A method of culture and composition that synergistically prolongs the in vitro survival time of neutrophils

Abstract

The application provides a culture method and composition for prolonging the in-vitro survival time of neutrophils. The composition provided by the application comprises prednisolone and beta-mercaptoethanol, which can prolong the survival time of neutrophils. The method provided by the application has low cost, simple operation, small interference to cell function, can guarantee an experimental window of 3-4 days, and has good application value.

Description

Technical Field

[0001] This invention relates to the field of in vitro cell culture, and more particularly to a culture method and composition for synergistically prolonging the in vitro survival time of neutrophils. Background Technology

[0002] The development and maturation of neutrophils is a delicate and complex process. Within the bone marrow, a hematopoietic organ, granulocyte precursor cells undergo a series of differentiation stages, eventually transforming into mature neutrophils. This maturation process can be divided into five key stages: first, the primitive granulocyte stage, characterized by a large nucleus; then, the promyelocyte stage, where the nucleus begins to segment; next, the metamyelocyte stage, where the nucleus becomes horseshoe-shaped; then, the metamyelocyte stage, where the degree of nucleus segmentation further increases; and finally, the band and polymorphonuclear granulocyte stage, where the nucleus morphology transforms into band or polymorphonuclear neutrophils, laying the foundation for the formation of mature polymorphonuclear neutrophils. In these successive 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 × 10⁻⁶ neutrophils daily. 11 These are neutrophils, which then undergo a maturation process.

[0003] Neutrophils are a classic type of immune cell, generally considered to have a short lifespan. Their survival time in blood circulation is approximately 18.5 hours, while their in vitro survival time is only a few hours. The inability to store them in vitro significantly limits in vitro research on neutrophils. Theoretically, infusing neutrophils into the body could help clear bacteria and reduce organ damage caused by infection. Improving or prolonging the in vitro survival time of neutrophils holds great promise for clinical and research applications.

[0004] Existing methods for prolonging neutrophil lifespan have limitations. Currently, common methods include adding cytokines (such as G-CSF and GM-CSF) or using glucocorticoids (such as dexamethasone) to extend the in vitro lifespan of neutrophils. However, these methods have the following drawbacks: ① High cost: Cytokines are expensive, increasing experimental costs; ② Potential impact 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 effectiveness: The lifespan extension effect is limited, making it difficult to meet the needs 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 once, raising the implementation threshold. It also affects multiple metabolic pathways, hindering subsequent experiments. Currently, there is no simple, low-cost method that can significantly prolong the in vitro lifespan of neutrophils, limiting the development of related research.

[0005] Therefore, finding a simple, low-cost, and efficient method to prolong the in vitro survival time of neutrophils would be of great significance for biological research on neutrophils and their clinical applications. Summary of the Invention

[0006] In view of this, in order to overcome the shortcomings 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, the composition comprising prednisolone and β-mercaptoethanol.

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

[0009] In this invention, prolonging the survival time of neutrophils 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 prolongation of neutrophil survival time includes increasing the viability or function of 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% 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 one 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 one specific embodiment, the molar concentration of prednisolone is 10 µM.

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

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

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

[0018] In this invention, the pharmaceutically acceptable carriers and / or excipients include, but are not limited to, diluents, binders, surfactants, humectants, adsorbents, lubricants, and / or disintegrants. The diluents include, but are not limited to, lactose, sodium chloride, glucose, urea, starch, and water; the binders include, but are not limited to, starch, pregelatinized starch, dextrin, maltodextrin, sucrose, gum arabic, gelatin, methylcellulose, carboxymethylcellulose, ethylcellulose, polyvinyl alcohol, polyethylene glycol, polyvinylpyrrolidone, alginate and alginates, xanthan gum, hydroxypropylcellulose, and hydroxypropyl methylcellulose; the surfactants include, but are not limited to, polyethylene oxide sorbitan fatty acid esters, sodium lauryl sulfate, glyceryl monostearate, and hexadecyl alcohol; the humectants include, but are not limited to, glycerol and starch; the adsorbents include, but are not limited to, starch, lactose, bentonite, silica gel, kaolin, and soap clay; and the lubricants include, but are not limited to, zinc stearate, glyceryl monostearate, polyethylene glycol, talc, calcium and magnesium stearate, polyethylene glycol, boric acid powder, hydrogenated vegetable oil, sodium stearate 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 / extending the survival time of neutrophils, the culture medium comprising basal 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 one specific embodiment, the molar concentration of prednisolone is 10 µM.

