Leukocyte product, frozen leukocyte, preparation method and application of leukocyte product and frozen leukocyte in preparation of medicine for inhibiting inflammation

Abstract

The invention relates to a leukocyte product, frozen leukocytes, a preparation method and application of the leukocyte product and the frozen leukocytes in preparation of medicines for inhibiting inflammation. The leukocyte product comprises one or more non-viable leukocytes from blood, the non-viable leukocytes carry cytokine receptors but basically do not have proliferation capacity, and the cytokine receptors can be combined with cytokines which can be combined with normal blood leukocytes. The inactive leukocytes may be frozen leukocytes. The frozen leukocytes can be obtained by quickly freezing in-vitro normal blood leukocytes, and the quick freezing treatment temperature is ultralow temperature. The inactive leukocytes or frozen leukocytes in the leukocyte product can recognize and be competitively combined with the cytokines with normal immune cells without additionally secreting new cytokines, so that the cascade amplification effect of inflammation cannot be caused. The leukocyte product or frozen leukocyte can play an immunosuppressive role, has no obvious toxic or side effect, and can be used for preparing medicines for inhibiting inflammation, such as medicines for inhibiting severe inflammation.

Description

Technical Field

[0001] The present application relates to the field of biomedicine technology, and in particular to a leukocyte product, frozen leukocytes, a preparation method, and the use of the leukocyte product in preparing drugs for inhibiting inflammation. The present application further relates to a leukocyte product, frozen leukocytes, a preparation method, and the use of the leukocyte product in preparing drugs for inhibiting severe inflammation. Background Art

[0002] Inflammation is the body's defensive response to the stimulation of various damaging factors. Under normal circumstances, after being invaded by pathogens, the body's macrophages will promptly identify and eliminate foreign bodies, while secreting inflammatory chemokines to recruit other immune cells in the blood circulation to jointly fight the infection. Once the pathogens are controlled, the body's inflammatory factor levels will decrease, and the patient will recover. However, if the pathogens are numerous and highly virulent, or if the patient has certain underlying diseases, the invasion and continuous stimulation of these viruses and bacteria will lead to an imbalance in the body's immune homeostasis, dysfunction, and a vicious cycle of immune hyperactivity, which is clinically manifested as cytokine storms, acute respiratory distress syndrome, and even respiratory failure. For severe inflammatory conditions, such as severe pneumonia, timely suppression of uncontrolled inflammatory responses is a necessary measure to reduce mortality.

[0003] Regarding the control of inflammation in clinical practice, inflammatory immunosuppressants such as glucocorticoids are important therapeutic drugs that can reduce the function of immune cells, relieve inflammatory stress, and reduce the secretion of inflammatory cytokines. Especially for some patients with severe inflammation who do not yet have effective drugs, immunosuppressive anti-inflammatory drugs are the first-line treatment options in clinical practice. However, glucocorticoids themselves have large toxic side effects. Excessive use can easily cause excessive suppression of the body's immune function, secondary infection, osteoporosis, femoral head necrosis and other side effects, and can even lead to disability in patients, seriously affecting their quality of life after recovery. Therefore, for the treatment and control of severe inflammation, the development of new, low-toxic inflammatory inhibitors is of great significance. Summary of the Invention

[0004] Based on this, the present application provides at least one leukocyte product, frozen leukocytes, preparation method, and use in the preparation of drugs for suppressing inflammation (e.g., drugs for suppressing severe inflammation). The leukocyte product and frozen leukocytes have the effect of suppressing severe inflammation without significant toxic side effects.

[0005] In a first aspect of the present application, a leukocyte product is provided, comprising one or more non-viable leukocytes from blood, wherein the non-viable leukocytes have the basic structure of leukocytes and carry cytokine receptors, but the non-viable leukocytes have substantially no proliferation ability;

[0006] The cytokine receptor is capable of binding to cytokines that can be bound by normal blood leukocytes.

[0007] In some embodiments, the cytokines to which normal blood leukocytes can bind include cytokines to which normal blood leukocytes can bind during an inflammatory response;

[0008] Optionally, the cytokines to which the normal blood leukocytes can bind include one or more of interleukin 1, interleukin 6, interleukin 8, interleukin 12, interleukin 17, tumor necrosis factor α, interferon-α, interferon-β, interferon-γ and monocyte chemoattractant protein-1;

[0009] Optionally, the cytokines to which normal blood leukocytes can bind include cytokines to which normal blood leukocytes can bind in severe inflammation;

[0010] Optionally, the cytokines that the normal blood leukocytes can bind to participate in the formation of a cytokine storm in the severe inflammation.

[0011] In some embodiments, the leukocyte product meets one or more of the following characteristics:

[0012] The non-viable leukocytes include one or more of monocytes, neutrophils and lymphocytes;

[0013] The normal blood leukocytes include one or more of monocytes, neutrophils and lymphocytes;

[0014] The non-viable leukocytes and the normal blood leukocytes together include one or more of monocytes, neutrophils and lymphocytes.

[0015] In some embodiments, the non-viable leukocytes include dead leukocytes;

[0016] Optionally, the non-viable leukocytes include dead cells of one or more leukocytes among monocytes, neutrophils and lymphocytes.

[0017] In some embodiments, the leukocyte product is a cell suspension, a cryoprotectant, or a cell puree;

[0018] Optionally, the leukocyte product is a pharmaceutical preparation.

[0019] In some embodiments, the non-viable leukocytes are the frozen leukocytes described in the second aspect of this application.

[0020] In a second aspect of the present application, there is provided a frozen leukocyte obtained by subjecting normal blood leukocytes isolated from the body to rapid freezing treatment, wherein the rapid freezing treatment is performed at a temperature of -50°C to -196°C;

[0021] Optionally, the frozen leukocytes include one or more of frozen monocytes, frozen neutrophils and frozen lymphocytes.

[0022] In some embodiments, the frozen leukocytes are the non-viable leukocytes included in the leukocyte product described in the first aspect of the present application.

[0023] In a third aspect of the present application, a method for preparing a leukocyte product or frozen leukocytes is provided, comprising the following steps:

[0024] Extract target leukocytes from isolated blood to prepare a leukocyte suspension;

[0025] The leukocyte suspension is subjected to rapid freezing treatment to obtain the leukocyte product or the frozen leukocytes, wherein the temperature for the rapid freezing treatment is -50°C to -196°C.

[0026] In some embodiments, the leukocyte product or the method for preparing frozen leukocytes satisfies one or more of the following characteristics:

[0027] The isolated blood is whole blood;

[0028] The isolated blood is subjected to anticoagulation treatment;

[0029] The step of extracting target leukocytes from the ex vivo blood to prepare a leukocyte suspension is achieved using Method A, Method B, or Method C; wherein Method A comprises the following steps: diluting the ex vivo blood, centrifuging, collecting blood cell sediment, rupturing the red blood cells with a red blood cell lysis solution, washing and centrifuging, collecting centrifugal sediment A, and resuspending the centrifugal sediment A to prepare the leukocyte suspension; Method B comprises the following steps: separating one or more target types of leukocytes from the ex vivo blood with a cell separation solution and preparing a leukocyte suspension in a desired ratio; optionally, Method B comprises the following steps: diluting the ex vivo blood, treating it with a cell separation solution, and horizontally centrifuging to form multiple liquid phase layers, the multiple liquid phase layers comprising an upper layer containing monocytes, a middle layer containing lymphocytes, and a bottom layer containing neutrophils and red blood cells, extracting at least one of the upper layer, the middle layer, and the bottom layer to obtain at least one target extraction layer, washing and centrifuging the at least one target extraction layer to obtain centrifugal sediments of the corresponding target extraction layers, and resuspending the centrifugal sediments of each target extraction layer in a desired ratio. The method comprises the steps of: extracting the blood from the blood sample and performing a leukocyte suspension resuspending the blood sample into the leukocyte suspension; wherein, when one of the target extraction layers is the upper layer, at least the upper layer is extracted, and then the extracted upper layer is washed and centrifuged to collect a centrifugal precipitate B1. In this case, the centrifugal precipitates of each target extraction layer include the centrifugal precipitate B1; when one of the target extraction layers is the middle layer, at least the middle layer is extracted, and then the extracted middle layer is washed and centrifuged to collect a centrifugal precipitate B2. In this case, the centrifugal precipitates of each target extraction layer include the centrifugal precipitate B2; when one of the target extraction layers is the bottom layer, at least the bottom layer is extracted, and then the extracted bottom layer is subjected to erythrocyte lysis treatment and centrifugation using an erythrocyte lysis solution to collect a centrifugal precipitate B31. The centrifugal precipitate B31 is washed and centrifuged to obtain a centrifugal precipitate B32. In this case, the centrifugal precipitates of each target extraction layer include the centrifugal precipitate B3; the method C comprises the following steps: subjecting the isolated blood to a blood component separator to separate the leukocyte components therein, obtaining one or more leukocytes of the target type, and preparing a leukocyte suspension according to a desired ratio;

