (1) Use of the composition of the first aspect of the present invention or the culture medium of the second aspect of the present invention in preparing a system, device or apparatus for preserving neutrophils / extending the survival time of neutrophils;

[0079] (2) Use of the system, device or apparatus described in the ninth aspect of the present invention for preserving neutrophils / extending neutrophil survival time.

[0080] The present invention has the following advantages and beneficial effects:

[0081] The present invention provides a culture method and composition for synergistically prolonging the survival time of neutrophils in vitro. The composition provided herein comprises prednisolone and β-mercaptoethanol, which synergistically prolong the survival time of neutrophils. The method provided herein is low-cost, simple to operate, minimally interferes with cell function, and maintains a 3-4 day experimental window, thus possessing excellent application value.

[0082] Extending the experimental window: Under normal culture conditions, the survival time of neutrophils (PMNs) is increased from 24 hours to ≥96 hours, solving the pain point of experimental interruption due to rapid apoptosis.

[0083] Biaxial synergy: King's formula was used to calculate the Q value after combination, and it was found that the Q values ​​were ≥1.15 at 24 h, 60 h, and 96 h, proving that Pred and β-ME played a synergistic role in prolonging the survival time of neutrophils in vitro.

[0084] Functionality is fully preserved: does not inhibit NETosis or ferroptosis induction, making it suitable for multi-pathway mechanism studies.

[0085] Low-cost and reproducible: All raw materials are commercially available and do not require a hypoxia chamber, recombinant proteins, or closed storage bags; the total cost for 24 wells is less than 0.2 USD, and cross-donor reproducibility is excellent. BRIEF DESCRIPTION OF THE DRAWINGS

[0086] Figure 1 Figure 24 is the result of neutrophil viability. Figure A is the result of neutrophil viability in each experimental group. Figure B is the result of neutrophil viability after different concentrations of Pred were combined with 50 μM β-ME.

[0087] Figure 2Figure 60 shows the viability of neutrophils. Figure A shows the viability of neutrophils in each experimental group. Figure B shows the viability of neutrophils after different concentrations of Pred were combined with 50 μM β-ME.

[0088] Figure 3 Figure 1 is the viability result of neutrophils at 96 hours. Figure A is the viability result of neutrophils in each experimental group. Figure B is the viability result of neutrophils after different concentrations of Pred are combined with 50 μM β-ME. DETAILED DESCRIPTION

[0089] The present invention will be further described below with reference to the following embodiments. The following description is merely a preferred embodiment of the present invention and does not limit the present invention in any other form. Any person skilled in the art may utilize the above disclosed technical content to make equivalent embodiments with equivalent variations. Any simple modification or equivalent variation of the following embodiments made in accordance with the technical essence of the present invention without departing from the content of the present invention shall fall within the scope of protection of the present invention.

[0090] Example 1

[0091] 1. Experimental Materials

[0092] The experimental materials are shown in Table 1:

[0093] Table 1 Experimental materials

[0094]

[0095] 2. Experimental methods

[0096] ① Neutrophil separation

[0097] Ficoll density gradient centrifugation → ammonium chloride lysis → PBS washing.

[0098] ② Group inoculation (24-well plate, 5 × 10 5 cells / well, 450µL / well). The drugs and dosages used in the groups are shown in Table 2:

[0099] Table 2 Drugs and dosages used in different groups

[0100]

[0101] ③ Culture conditions: as shown in Table 3

[0102] Table 3 Culture conditions

[0103]

[0104] ④Detection time points and indicators:

[0105] At 24 h, 60 h, and 96 h, the survival rate was detected by Annexin V-FITC / PI flow cytometry.

[0106] 3. Experimental results: The experimental results are shown in Table 4. Figure 1-Figure 3 As shown:

[0107] Table 4 Experimental results

[0108]

[0109] All three volunteers (HC1-HC3) showed the same trend; at 96 hours, the combined group showed a 211% improvement over the control group (ANOVA + Tukey P < 0.05). Q values ​​calculated using King's formula after the combined group were ≥1.15 at 24 hours, 60 hours, and 96 hours, demonstrating that Pred and β-ME exert a synergistic effect in prolonging neutrophil survival in vitro.

[0110] The present invention has many advantages compared with the prior art. The comparison results are shown in Table 5, Table 6, Table 7 and Table 8.