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

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

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

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

[0028] In some embodiments, the culture medium also includes serum. The serum may be of human origin or non-human origin.

[0029] Non-human serum sources include, but are 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 one specific implementation, the serum is preferably human serum.

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

[0032] In one specific implementation, the serum has a mass concentration of 10%.

[0033] In some embodiments, the culture medium also includes growth factors, hormones, adhesion factors, carrier proteins, cytokines, antibiotics, and buffer solutions.

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

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

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

[0037] A third aspect of the present invention provides a method for preserving neutrophils / extending the survival time of neutrophils, the method comprising culturing neutrophils with the composition described in the first aspect of the present invention or the culture medium described in the second aspect of the present invention.

[0038] In some embodiments, the density of the neutrophils is 5 × 10⁻⁶. 5 -20×10 5 cells / mL.

[0039] In one specific implementation, the density of the neutrophils is 11 × 10⁻⁶. 5 cells / mL.

[0040] In some implementations, the culture conditions are 30-40 °C.

[0041] In one specific implementation, the culture conditions are 37 °C.

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

[0043] In one specific implementation, the culture conditions are 5-21% CO2.

[0044] In some implementations, the culture conditions are 21% O2.

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

[0046] In one specific embodiment, the method for maintaining the molar concentration of β-mercaptoethanol at 50 µM includes replenishing β-mercaptoethanol every 12-36 h.

[0047] In one specific embodiment, the method for maintaining the molar concentration of β-mercaptoethanol at 50 µM includes replenishing β-mercaptoethanol every 24 h.

[0048] In some embodiments, the method further includes a neutrophil isolation and purification step.

[0049] In some implementations, the separation and purification steps include density gradient centrifugation, lysis of red blood cells, and washing.

[0050] In some implementations, the density gradient centrifugation is performed using Ficoll-Paque™ PLUS / Lymphoprep™ or a polysucrose-diatrizoate meglumine specific stratification solution.

[0051] In some implementations, the polysucrose-diatrizoate meglumine specific stratification solution includes, but is not limited to, Polymorphprep™, Histopaque®-1077 / 1119 combination, and Percoll®.

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

[0053] In some implementations, the lysing of red blood cells is performed using red blood cell lysis buffer.

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

[0055] In one specific implementation, the erythrocyte lysis buffer is selected from ammonium chloride.

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

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

[0058] In some implementations, the washing is selected from PBS.

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

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

[0061] In this invention, non-human mammals refer to all members of the class Mammalia other than 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 rodent order (e.g., mice, rats, squirrels, beavers, groundhogs, voles, hamsters, guinea pigs, and spiny guinea pigs), including any offspring derived therefrom.

[0062] In some implementations, the cell culture container used in neutrophil culture is not particularly limited as long as it is capable of culturing neutrophils, including but not limited to bottles, tissue culture bottles, dishes, petri dishes, tissue culture dishes, multi-plate dishes, microplates, microwell plates, multi-plate trays, multiwell plates, microscope slides, chamber slides, culture dishes, tubes, trays, culture bags, or roller bottles.

[0063] Cell culture containers can be either cell-adhesive or cell-non-adhesive, depending on the intended purpose. Cell-adhesive cell culture containers are designed to increase the adhesion between the surface of the cell culture container and the cells, and are coated with any cell support matrix such as extracellular matrix (ECM). The cell support matrix can be any material intended to promote the adhesion of neutrophils.

[0064] The fourth aspect of the present invention provides the 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 the preservation of neutrophils / prolongation of neutrophil survival time.

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

[0066] A sixth aspect of the present invention provides a cell population comprising the neutrophils described in the fifth aspect of the present invention.