[0030] The leukocyte suspension comprises a resuspension and the extracted target leukocytes; wherein, the resuspension is composed of, by volume percentage, 0% to 90% cell culture medium, 0% to 90% serum, and 0% to 30% dimethyl sulfoxide, and the resuspension comprises at least one of the cell culture medium and the serum;

[0031] The rapid freezing process is achieved by using at least one of a freezing medium and a freezing device, wherein the freezing medium includes at least one of liquid nitrogen, liquid carbon dioxide, and liquefied air, and the freezing device includes an ultra-low temperature refrigerator; the ultra-low temperature refrigerator can provide at least one temperature between -50°C and -196°C;

[0032] After the rapid freezing treatment and before the leukocyte product or the frozen leukocytes are obtained, the method further comprises thawing the frozen product obtained by the rapid freezing treatment, wherein the thawing temperature is 4° C. to 37° C.;

[0033] The leukocyte product or frozen leukocytes is prepared by mixing prepared frozen leukocytes of different types in a desired ratio.

[0034] In some embodiments, the leukocyte product or the method for preparing frozen leukocytes satisfies one or more of the following characteristics:

[0035] In the step of diluting the isolated blood, the diluent is phosphate buffer or physiological saline, and the amount of the diluent is 3 to 20 times the volume of the isolated blood;

[0036] The centrifugal precipitate of each target extraction layer is a mixture of centrifugal precipitates of multiple target extraction layers or a centrifugal precipitate of one target extraction layer;

[0037] After the rapid freezing treatment and before the leukocyte product or the frozen leukocytes are obtained, the method further comprises thawing and centrifuging the frozen product obtained by the rapid freezing treatment, with or without a washing step after thawing and before centrifugation;

[0038] Each washing step independently adopts the following washing method: the washing solution is phosphate buffer or physiological saline, the amount of the washing solution is 3 to 20 times the volume of the washed object, and the number of washing times is 1 to 5 times;

[0039] Each centrifugation step independently adopts the following centrifugation mode: centrifugation speed is 200g~800g, and centrifugation time is 2min~10min;

[0040] The temperature for performing the rethawing is 37°C;

[0041] The prepared leukocyte product is the leukocyte product described in the first aspect of the present application;

[0042] The prepared frozen leukocytes are the frozen leukocytes described in the second aspect of the present application.

[0043] In a fourth aspect of the present application, there is provided a use of the leukocyte product described in the first aspect of the present application, or the frozen leukocytes described in the second aspect of the present application, or the leukocyte product or frozen leukocytes prepared by the preparation method described in the third aspect of the present application in the preparation of a drug for inhibiting inflammation;

[0044] Optionally, the drug for suppressing inflammation is a drug for suppressing severe inflammation;

[0045] Further optionally, the severe inflammation is severe pneumonia;

[0046] Optionally, the drug that suppresses inflammation is used to control cytokine storm.

[0047] In patients with severe inflammation, the "uncontrolled" positive feedback loop between immune cells and cytokines can lead to the activation of a large number of inflammatory cells and the secretion of cytokines, potentially causing a cytokine storm and the patient's death. Based on this, the white blood cell products provided herein include one or more abiotic white blood cells from blood. These abiotic white blood cells carry cytokine receptors that can bind to normal blood white blood cells, but have essentially no proliferation capacity. The frozen white blood cells provided herein are obtained by rapidly freezing isolated normal blood white blood cells (at ultra-low temperatures, such as -50°C to -196°C). The abiotic white blood cells can be the frozen white blood cells described above. These abiotic white blood cells or frozen white blood cells can serve as an immunosuppressant derived from the body itself. These abiotic white blood cells or frozen white blood cells can act as "disguises" of normal immune cells, capable of recognizing and competitively binding to cytokines with normal immune cells, thereby reducing or severing the connection between normal immune cells and cytokines. These anergic or frozen leukocytes can function as cytokine-antibody hybrids without generating feedback loops. Specifically, after binding to cytokines, the anergic or frozen leukocytes themselves do not secrete additional cytokines, thus preventing the inflammatory cascade. This can mitigate the positive feedback loop between normal immune cells and cytokines. Consequently, these anergic or frozen leukocyte products can exert immunosuppressive effects. Compared to traditional immunosuppressants such as glucocorticoids, the anti-inflammatory mechanism of these anergic or frozen leukocyte products does not rely on suppressing the activity and phagocytic capacity of immune cells themselves, thus preventing subsequent bacterial and viral clearance. Furthermore, the anergic or frozen leukocytes can be derived from the patient's blood, thus embodying the concept of autologous therapy and resulting in no significant toxic side effects.

[0048] The leukocyte product or frozen leukocyte can be used to prepare drugs for suppressing inflammation, such as drugs for suppressing severe inflammation, and can be used to suppress severe pneumonia.

[0049] The leukocyte product or frozen leukocytes can be prepared by a method comprising the following steps: extracting target leukocytes from ex vivo blood to prepare a leukocyte suspension; and rapidly freezing the leukocyte suspension at an ultra-low temperature (e.g., -50°C to -196°C) to produce the leukocyte product or frozen leukocytes. This preparation method has few steps and is simple to operate. The resulting leukocyte product or frozen leukocytes can be used as a novel, low-toxic immunosuppressant. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] In order to more clearly illustrate the technical solutions in the embodiments and examples of this application and to provide a more complete understanding of the application and its beneficial effects, the following briefly introduces the drawings required for use in the description of the embodiments or examples. Obviously, the drawings described below are only some embodiments of this application. Those skilled in the art can also derive other drawings based on these drawings without inventive effort.

[0051] Figure 1 This is a schematic diagram of the preparation process of a leukocyte product in one embodiment of the present application.

[0052] Figure 2 This is a schematic diagram of the preparation process of frozen leukocytes in one embodiment of the present application.

[0053] Figure 3 This is the result of structural characterization of frozen leukocytes in one embodiment of the present application, which was performed using a laser confocal microscope.

[0054] Figure 4 The frozen leukocyte structure characterization results in one embodiment of the present application were characterized using a scanning electron microscope.

[0055] Figure 5 This is the CCK8 test result of the in vitro proliferation of frozen leukocytes in one embodiment of the present application.

[0056] Figure 6 This is the expression result of cytokine receptors in frozen leukocytes in one embodiment of the present application. The cytokine receptors were fluorescently labeled and characterized by flow cytometry after antibody incubation.

[0057] Figure 7 This is the result of the frozen leukocyte cytokine adsorption test in one embodiment of the present application, corresponding to the laser confocal image after the frozen leukocytes were incubated with FAM (fluorescein, maleimidized fluorescein) labeled interleukin 6 (IL-6).

[0058] Figure 8 This is the result of cytokine secretion of frozen leukocytes after lipopolysaccharide stimulation in one embodiment of the present application.

[0059] Figure 9This is the in vitro anti-inflammatory efficacy result of frozen leukocytes in one embodiment of the present application.

[0060] Figure 10 The in vitro anti-inflammatory efficacy results of different frozen leukocytes in one embodiment of the present application are shown.

[0061] Figure 11 This is the in vivo anti-inflammatory efficacy result of frozen leukocytes in one embodiment of the present application, targeting a severe pneumonia model. DETAILED DESCRIPTION

[0062] The present application will be described in further detail below in conjunction with the accompanying drawings, embodiments and examples. It should be understood that these embodiments and examples are only used to illustrate the present application and are not used to limit the scope of the present application. The purpose of providing these embodiments and examples is to make the understanding of the present application disclosure more thorough and comprehensive. It should also be understood that the present application can be implemented in many different forms and is not limited to the embodiments and examples described herein. Those skilled in the art can make various changes or modifications without violating the connotation of the present application, and the equivalent forms obtained also fall within the protection scope of the present application. For example, the features described or described as part of one embodiment can be combined in another embodiment in a suitable manner to produce a new embodiment. In addition, in the description below, a large amount of details are given in order to provide a more comprehensive understanding of the present application. It should be understood that the present application can be implemented without one or more of these details.

[0063] In this application, “multiple”, “multiple”, “multiple times”, “multiples”, “several”, etc., unless otherwise specified, refer to a quantity greater than or equal to 2. For example, “one or more” means one or more than or equal to two.