[0111] Table 5 Advantages of the present invention compared with the prior art

[0112]

[0113] Table 6 Advantages of the present invention compared with the prior art

[0114]

[0115] Experimental data proves that at the same cost and simplicity, the Pred + β-ME system extends the "experimental lifespan" of neutrophils from less than 3 days to more than 4 days, which is difficult to achieve with any existing single factor or protein factor solution.

[0116] Table 7 Disadvantages of the prior art compared with this application

[0117]

[0118] Table 8 Disadvantages of the prior art compared with this application

[0119]

[0120] The above embodiments are only provided for understanding the method and core concept of the present invention. It should be noted that, without departing from the principles of the present invention, a number of improvements and modifications may be made to the present invention by a person skilled in the art, and such improvements and modifications shall fall within the scope of protection of the claims of the present invention.

Claims

1. A composition for preserving neutrophils / extending neutrophil survival time, characterized in that: The composition includes prednisolone and beta-mercaptoethanol; Preferably, the molar ratio of prednisolone to β-mercaptoethanol is (0.1-10): (20-100); Preferably, the molar ratio of prednisolone to β-mercaptoethanol is 1:

5.

2. A culture medium for preserving neutrophils / prolonging the survival time of neutrophils, characterized in that: The culture medium comprises a basal culture medium, prednisolone and β-mercaptoethanol; Preferably, the molar ratio of prednisolone to β-mercaptoethanol is (0.1-10): (20-100); Preferably, the molar ratio of prednisolone to β-mercaptoethanol is 1:5; Preferably, the molar concentration of prednisolone is 0.1-10 μM; Preferably, the molar concentration of prednisolone is 10 μM; Preferably, the molar concentration of β-mercaptoethanol is 20–100 μM; Preferably, the molar concentration of β-mercaptoethanol is 50 μM; Preferably, the basal culture medium comprises RPMI-1640, IMDM or DMEM; Preferably, the basal culture medium is selected from RPMI-1640; Preferably, the culture medium further comprises serum; Preferably, the mass concentration of the serum is 5-20%; Preferably, the mass concentration of the serum is 10%.

3. A method for preserving neutrophils / extending neutrophil survival time, characterized in that: The method comprises administering the composition of claim 1 or the culture medium of claim 2 to culture neutrophils; Preferably, the density of the neutrophils is 5×10 5 -20×10 5 cells / mL; Preferably, the density of the neutrophils is 11×10 5 cells / mL; Preferably, the culture condition is 30-40°C; Preferably, the culture condition is 37°C; Preferably, the culture conditions are 1-30% CO2; Preferably, the culture conditions are 5-21% CO2; Preferably, the culture condition is 21% O2; Preferably, the method further comprises maintaining the molar concentration of β-mercaptoethanol at 50 μM; Preferably, the method further comprises a step of separating and purifying neutrophils; Preferably, the separation and purification steps include density gradient centrifugation, lysis of red blood cells, and washing.

4. Use of the composition according to claim 1 or the culture medium according to claim 2 in preserving neutrophils / prolonging the survival time of neutrophils.

5. A neutrophil, characterized in that: The neutrophils are preserved by the method of claim 3.

6. A cell population, characterized in that The cell population includes the neutrophils according to claim 5.

7. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the neutrophil of claim 5 or the cell population of claim 6; Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or excipient.

8. Use of the neutrophil according to claim 5, the cell population according to claim 6, or the pharmaceutical composition according to claim 7 in the preparation of a pharmaceutical composition for treating, preventing, or treating a disease, wherein the disease includes a neutropenia-related disease or condition, or an autoimmune disease; Preferably, the neutropenia-related diseases or disorders include myeloablative hypoplasia, viral infection, typhoid fever, paratyphoid fever, certain parasitic diseases, hyperthyroidism, and hypersplenism; Preferably, the autoimmune diseases include systemic lupus erythematosus, rheumatoid arthritis, Sjögren's syndrome, systemic vasculitis, and systemic sclerosis.

9. A system, device or apparatus for preserving neutrophils / extending neutrophil survival time, characterized in that: The system, device or apparatus performs the method according to claim 3 when running; Preferably, the system, device or apparatus comprises: Mixed unit: Neutrophils are mixed with prednisolone and β-mercaptoethanol; Culture unit: Cultivate the mixed cells.

10. Any of the following applications: (1) Use of the composition according to claim 1 or the culture medium according to claim 2 in preparing a system, device or apparatus for preserving neutrophils / extending neutrophil survival time; (2) Use of the system, device or apparatus according to claim 9 for preserving neutrophils / extending neutrophil survival time.

Images