[0067] A seventh aspect of the present invention provides a pharmaceutical composition comprising neutrophils as described in the fifth aspect of the present invention or a cell population as described in the sixth aspect of the present invention.

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

[0069] In this invention, the pharmaceutically acceptable carriers and / or excipients include, but are not limited to, diluents, binders, surfactants, humectants, adsorbents, lubricants, and / or disintegrants. The diluents include, but are not limited to, lactose, sodium chloride, glucose, urea, starch, and water; the binders include, but are not limited to, starch, pregelatinized starch, dextrin, maltodextrin, sucrose, gum arabic, gelatin, methylcellulose, carboxymethylcellulose, ethylcellulose, polyvinyl alcohol, polyethylene glycol, polyvinylpyrrolidone, alginate and alginates, xanthan gum, hydroxypropylcellulose, and hydroxypropyl methylcellulose; the surfactants include, but are not limited to, polyethylene oxide sorbitan fatty acid esters, sodium lauryl sulfate, glyceryl monostearate, and hexadecyl alcohol; the humectants include, but are not limited to, glycerol and starch; the adsorbents include, but are not limited to, starch, lactose, bentonite, silica gel, kaolin, and soap clay; and the lubricants include, but are not limited to, zinc stearate, glyceryl monostearate, polyethylene glycol, talc, calcium and magnesium stearate, polyethylene glycol, boric acid powder, hydrogenated vegetable oil, sodium stearate 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, including neutropenia-related diseases or conditions and autoimmune diseases.

[0071] In some implementations, the neutropenia-related diseases or conditions include, but are not limited to, myelodylocytosis, viral infections, typhoid fever, paratyphoid fever, certain parasitic diseases, hyperthyroidism, and hypersplenism.

[0072] In some implementations, the autoimmune diseases include, but are not limited to, systemic lupus erythematosus, rheumatoid arthritis, Sjögren's syndrome, systemic vasculitis, and systemic sclerosis.

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

[0074] The systems, apparatus, or devices described in some embodiments include:

[0075] Mixing unit: Neutrophils are mixed with prednisolone and β-mercaptoethanol.

[0076] Culture unit: Cultured mixed cells.

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

[0078] (1) The 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 the preparation of a system, apparatus or device for preserving neutrophils / extending the survival time of neutrophils;

[0079] (2) The application of the system, apparatus or device described in the ninth aspect of the present invention in the preservation of neutrophils / prolongation of neutrophil survival time.

[0080] The advantages and beneficial effects of this invention are as follows:

[0081] This invention provides a culture method and composition for synergistically prolonging the in vitro survival time of neutrophils. The composition provided by this invention includes prednisolone and β-mercaptoethanol, which synergistically prolong the survival time of neutrophils. The method provided by this invention is low-cost, simple to operate, and has minimal interference with cell function, ensuring an experimental window of 3-4 days, and has good application value.

[0082] Extended experimental window: Under normal culture conditions, the survival time of neutrophils (PMN) was increased from 24 h to ≥96 h, solving the problem of experimental interruption caused by rapid apoptosis.

[0083] Biaxial synergy: The Q value after combination was calculated using the King's formula. It was found that the Q value was ≥1.15 at 24 h, 60 h, and 96 h, which proved that Pred and β-ME played a synergistic role in prolonging the in vitro survival time of neutrophils.

[0084] Functionally intact: It does not inhibit NETosis or ferroptosis induction, making it suitable for multi-pathway mechanism research.

[0085] Low cost and reproducible: All raw materials are commercially available and universal, requiring no hypoxia chamber, recombinant protein, or sealed storage bags; total cost of 24 wells is <0.2 USD, with excellent reproducibility across suppliers. Attached Figure Description

[0086] Figure 1 The results show the neutrophil viability at 24 h. Figure A shows the neutrophil viability in each experimental group, and Figure B shows the neutrophil viability after combining different concentrations of Pred with 50 µM β-ME.