[0064] In this application, unless otherwise specified, "one or more" refers to any one of the listed items or any combination of the listed items. Similarly, "one or more" and other similar expressions that refer to "one or more" are also understood in the same way unless otherwise specified.

[0065] In this application, the word "suitable" in "suitable combination", "combining in a suitable manner", "suitable manner", "any suitable manner", etc., shall be based on the ability to implement the technical solution of this application, solve the technical problems of this application, and achieve the expected technical effects of this application.

[0066] In this application, the terms "preferred," "better," "more preferable," "suitable," "comparatively better," and "preferable" are used solely to describe preferred implementations or examples, and should not be construed as limiting the scope of protection of this application. If multiple "preferred" terms appear in a technical solution, each "preferred" term is considered independent unless otherwise specified and there are no contradictions or mutual constraints.

[0067] In this application, terms such as "further," "further," "particularly," "for example," "such as," "example," and "for instance" are used for descriptive purposes to indicate that the preceding and following technical solutions are related in terms of their coverage. However, they should not be construed as limiting the preceding technical solution or the scope of protection of this application. In this application, unless otherwise specified, "A (such as B)" means that B is a non-limiting example of A, and it should be understood that A is not limited to B.

[0068] In this application, the terms "optionally," "optional," and "optional" mean optional, that is, they refer to either option selected from the two parallel options of "yes" or "no." If multiple "options" appear in a technical solution, unless otherwise specified and there are no contradictions or mutual constraints, each "optional" is independent. Unless otherwise specified, the descriptions "optionally include," "optionally include," etc. in this application, taking "optionally include" as an example, mean "may include or not include."

[0069] As used herein, the terms "comprising," "including," and "include" are synonymous and are inclusive or open-ended, not excluding additional, unrecited members or features. Examples of members or features include materials or components, structures, elements, and instruments. Non-limiting examples of members or features include actions, conditions for the occurrence of actions, timing, and states.

[0070] In this application, the technical features or technical solutions described in open language include closed technical features or technical solutions composed of the listed contents, and also include open technical features or technical solutions containing the listed contents.

[0071] The temperature parameters in this application, unless otherwise specified, allow for both constant temperature treatment and temperature fluctuations within a certain temperature range. It should be understood that constant temperature treatment allows for temperature fluctuations within the accuracy range of instrument control, for example, within a range of ±2°C or ±1°C.

[0072] In this application, when referring to a data range, if the unit is followed only by the right endpoint, it means that the units of the left and right endpoints are the same. For example, "3~5h" means that the units of the left endpoint "3" and the right endpoint "5" are both hours, which has the same meaning as "3h~5h". Similar descriptions of other parameters such as temperature and dosage also apply to this understanding.

[0073] In this application, if a method flow involves multiple steps, unless otherwise specified herein, there is no strict order restriction on the execution of these steps, and they can be executed in an order other than the order described. Moreover, any step can include multiple sub-steps or multiple stages, and these sub-steps or stages do not necessarily need to be completed at the same time, but can be executed at different times, and their execution order is not necessarily sequential, but can be executed in turn, alternating, or simultaneously with other steps or parts of sub-steps or stages of other steps.

[0074] In this application, exemplary descriptions such as "in some embodiments (or examples)" and "in one embodiment (or example)" may include but are not limited to the following meanings: these solutions can be combined with other solutions in a suitable manner to form new technical solutions.

[0075] In this application, when referring to a numerical interval (i.e., a numerical range), unless otherwise specified, the distribution of the optional numerical values ​​within the numerical interval is deemed to be continuous and includes the two numerical endpoints of the numerical interval (i.e., the minimum and maximum values), as well as every numerical value between these two numerical endpoints. Unless otherwise specified, when a numerical interval refers only to integers within the numerical interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints, which is equivalent to directly listing every integer. When multiple numerical ranges are provided to describe a feature or characteristic, these numerical ranges can be combined. In other words, unless otherwise specified, the numerical ranges disclosed herein should be understood to include any and all subranges included therein. The "numerical value" in the numerical interval can be any quantitative value, such as a number, percentage, ratio, etc. "Numerical interval" allows for a broad range of numerical interval types including percentage intervals, ratio intervals, and ratio intervals.

[0076] In this application, the terms "first," "second," "third," etc. in "the first aspect," "the second aspect," "the first aspect," "the fourth aspect," etc. are used for descriptive purposes only and should not be understood as indicating or implying relative importance or quantity, nor should they be understood as implicitly indicating the importance or quantity of the technical features indicated. Furthermore, "first," "second," "third," etc. serve only as non-exhaustive enumeration and description and should be understood not to constitute a closed-ended limitation on quantity.

[0077] In this application, unless otherwise specified, % (v / v) means volume percentage.

[0078] In this application, unless otherwise specified, "cytokine" refers to a cytokine that can specifically bind to a cytokine receptor carried by normal immune cells to exert an immunological function or effect.

[0079] In the present application, unless otherwise specified, the "binding" between normal blood leukocytes and cytokines refers to the binding between the cytokine receptors carried by normal blood leukocytes and the corresponding cytokines.

[0080] In patients with severe inflammation, the "out-of-control" positive feedback between immune cells and cytokines can lead to the activation of a large number of inflammatory cells and the secretion of cytokines, which may cause the occurrence of cytokine storm and the death of the patient.

[0081] Based on this, the present application relates to a leukocyte product, frozen leukocytes, a preparation method and their use in the preparation of drugs for inhibiting inflammation (such as drugs for inhibiting severe inflammation).

[0082] The leukocyte product provided herein includes one or more avital leukocytes derived from blood. These avital leukocytes carry cytokine receptors that can bind to normal leukocytes, but possess essentially no proliferative capacity. The frozen leukocytes provided herein are obtained by subjecting isolated normal leukocytes to a rapid freezing treatment (at an ultra-low temperature, such as -50°C to -196°C). The avital leukocytes may be the aforementioned frozen leukocytes. These avital or frozen leukocytes can serve as an immunosuppressant derived from the body itself. They can act as "disguises" of normal immune cells, capable of recognizing and competitively binding cytokines with normal immune cells, thereby reducing or severing the connection between normal immune cells and cytokines. These avital or frozen leukocytes can function as cytokine hybrid antibodies without generating feedback loops. That is, after binding to cytokines, the avital leukocytes (or frozen leukocytes) do not secrete additional cytokines, thus preventing the inflammatory cascade. This can mitigate the positive feedback loop between normal immune cells and cytokines. Therefore, the leukocyte product or frozen leukocytes can exert an immunosuppressive effect.

[0083] Compared with traditional immunosuppressants such as glucocorticoids, the anti-inflammatory mechanism of the aforementioned white blood cell products or frozen white blood cells does not rely on suppressing the activity and phagocytic ability of the immune cells themselves, and will not affect the subsequent clearance of bacteria and viruses. Moreover, the source of inactive white blood cells or frozen white blood cells can be the patient's blood, which can fall within the scope of autologous treatment, and therefore has no obvious toxic side effects.

[0084] The leukocyte product or frozen leukocyte can be used to prepare drugs for suppressing inflammation, such as drugs for suppressing severe inflammation, and can be used to suppress severe pneumonia.

[0085] In a first aspect of the present application, a leukocyte product is provided.

[0086] In this application, unless otherwise specified, "leukocyte product" refers to an article comprising non-viable leukocytes. The leukocyte product may be a pharmaceutical preparation, also referred to as a "leukocyte preparation," which at least comprises non-viable leukocytes and may further comprise a pharmaceutically acceptable carrier.

[0087] In this application, unless otherwise specified, "pharmaceutical preparation" refers to a drug product formulated according to specific dosage form requirements and intended for final administration to a subject. The leukocyte preparation herein may be a liquid preparation, for example, a cell suspension containing non-viable leukocytes. Prior to use as a liquid preparation, the leukocyte preparation may be refrigerated at 4°C or frozen at, but not limited to, -20°C to -80°C. Other suitable storage temperatures are also acceptable, as long as the non-viable leukocytes substantially maintain their aforementioned functions. A cryopreserved liquid preparation can be reconstituted at an appropriate temperature (e.g., 4°C to 37°C, further preferably 37°C) upon use. "Appropriate temperature" herein refers to a temperature that facilitates the effects of the non-viable leukocytes in the liquid preparation. Depending on the type of pharmaceutical preparation, an appropriate administration method may be selected. For example, injection may be used for a liquid preparation, but this is not limited to such a method.

[0088] In this application, unless otherwise indicated, "pharmaceutically acceptable" means suitable for administration to a patient within the scope of sound medical judgment and commensurate with a reasonable benefit / risk ratio.

[0089] In this application, unless otherwise specified, "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition or vehicle, for example, a diluent, a resuspension, etc. mentioned in the context of this article, but is not limited thereto.

[0090] In some embodiments, the white blood cell product includes one or more abiotic white blood cells derived from blood. These abiotic white blood cells carry cytokine receptors but lack substantial proliferative capacity. These cytokine receptors are capable of binding to cytokines that are also bound by normal white blood cells. The abiotic white blood cells in this white blood cell product are capable of recognizing and competitively binding to cytokines with normal immune cells, without secreting additional cytokines and thus without inducing an inflammatory cascade. This white blood cell product or abiotic white blood cells can exert an immunosuppressive effect without significant toxic side effects and can be used to prepare drugs for suppressing inflammation, such as those for severe inflammation.

[0091] In this application, unless otherwise specified, "non-viable leukocytes" in leukocyte products are derived from blood. Compared to leukocytes from other parts of the body, blood-derived leukocytes are primarily composed of monocytes, granulocytes, and lymphocytes, but contain very low levels of common immune cells found in tissues, such as mature macrophages and dendritic cells. Non-viable leukocytes may include one or more of monocytes, neutrophils, and lymphocytes.

[0092] In this application, unless otherwise specified, "non-viable leukocytes" are normal blood leukocytes that carry cytokine receptors but have been treated to render them essentially incapable of proliferation. Non-viable leukocytes may carry receptors for one or more cytokines, including interleukin-1, interleukin-6, interleukin-8, and tumor necrosis factor-α.

[0093] In this application, unless otherwise specified, "normal blood leukocytes" refer to normal leukocytes in the blood, which can function normally as leukocytes, can undergo normal cell proliferation, and can secrete cytokines. Typically, normal blood leukocytes carry cytokine receptors on their cell surface and have the ability to bind to cytokines. Taking inflammatory cytokines in an inflammatory response as an example, when an inflammatory response occurs, normal blood leukocytes bind to inflammatory cytokines through cytokine receptors located on the cell surface, stimulating the leukocytes to secrete cytokines and rapidly proliferate, producing an inflammatory cascade amplification effect; and for the "non-viable leukocytes" in this application, they can bind to inflammatory cytokines through the cytokine receptors carried on their surface, forming a competitive effect with normal blood leukocytes, thereby reducing or cutting off the binding effect of normal blood leukocytes on inflammatory cytokines, but non-viable cells have no proliferation ability and do not secrete new cytokines under the stimulation of an inflammatory response, and do not produce an inflammatory cascade amplification effect.

[0094] In this application, unless otherwise specified, "substantially lacking proliferation ability" means no cell proliferation occurs during an observation period of at least 3 days (72 hours). Generally, it can be assumed that non-viable leukocytes with "substantially lacking proliferation ability" also cannot secrete cytokines. For example, the frozen leukocytes in Example 10 do not secrete cytokines such as interleukin 6 (IL-6), interleukin 8 (IL-8), interleukin 1 (IL-1), and tumor necrosis factor α (TNF-α). It should be noted that "substantially lacking proliferation ability" can mean a complete lack of proliferation ability or a state of extremely low proliferation ability, such as a dead state or a state of "shock." Accordingly, non-viable leukocytes can be dead leukocytes or leukocytes in a state of shock.

[0095] In the present application, unless otherwise specified, "non-viable leukocytes carry cytokine receptors" means that the cytokine receptors are at least located on the surface of the non-viable leukocytes.

[0096] In the present application, unless otherwise specified, the cytokine receptors carried by non-viable leukocytes "are capable of binding to cytokines that normal blood leukocytes can bind to", thereby forming a competitive effect with normal blood leukocytes, competitively binding to cytokines and exerting an immunosuppressive effect.

[0097] In this application, unless otherwise specified, "non-viable leukocytes" still possess a basic cellular morphology, including at least a cell membrane and at least one cytokine receptor located on the cell membrane. However, compared to the cell membrane of normal blood leukocytes, the cell membrane of non-viable leukocytes has increased permeability. Non-viable leukocytes can be frozen leukocytes. Frozen leukocytes can be obtained by subjecting normal blood leukocytes to a rapid freezing treatment at ultra-low temperatures, such as -50°C to -196°C. After the aforementioned rapid freezing treatment, the leukocyte cell membrane loses its selective permeability, and even portions of the cell membrane and organelle membranes may be ruptured and damaged.

[0098] In this application, unless otherwise specified, "non-viable leukocytes have the basic structure of leukocytes" means that non-viable leukocytes still retain the basic nucleus-membrane morphology. However, the cell membrane has lost its selective permeability, allowing for damage to the membrane structure in some areas.

[0099] In this application, unless otherwise specified, "rapid freezing treatment" refers to freezing treatment at ultra-low temperature. Due to the use of ultra-low temperature, the freezing speed is very fast. The ultra-low temperature used for rapid freezing treatment can be -50°C to -196°C. The ultra-low temperature used for rapid freezing treatment can be achieved by using one or more of the following freezing media: liquid nitrogen, liquid carbon dioxide, liquefied air, ultra-low temperature refrigerator, etc. Generally, an "ultra-low temperature refrigerator" can provide at least one temperature between -50°C and -196°C, optionally, at least one temperature between -50°C and -150°C, for example, it can provide any one of the following temperatures or at least one temperature in the range consisting of any two of the following temperatures: -50°C, -55°C, -60°C, -65°C, -70°C, -75°C, -80°C, -100°C, -120°C, -140°C, -150°C, -180°C, -196°C, etc.

[0100] In this application, when referring to "ultra-low temperature", unless otherwise specified, ultra-low temperature may be -50°C to -196°C, or any of the following temperatures or a range consisting of any two of the following temperatures: -50°C, -55°C, -60°C, -65°C, -70°C, -75°C, -80°C, -100°C, -120°C, -140°C, -150°C, -160°C, -180°C, -196°C, etc. The description of ultra-low temperature here is applicable at least to ultra-low temperature conditions for rapid freezing treatment.

[0101] In some embodiments, the leukocyte preparation comprises one or more non-viable leukocytes from blood, wherein the non-viable leukocytes have the basic structure of leukocytes and carry cytokine receptors but have substantially no proliferation capacity; wherein the cytokine receptors can bind to cytokines that normal blood leukocytes can bind to.

[0102] In some embodiments, the cytokines to which normal blood leukocytes can bind include cytokines to which normal blood leukocytes can bind during an inflammatory response.

[0103] Without limitation, cytokines that normal leukocytes can bind to may include one or more of interleukin 1 (IL-1), interleukin 6 (IL-6), interleukin 8 (IL-8), tumor necrosis factor α (TNF-α), interferon-α (IFN-α), interferon-β (IFN-β), interferon-γ (IFN-γ), and monocyte chemoattractant protein-1 (MCP-1), but are not limited thereto. In some embodiments, cytokines that normal leukocytes can bind to include one or more of interleukin 1 (IL-1), interleukin 6 (IL-6), interleukin 8 (IL-8), tumor necrosis factor α (TNF-α), and the like.

[0104] In some embodiments, the cytokines to which normal blood leukocytes can bind include cytokines to which normal blood leukocytes can bind in severe inflammation.

[0105] In some embodiments, cytokines that can be bound by normal blood leukocytes participate in the formation of a cytokine storm during severe inflammation.

[0106] In some embodiments, the non-viable leukocytes include one or more of monocytes, neutrophils, and lymphocytes. In some embodiments, the non-viable leukocytes include monocytes, which may further be monocytes. In some embodiments, the non-viable leukocytes include neutrophils, which may further be neutrophils. In some embodiments, the non-viable leukocytes include lymphocytes, which may further be lymphocytes.

[0107] In some embodiments, normal blood white blood cells include one or more of monocytes, neutrophils, and lymphocytes. In some embodiments, normal blood white blood cells include monocytes, and may further be monocytes. In some embodiments, normal blood white blood cells include neutrophils, and may further be neutrophils. In some embodiments, normal blood white blood cells include lymphocytes, and may further be lymphocytes. In some embodiments, normal blood white blood cells are all types of white blood cells in whole blood.

[0108] In some embodiments, the non-viable leukocytes include at least one of the same species of leukocytes as the normal blood leukocytes.

[0109] In some embodiments, the non-viable leukocytes together with normal blood leukocytes include one or more of monocytes, neutrophils, and lymphocytes.

[0110] In some embodiments, the non-viable leukocytes are of the same leukocyte type as normal blood leukocytes.

[0111] In some embodiments, non-viable leukocytes include all types of leukocytes in whole blood.

[0112] In some embodiments, the non-viable leukocytes are of the same leukocyte type as is present in whole blood.