[0087] Figure 2The results show the neutrophil viability at 60 h. Figure A shows the neutrophil viability in each experimental group, and Figure B shows the neutrophil viability after combining different concentrations of Pred with 50 µM β-ME.

[0088] Figure 3 The results show the neutrophil viability at 96 h. Figure A shows the neutrophil viability in each experimental group, and Figure B shows the neutrophil viability after combining different concentrations of Pred with 50 µM β-ME. Detailed Implementation

[0089] The present invention will be further described below with reference to embodiments. The following description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any other way. Any person skilled in the art may make equivalent modifications to the disclosed technical content to create equivalent embodiments. Any simple modifications or equivalent changes made to the following embodiments based on the technical essence of the present invention without departing from the scope of the invention are all within the protection scope 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 isolation

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

[0098] ② Group inoculation (24-well plate, 5 × 10⁻⁶) 5 The drugs and dosages used for grouping (cells / well, 450µL / well) are shown in Table 2.

[0099] Table 2. Drugs and dosages used in each group

[0100]

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

[0102] Table 3 Cultivation conditions

[0103]

[0104] ④ Detection time points and indicators:

[0105] Survival rates were assessed using Annexin V-FITC / PI flow cytometry at 24 h, 60 h, and 96 h.

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

[0107] Table 4 Experimental Results

[0108]

[0109] All three volunteers (HC1-HC3) showed the same trend; at 96 h, the combined group showed a 211% increase compared to the control group, ANOVA + Tukey P < 0.05. The Q-values ​​calculated using the King's formula showed that at 24 h, 60 h, and 96 h, the Q-values ​​were ≥1.15, demonstrating that Pred and β-ME play a synergistic role in prolonging the in vitro survival time of neutrophils.

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

[0111] Table 5. Advantages of the method in this application compared with the prior art.

[0112]

[0113] Table 6. Advantages of the method in this application compared with the prior art.

[0114]

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

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

[0117]

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

[0119]

[0120] The above description of the embodiments is only for understanding the method and core ideas of the present invention. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from the principles of the invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. A composition for prolonging the survival time of mature neutrophils, characterized in that, The composition consists of prednisolone, β-mercaptoethanol, basal culture medium and human serum, wherein the molar ratio of prednisolone to β-mercaptoethanol is 1:5 and the mass concentration of human serum is 5-20%.

2. The composition according to claim 1, characterized in that, The molar concentration of prednisolone is 0.1-10 µM.

3. The composition according to claim 2, characterized in that, The molar concentration of prednisolone is 10 µM.

4. The composition according to claim 1, characterized in that, The molar concentration of the β-mercaptoethanol is 20-100 µM.

5. The composition according to claim 4, characterized in that, The molar concentration of the β-mercaptoethanol is 50 µM.

6. The composition according to claim 1, characterized in that, The basal culture medium is selected from one or more of RPMI-1640, IMDM, or DMEM.

7. The composition according to claim 6, characterized in that, The basal culture medium was RPMI-1640.

8. The composition according to claim 1, characterized in that, The serum concentration was 10%.

9. A method for prolonging the survival time of mature neutrophils, characterized in that, The method includes using the composition according to any one of claims 1-8 to preserve mature neutrophils, wherein the density of the mature neutrophils is 5 × 10⁻⁶. 5 -20×10 5 The cells / mL storage temperature is 30-40 ℃.

10. The method according to claim 9, characterized in that, The density of the mature neutrophils was 11 × 10⁻⁶. 5 cells / mL.

11. The method according to claim 9, characterized in that, The storage temperature is 37°C.

12. The method according to claim 9, characterized in that, The storage conditions are 1-30% O2.

13. The method according to claim 12, characterized in that, The storage conditions are 5-21% O2.

14. The method according to claim 13, characterized in that, The storage conditions are 21% O2.

15. The method according to claim 9, characterized in that, The method also includes a step of isolating and purifying neutrophils.

16. The method according to claim 15, characterized in that, The separation and purification steps include density gradient centrifugation, lysis of red blood cells, and washing.

17. The use of the composition according to any one of claims 1-8 in prolonging the survival time of mature neutrophils.

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