[0113] In some embodiments, the non-viable leukocytes include dead leukocytes. Without limitation, the non-viable leukocytes may include dead cells of one or more of monocytes, neutrophils, and lymphocytes.

[0114] Without limitation, the leukocyte preparation can be a cell suspension, a cryopreservative, or a cell puree.

[0115] In this application, unless otherwise specified, the term "freezing agent" for leukocyte products refers to the frozen form of a cell suspension. The preparation and storage temperatures of the freezing agent can independently be cryogenic or ultracold. Non-limiting examples of "ultracold" temperatures that can be used during preparation include -50°C to -196°C, as defined above. Non-limiting examples of "low temperature" that can be used during storage include 4°C to -80°C, such as any one of the following temperatures, or a range consisting of any two of the following: 4°C, 2°C, 1°C, 0°C, -4°C, -5°C, -10°C, -15°C, -20°C, -25°C, -30°C, -40°C, -50°C, -60°C, -70°C, -80°C, etc. Leukocyte products should be stored in a manner (e.g., storage temperature) that best preserves their immunosuppressive effects. Storage can be performed in a manner that is more conducive to maintaining cytokine receptor activity. Generally, lower storage temperatures extend shelf life.

[0116] In this application, unless otherwise specified, "cell sludge" of a leukocyte product refers to the wet cell sediment collected by centrifuging a cell suspension. The cell suspension may be washed prior to centrifugation. The washing solution used may be phosphate buffered saline or physiological saline. The amount of washing solution used may be 3 to 20 times (e.g., 3, 4, 5, 8, 10, 15, 16, or 20 times) the volume of the material being washed. The number of washes may be 1 to 5 (e.g., 1, 2, 3, 4, or 5 times), but is not limited thereto.

[0117] In some embodiments, the leukocyte product is a pharmaceutical preparation. In this case, the dosage form of the leukocyte product can be a liquid preparation, and the above description can also be referred to.

[0118] In some embodiments, at least one or at least a portion of the non-viable leukocytes can be obtained by rapid freezing treatment, wherein the rapid freezing treatment temperature is ultra-low temperature, such as -50°C to -196°C.

[0119] In some embodiments, the non-viable leukocytes are the frozen leukocytes described in the second aspect of this application.

[0120] In some embodiments, the leukocyte product or the inactive leukocyte can be prepared using the method described in the third aspect of the present application.

[0121] In some embodiments, a leukocyte product or avital leukocytes can be prepared using a method comprising the following steps: extracting target leukocytes from ex vivo blood to prepare a leukocyte suspension; and rapidly freezing the leukocyte suspension at an ultra-low temperature (e.g., -50°C to -196°C) to produce the leukocyte product or avital leukocytes. See also the third aspect of this application.

[0122] In yet another aspect of the present application, the non-viable leukocytes described in the first aspect of the present application are provided.

[0123] The method provided in the third aspect of the present application can be used to prepare leukocyte products or inactive leukocytes, but is not limited thereto. Leukocyte products or inactive leukocytes obtained in other possible ways are within the scope of the present application as long as they meet the corresponding features in any embodiment or example.

[0124] In a second aspect of the present application, frozen leukocytes are provided. These frozen leukocytes can serve as the inert leukocytes described in the first aspect of the present application. These frozen leukocytes can recognize and competitively bind cytokines with normal immune cells, do not secrete additional cytokines, and do not induce inflammatory cascades. These frozen leukocytes can exert immunosuppressive effects without significant toxic side effects and can be used to prepare drugs for suppressing inflammation, such as drugs for suppressing severe inflammation.

[0125] In some embodiments, a frozen leukocyte is provided, which is obtained by subjecting normal blood leukocytes isolated from the body to rapid freezing treatment, wherein the rapid freezing treatment is performed at an ultra-low temperature, for example, -50°C to -196°C.

[0126] In this application, unless otherwise specified, "frozen leukocytes" refer to leukocytes obtained by rapid freezing treatment, and therefore may also be referred to as "frozen leukocytes".

[0127] In the context of this application, unless otherwise specified, "frozen monocytes" may refer to frozen monocytes, "frozen lymphocytes" may refer to frozen lymphocytes, and "frozen neutrophils" may refer to frozen neutrophils. Unless otherwise specified, the frozen monocytes, frozen monocytes, frozen lymphocytes, frozen lymphocytes, frozen neutrophils, and frozen neutrophils referred to herein are all within the scope of "frozen white blood cells" in this application.

[0128] In some embodiments, the frozen leukocytes are the non-viable leukocytes included in the leukocyte product described in the first aspect of the present application.

[0129] In some embodiments, the frozen white blood cells may include one or more of monocytes, neutrophils, and lymphocytes. In some embodiments, the frozen white blood cells include monocytes, and may further be frozen monocytes. In some embodiments, the frozen white blood cells include neutrophils, and may further be frozen neutrophils. In some embodiments, the frozen white blood cells include lymphocytes, and may further be frozen lymphocytes.

[0130] In some embodiments, the frozen white blood cells may include one or more of frozen monocytes, frozen neutrophils, and frozen lymphocytes. The method of obtaining the white blood cells is not particularly limited. The target white blood cell types may be collected at a desired ratio and then rapidly frozen simultaneously. Alternatively, different types of frozen white blood cells may be prepared separately and then mixed at a desired ratio, but the present invention is not limited thereto.

[0131] In some embodiments, the frozen leukocytes may be a combination of multiple frozen leukocytes, including but not limited to at least two of frozen monocytes, frozen neutrophils, and frozen lymphocytes.

[0132] In some embodiments, frozen leukocytes can be obtained by subjecting leukocytes to a single rapid freezing process, or by combining different types of frozen leukocytes obtained by separate rapid freezing processes. In the latter case, the different types of frozen leukocytes can be combined to obtain new frozen leukocytes, which can also be referred to as mixed frozen leukocytes.

[0133] In the third aspect of the present application, a method for preparing a leukocyte product or frozen leukocytes is provided, which can be used to prepare the leukocyte product described in the first aspect of the present application or the frozen leukocytes described in the second aspect of the present application.

[0134] In the third aspect of the present application, reference may be made to the method for preparing frozen leukocytes described in the second aspect of the present application.

[0135] In some embodiments, a method for preparing a leukocyte product or frozen leukocytes is provided (see Figure 1), which includes the following steps:

[0136] S100: Extract target leukocytes from isolated blood to prepare a leukocyte suspension;

[0137] S200: The leukocyte suspension is rapidly frozen to obtain a leukocyte product or frozen leukocytes (the aforementioned inactive leukocytes may also be obtained).

[0138] In step S200, the temperature for rapid freezing can be ultra-low temperature, which can be -50°C to -196°C. Please refer to the above description. In some embodiments, the leukocyte suspension is rapidly frozen under ultra-low temperature conditions.

[0139] The preparation method has few steps and is simple to operate. The prepared leukocyte products or frozen leukocytes can be used as a new type of low-toxic immunosuppressant.

[0140] The prepared target product (eg, leukocyte product or frozen leukocytes or non-viable leukocytes) may include one or more types of leukocytes.

[0141] When the target product includes only one type of leukocytes, the target leukocytes are extracted from the blood in vitro, prepared into a leukocyte suspension, and subjected to rapid freezing treatment to obtain the target product.

[0142] When the target product includes two or more types of leukocytes, the different types of leukocyte products can be prepared separately and then mixed in the desired ratio. Alternatively, all types of leukocytes can be extracted from ex vivo blood (e.g., whole blood) and then rapidly frozen simultaneously to prepare the target product. Alternatively, some types of leukocytes can be extracted from ex vivo blood (e.g., whole blood) and then mixed in the desired ratio and rapidly frozen to prepare the target product. For example, if the target product is frozen leukocytes, the target product can be obtained by combining different types of frozen leukocytes prepared separately by rapid freezing in the desired ratio.

[0143] In some embodiments, the leukocyte preparation or frozen leukocytes is prepared by mixing prepared frozen leukocytes of different types in a desired ratio.

[0144] In some embodiments, target leukocytes are extracted from ex vivo blood using the following method 1, method 2, or method 3:

[0145] Method 1: Ex vivo blood (e.g., whole blood) is centrifuged, the blood cell pellet is collected, the red blood cells are ruptured with an erythrocyte lysis buffer, washed, and centrifuged, and the white blood cell pellet is collected to obtain the target white blood cells (also known as "total white blood cells," which essentially includes all types of white blood cells in the ex vivo blood (e.g., whole blood). This method requires few steps, is simple to operate, and is suitable for industrial applications.

[0146] Method 2: Ex vivo blood (e.g., whole blood) is centrifuged, the blood cell pellet is collected, the red blood cells are ruptured with an erythrocyte lysis buffer, washed, and centrifuged, and the white blood cell pellet is collected. White blood cell separation is then used to obtain a specific type of white blood cell as the target white blood cell. This method allows for the selective preparation of specific frozen white blood cell types based on functional requirements.

[0147] Method 3: Ex vivo blood (e.g., whole blood) is centrifuged, the blood cell pellet is collected, the red blood cells are ruptured with an erythrocyte lysis buffer, washed and centrifuged, the white blood cell pellet is collected, and a leukocyte separation method is used to obtain specific types of white blood cells. The different types of white blood cells are then mixed in the desired ratio to obtain the target white blood cells. This method can be used to combine different types of frozen white blood cells in a specific ratio to meet multifunctional needs.

[0148] In the present application, the leukocyte separation method can employ existing technical means in the art, including but not limited to conventional technical means. As a non-limiting example, it can be implemented using a blood cell separator, a blood cell separation solution (such as Percoll cell separation solution), separation magnetic beads, etc. This is achievable by those skilled in the art.

[0149] S100: Extract target white blood cells and prepare a white blood cell suspension.

[0150] In step S100 , target leukocytes are extracted from blood in vitro to prepare a leukocyte suspension.

[0151] In some embodiments, the ex vivo blood is whole blood.

[0152] In some embodiments, the ex vivo blood is anticoagulated.

[0153] In some embodiments, the leukocyte suspension includes a resuspension and the extracted leukocytes of the target species. In a non-limiting manner, the composition of the resuspension can be: 0% to 90% cell culture medium, 0% to 90% serum and 0% to 30% dimethyl sulfoxide (DMSO) by volume percentage, and the resuspension includes at least one of the cell culture medium and serum. For example, the resuspension can be made of only cell culture medium, only serum, a combination of cell culture medium and DMSO, a combination of serum and DMSO, or a combination of cell culture medium, serum and DMSO. Those skilled in the art can select the composition of the appropriate resuspension according to the type and quantity of leukocytes to be extracted, for example, they can select the appropriate type and amount of cell culture medium, the appropriate type and amount of serum, and the appropriate amount of DMSO. As non-limiting examples, the cell culture medium can be any one of DMEM, RPMI 1640, MEM, DMEM / F12, M199, IMDM, L15, etc., and the serum can be any one of fetal bovine serum, calf serum, newborn bovine serum, fetal bovine serum substitute, other animal serum, synthetic serum substitute, etc.

[0154] In some embodiments, the leukocyte suspension primarily comprises a resuspension and the extracted leukocytes of the target species. For example, when the leukocytes of the target species in the form of a centrifugal pellet are mixed with the resuspension, the resulting mixture primarily comprises the leukocytes of the target species and the resuspension.

[0155] When the resuspension contains cell culture medium, the volume percentage of the cell culture medium in the resuspension can be any of the following percentages or an interval consisting of any two of the following percentages: 0%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, etc.

[0156] When the resuspension contains serum, the volume percentage of serum in the resuspension can be any of the following percentages or an interval consisting of any two of the following percentages: 0%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, etc. When the resuspension contains serum, the efficiency of leukocyte extraction is high, and a larger number of non-viable leukocytes or frozen leukocytes can be obtained.

[0157] When DMSO is included in the resuspension, the volume percentage of DMSO in the resuspension can be any of the following percentages, or a range consisting of any two of the following percentages: 0%, 5%, 10%, 15%, 20%, 25%, 30%, etc. Adding DMSO to the resuspension can help increase the number of non-viable or frozen leukocytes recovered. By adjusting the DMSO dosage within a suitable range, protein denaturation can be avoided, as well as cell disruption that could compromise the basic cellular morphology.

[0158] In some embodiments, the resuspension comprises a cell culture medium and serum in a volume ratio of (3-5):1, and can further consist of a cell culture medium and serum in a volume ratio of (3-5):1. The volume ratio of cell culture medium to serum can be any of the following ratios or an interval consisting of any two of the following ratios: 3:1, 3.5:1, 4:1, 4.5:1, 5:1, etc.

[0159] In some embodiments, the resuspension is composed of cell culture medium and serum in a volume ratio of 4:1. In this case, the resuspension contains 80% cell culture medium and 20% serum, calculated by volume percentage.

[0160] In some embodiments, in step S100 , the step of extracting target leukocytes from ex vivo blood (eg, whole blood) to prepare a leukocyte suspension is achieved using method A, method B, or method C.

[0161] In some embodiments, method A may include the following steps: diluting isolated blood (e.g., whole blood), centrifuging, collecting a blood cell pellet, disrupting red blood cells with a red blood cell lysis buffer, washing, and centrifuging, collecting a centrifugal pellet A, and resuspending the centrifugal pellet A into a white blood cell suspension. This method has few steps, is simple to operate, and is suitable for industrial application.

[0162] In some embodiments, method B may include the following steps: using a cell separation solution to separate one or more types of leukocytes of a target type from blood isolated from the body, and preparing a leukocyte suspension in a desired ratio.

[0163] In the present application, unless otherwise specified, in the step of extracting target leukocytes from ex vivo blood or in step S100, "preparing a leukocyte suspension in accordance with the required proportion" means preparing a leukocyte suspension of a specific type of collected leukocytes (the number of the specific type of leukocytes accounts for 100%), or preparing a leukocyte suspension of multiple specific types of leukocytes collected in different ways in accordance with the required proportion.

[0164] In some embodiments, method B may include the following steps: diluting ex vivo blood (e.g., whole blood), treating it with a cell separation fluid (e.g., Percoll cell separation fluid), and performing horizontal centrifugation to form multiple liquid layers, including an upper layer containing monocytes, a middle layer containing lymphocytes, and a bottom layer containing neutrophils and red blood cells; extracting at least one of the upper, middle, and bottom layers to obtain at least one target extraction layer; washing and centrifuging the at least one target extraction layer to obtain a centrifugal precipitate of the corresponding target extraction layer; and resuspending the centrifugal precipitate of each target extraction layer into a white blood cell suspension according to a desired ratio. This method can achieve the selective combination of specific types of frozen white blood cells.

[0165] In some embodiments, in approach B,

[0166] When one of the target extraction layers is the upper layer, at least the upper layer is extracted, and then the extracted upper layer is washed and centrifuged to collect the centrifugal precipitate B1. At this time, the centrifugal precipitates of each target extraction layer include the centrifugal precipitate B1.

[0167] When one of the target extraction layers is the middle layer, at least the middle layer is extracted, and then the extracted middle layer is washed and centrifuged to collect the centrifugal precipitate B2. At this time, the centrifugal precipitates of each target extraction layer include the centrifugal precipitate B2.

[0168] When one of the target extraction layers is the bottom layer, at least the bottom layer is extracted, and then the extracted bottom layer is subjected to red blood cell rupture and centrifugation using red blood cell lysis solution, and the centrifugal precipitate B31 is collected. The centrifugal precipitate B31 is washed and centrifuged to obtain the centrifugal precipitate B32. At this time, the centrifugal precipitates of each target extraction layer include the centrifugal precipitate B3.

[0169] In some embodiments, method C may include the following steps: processing the ex vivo blood through a blood component separator to separate the white blood cell components therein, obtain one or more target types of white blood cells, and prepare a white blood cell suspension according to the required ratio.

[0170] In some embodiments, during the step of diluting the isolated blood, the diluent may be phosphate buffered saline or physiological saline. Without limitation, the amount of the diluent may be 3 to 20 times the volume of the isolated blood, for example, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 16, 18, 20, etc., or may be any range consisting of any two of the foregoing multiples.

[0171] In some embodiments, the centrifugal precipitate of each target extraction layer is a mixture of centrifugal precipitates of multiple target extraction layers or is the centrifugal precipitate of one target extraction layer.

[0172] S200: Rapid freezing process.

[0173] In step S200, the leukocyte suspension is rapidly frozen to obtain a leukocyte product or inactive leukocytes or frozen leukocytes.

[0174] In some embodiments, the rapid freezing process can be achieved using at least one of a freezing medium and a freezing device. Without limitation, the freezing medium can include at least one of liquid nitrogen, liquid carbon dioxide, liquefied air, and the like. The freezing device can include, but is not limited to, an ultra-low temperature freezer. As an example, an ultra-low temperature freezer can provide at least one temperature between -50°C and -196°C, and optionally at least one temperature between -50°C and -150°C, as described above.

[0175] In some embodiments, after the rapid freezing treatment and before the leukocyte product or frozen leukocytes are prepared, the frozen product obtained by the rapid freezing treatment is further thawed. Without limitation, the thawing temperature can be 4°C to 37°C, or any one of the following temperatures or an interval consisting of any two of the following temperatures: 4°C, 5°C, 6°C, 8°C, 10°C, 12°C, 15°C, 16°C, 18°C, 20°C, 22°C, 24°C, 25°C, 26°C, 28°C, 30°C, 32°C, 34°C, 35°C, 36°C, 37°C, etc. In some embodiments, the thawing temperature is 37°C.

[0176] In some embodiments, after the rapid freezing treatment and before the preparation of the leukocyte product or the frozen leukocytes, the frozen material obtained by the rapid freezing treatment is further thawed and centrifuged. Furthermore, a washing step is included or not included after the thawing and before the centrifugation.

[0177] In some embodiments, in step S100 and step S200, each washing step can independently adopt the following washing method: the washing solution can be phosphate buffered saline or physiological saline, the amount of washing solution can be 3 to 20 times the volume of the object being washed, and the number of washes is 1 to 5 times (e.g., 1, 2, 3, 4, or 5 times). The amount of washing solution can be 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 16, 18, 20 times the volume of the object being washed, etc., and can also be a range consisting of any two of the foregoing multiples.

[0178] In some embodiments, in step S100 and step S200, each centrifugation step independently employs the following centrifugation method: a centrifugation speed of 200 g to 800 g, and a centrifugation time of 2 min to 10 min. Without limitation, the centrifugation speed may be any one of the following speeds, or a range consisting of any two of the following speeds: 200 g, 250 g, 300 g, 350 g, 450 g, 500 g, 550 g, 600 g, 650 g, 700 g, 750 g, 800 g, etc. Without limitation, the centrifugation time may be any one of the following durations, or a range consisting of any two of the following durations: 2 min, 3 min, 4 min, 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, etc.

[0179] In some embodiments, a method for preparing frozen leukocytes may include the following steps: extracting target leukocytes from ex vivo blood (e.g., whole blood) to prepare a leukocyte suspension; rapidly freezing the leukocyte suspension at ultra-low temperatures (to render the leukocytes inactive, e.g., to cause leukocyte death) to obtain frozen cells; thawing the frozen cells, washing, centrifuging, and collecting the cell pellet to obtain the frozen leukocytes. For details on how to implement the rapid freezing process, please refer to the relevant description above.

[0180] In some embodiments, the method for preparing frozen monocytes may include the following steps: extracting monocytes (the target white blood cells are monocytes) from ex vivo blood (such as whole blood), preparing a monocyte suspension (as a white blood cell suspension), and rapidly freezing the monocyte suspension to prepare frozen monocytes.

[0181] In some embodiments, the method for preparing frozen monocytes may include the following steps: diluting ex vivo blood (such as whole blood), treating it with a cell separation fluid (such as Percoll cell separation fluid), performing horizontal centrifugation to form multiple liquid layers, the multiple liquid layers including at least an upper layer containing monocytes, extracting the upper layer to obtain a mononuclear cell extract, washing and centrifuging the mononuclear cell extract to obtain a mononuclear cell precipitate (centrifugal precipitate B1), resuspending the mononuclear cell precipitate to form a mononuclear cell suspension, rapidly freezing the mononuclear cell suspension to obtain frozen monocytes, rethawing the frozen monocytes, washing, centrifuging, and collecting the centrifugal precipitate to obtain frozen monocytes.

[0182] In some embodiments, the method for preparing frozen lymphocytes may include the following steps: extracting lymphocytes (the target white blood cells are lymphocytes) from ex vivo blood (such as whole blood), preparing a lymphocyte suspension (as a white blood cell suspension), and rapidly freezing the lymphocyte suspension to obtain frozen lymphocytes.

[0183] In some embodiments, the method for preparing frozen lymphocytes may include the following steps: diluting isolated blood (such as whole blood), treating it with a cell separation fluid (such as Percoll cell separation fluid), performing horizontal centrifugation to form multiple liquid layers, the multiple liquid layers including at least a middle layer containing lymphocytes, extracting the middle layer to obtain a lymphocyte extract, washing and centrifuging the lymphocyte extract to obtain a lymphocyte precipitate (centrifugal precipitate B2), resuspending the lymphocyte precipitate to form a lymphocyte suspension, rapidly freezing the lymphocyte suspension to obtain frozen lymphocytes, rethawing the frozen lymphocytes, washing, and centrifuging them, and collecting the centrifugal precipitate to obtain frozen lymphocytes.

[0184] In some embodiments, the method for preparing frozen neutrophils may include the following steps: extracting neutrophils (the target white blood cells are neutrophils) from ex vivo blood (such as whole blood), preparing a neutrophil suspension (as a white blood cell suspension), and rapidly freezing the neutrophil suspension to obtain frozen neutrophils.

[0185] In some embodiments, the method for preparing frozen neutrophils may include the following steps: diluting ex vivo blood (such as whole blood), treating it with a cell separation fluid (such as Percoll cell separation fluid), and performing horizontal centrifugation to form multiple liquid layers, the multiple liquid layers including at least a bottom layer containing neutrophils and red blood cells, extracting the bottom layer, then using a red blood cell lysis fluid to rupture the red blood cells and centrifuge the extracted bottom layer, collecting the centrifugal precipitate B31, washing and centrifuging the centrifugal precipitate B31 to obtain a neutrophil precipitate (centrifugal precipitate B32); resuspending the neutrophil precipitate to form a neutrophil suspension, rapidly freezing the neutrophil suspension to obtain frozen neutrophils, rethawing the frozen neutrophils, washing, centrifuging, and collecting the centrifugal precipitate to obtain frozen neutrophils.

[0186] In some embodiments, the leukocyte product prepared in the third aspect of the present application is the leukocyte product of the first aspect of the present application.

[0187] In some embodiments, the frozen leukocytes prepared in the third aspect of the present application are the frozen leukocytes in the second aspect of the present application.

[0188] It will be appreciated that this method can also be used to prepare the non-viable leukocytes described in the first aspect.

[0189] In some embodiments, a method for preparing non-viable leukocytes is provided, comprising the following steps:

[0190] S100: Extract target leukocytes from isolated blood to prepare a leukocyte suspension;

[0191] S200: The leukocyte suspension is rapidly frozen to obtain inactive leukocytes.

[0192] In the fourth aspect of the present application, there is provided an application of the leukocyte product in the first aspect of the present application, or the aforementioned non-viable leukocytes, or the frozen leukocytes in the second aspect of the present application, or the leukocyte product or frozen leukocytes prepared by the preparation method in the third aspect of the present application, which application at least includes application in the preparation of drugs for inhibiting inflammation, such as at least application in the preparation of drugs for inhibiting severe inflammation.

[0193] Without limitation, the application may include at least one of suppressing the body's inflammatory response and controlling cytokine storm.

[0194] In some embodiments, drugs that suppress inflammation are used to control cytokine storm.

[0195] In some embodiments, the inflammation is severe inflammation, and in this case, the drug that suppresses inflammation is a drug that suppresses severe inflammation. Further, the drug that suppresses severe inflammation is used to control cytokine storm.

[0196] It is generally known to those skilled in the art that severe inflammation (such as severe pneumonia) is different from ordinary inflammation. If left untreated, there is a higher risk of death.

[0197] In some embodiments, the severe inflammatory disease is severe pneumonia.

[0198] In the fifth aspect of the present application, a method for inhibiting inflammation is also provided, which can provide the use of the leukocyte product in the first aspect of the present application, or the aforementioned non-viable leukocytes, or the frozen leukocytes in the second aspect of the present application, or the leukocyte product or frozen leukocytes prepared by the preparation method in the third aspect of the present application in inhibiting inflammation.

[0199] In some embodiments, the method for inhibiting inflammation or the use thereof in inhibiting inflammation comprises: administering to a subject a therapeutically effective amount of a functional component, the functional component comprising at least one of the leukocyte product of the first aspect of the present application, the aforementioned non-viable leukocytes, the frozen leukocytes of the second aspect of the present application, and the leukocyte product and frozen leukocytes prepared by the preparation method of the third aspect of the present application; further, the functional component is the leukocyte product of the first aspect of the present application, or the aforementioned non-viable leukocytes, or the frozen leukocytes of the second aspect of the present application, or the leukocyte product or frozen leukocytes prepared by the preparation method of the third aspect of the present application.

[0200] In some embodiments, the inhibition of inflammation is the inhibition of severe inflammation, and further can be severe pneumonia. The definition of severe inflammation can be found in the fourth aspect of this application.

[0201] In this application, unless otherwise specified, a subject refers to an animal, preferably a mammal, and further can be a human. The term "mammal" primarily refers to warm-blooded vertebrate mammals, including but not limited to cats, dogs, rabbits, bears, foxes, wolves, monkeys, deer, mice, pigs, cattle, sheep, horses, and humans.

[0202] In some embodiments, the subject is a human or a mouse. In some embodiments, the subject is a human.

[0203] In this application, "therapeutically effective amount" refers to the amount of a pharmaceutically active ingredient that will elicit a biological or medical response in an individual for a disease, disorder and / or symptom, such as an amount that produces a pharmacologically positive effect in an individual.

[0204] The embodiments of the present application will be described in detail below in conjunction with some examples. It should be understood that these examples are only used to illustrate the present application and are not intended to limit the scope of the present application. The experimental methods for which the conditions are not specified in the following examples are preferably referred to the guidance provided in the present application, and can also be based on the experimental manuals or conventional conditions in this area, or according to the conditions recommended by the manufacturer, or with reference to experimental methods known in the art.

[0205] In the following examples, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument testing accuracy or operating accuracy are allowed.

[0206] In the following examples, the dosage multiples of the diluent or the washing solution, unless otherwise specified, refer to the volume multiples.

[0207] In the following examples, unless otherwise specified, “equal dilution” refers to diluting the substance to be diluted to 50% of its original volume with the diluent at a ratio of 1:1.

[0208] 1. Preparation of frozen leukocytes (a type of non-viable leukocytes) and leukocyte preparations

[0209] Example 1. Preparation of frozen leukocytes

[0210] After collecting blood (whole blood, anticoagulated), dilute the whole blood with 10x volume of phosphate buffer or normal saline. Centrifuge at 700g for 8 minutes, and retain the cell pellet at the bottom of the centrifuge tube. Add an appropriate amount of red blood cell lysis buffer to rupture the red blood cells. Centrifuge at 450g for 5 minutes. The pellet at the bottom of the centrifuge tube is the white blood cells (white blood cells that have not been rapidly frozen) in the whole blood. Wash the white blood cells twice with 10x volume of phosphate buffer or normal saline, centrifuge, and set aside.

[0211] The white blood cells are suspended in a cell suspension (the composition of the resuspension is: 80% cell culture medium, 20% serum, by volume) using a resuspension solution, and quickly immersed in liquid nitrogen for rapid freezing (i.e., rapid freezing treatment). Before use, the cells are thawed at 37 degrees Celsius (37°C), washed with 10 times the volume of phosphate buffer or saline, and centrifuged at 250g for 3 minutes to obtain frozen white blood cells. In this example, the preparation flow chart of frozen white blood cells can be found in Figure 2 .

[0212] Example 2. Preparation of frozen monocytes

[0213] After blood collection (whole blood, anticoagulated), the whole blood was diluted with an equal volume of phosphate buffer in a clean bench. Percoll stock solution (100%, by volume percentage) was prepared according to a volume ratio of Percoll stock solution to 0.85% NaCl of 9:1. The Percoll stock solution was diluted with phosphate buffer to prepare 25% and 62% Percoll suspensions.

[0214] Place the pre-prepared 62% and 25% Percoll suspensions in the centrifuge tube from bottom to top, slowly add the equally diluted whole blood to the Percoll cell separation solution, and centrifuge horizontally at 400g, 25°C, for 20 minutes. Set the tube to slowly descend at the end to avoid sudden stops. At the end of the centrifugation, the cells are layered, with plasma on the surface, neutrophils and red blood cells at the bottom, monocytes on the upper layer, and lymphocytes in the middle layer.

[0215] The top layer of cells was aspirated and transferred to a new centrifuge tube. 10 times the amount of phosphate buffer was added to mix and wash thoroughly. After centrifugation, the cells were suspended in a cell suspension (the composition of the resuspension was 80% cell culture medium and 20% serum, by volume percentage). The cells were quickly immersed in liquid nitrogen and frozen. The cells were thawed at 37°C before use and centrifuged at 250 g for 3 min to obtain frozen mononuclear cells.

[0216] Example 3. Preparation of frozen lymphocytes

[0217] After blood collection (whole blood, anticoagulated), the whole blood was diluted with an equal volume of phosphate buffer in a clean bench. A Percoll stock solution (100%, by volume percentage) was prepared at a volume ratio of 9:1 between the Percoll stock solution and 0.85% NaCl. The Percoll stock solution was diluted with phosphate buffer to prepare 25% and 62% Percoll suspensions.

[0218] Place the pre-prepared 62% and 25% Percoll suspensions in the centrifuge tube from bottom to top, slowly add the equally diluted whole blood to the Percoll cell separation solution, and centrifuge horizontally at 400g, 25°C, for 20 minutes. Set the tube to slowly descend at the end to avoid sudden stops. At the end of the centrifugation, the cells are layered, with plasma on the surface, neutrophils and red blood cells at the bottom, monocytes on the upper layer, and lymphocytes in the middle layer.

[0219] The middle layer cells were aspirated and transferred to a new centrifuge tube. 10 times the amount of phosphate buffer was added for thorough mixing and washing. After centrifugation, the cells were suspended in a cell suspension (the composition of the resuspension was 80% cell culture medium and 20% serum, by volume percentage). The cells were quickly immersed in liquid nitrogen for freezing. The cells were thawed at 37°C before use and centrifuged at 300 g for 3 min to obtain frozen lymphocytes.

[0220] Example 4. Preparation of frozen neutrophils

[0221] After blood collection (whole blood, anticoagulated), the whole blood was diluted with an equal volume of phosphate buffer in a clean bench. A Percoll stock solution (100%, by volume percentage) was prepared at a volume ratio of 9:1 between the Percoll stock solution and 0.85% NaCl. The Percoll stock solution was diluted with phosphate buffer to prepare 25% and 62% Percoll suspensions.

[0222] Place the pre-prepared 62% and 25% Percoll suspensions in the centrifuge tube from bottom to top, slowly add the equally diluted whole blood to the Percoll cell separation solution, and centrifuge horizontally at 400g, 25°C, for 20 minutes. Set the tube to slowly descend at the end to avoid sudden stops. At the end of the centrifugation, the cells are layered, with plasma on the surface, neutrophils and red blood cells at the bottom, monocytes on the upper layer, and lymphocytes in the middle layer.

[0223] Add an appropriate amount of red blood cell lysis solution diluted with 10 times the amount of deionized water to the cells at the bottom layer to rupture the red blood cells. After centrifugation at 450g for 5 minutes, add 10 times the amount of phosphate buffer to the precipitate at the bottom of the centrifuge tube, mix thoroughly, wash, and centrifuge to obtain neutrophils.

[0224] The neutrophils isolated above were suspended in a cell suspension (the composition of the resuspension was: 80% cell culture medium and 20% serum, by volume percentage), and quickly immersed in liquid nitrogen for freezing. Before use, the cells were thawed at 37°C and centrifuged at 250 g for 3 minutes to obtain frozen neutrophils.

[0225] Example 5. Different types of frozen leukocytes were mixed in proportion to prepare mixed frozen leukocytes.

[0226] The different types of frozen leukocytes prepared in Example 2, Example 3, and Example 4 were mixed at a cell number ratio of 1:4:5 to obtain mixed frozen leukocytes.

[0227] Example 6. Characterization of frozen leukocyte structure

[0228] Instrument model and scanning parameters: The laser confocal characterization was performed using the Keyence All-in-One Fluorescence Microscopy System BZ-X800 instrument with the following shooting parameters: resolution 1920×1440, grayscale level 48 bit; the scanning electron microscope used the ultra-high resolution scanning electron microscope Regulus (Hitachi), model Regulus-8100, with the following scanning parameters: acceleration voltage 10 kV, working distance 8.4 mm, SE (UL) mode.

[0229] Laser confocal microscopy characterization: Rhodamine-labeled phalloidin (red fluorescent reagent) and Hoechst 33342 (blue fluorescent reagent) were used to stain the cytoskeleton F-actin and cell nucleus in frozen leukocytes (Example 1), and the morphological structure was observed using a laser confocal microscopy (see Figure 3 ). Figure 3 From left to right, the bright field image, the cell nucleus staining result, the cytoskeleton staining result, and the superposition effect of the cell nucleus and cytoskeleton counterstaining (merged image) are shown.

[0230] Scanning electron microscopy (SEM): Frozen leukocytes (Example 1) were centrifuged and washed, then added to a 2.5% (v / v) glutaraldehyde solution and fixed at 4°C for 12 h. The upper glutaraldehyde solution was centrifuged and discarded. After washing with phosphate buffer, the cells were fixed with 1% (v / v) osmium phosphate for 2 h. After fixation, the cells were washed three times with phosphate buffer (PBS). The cells were dehydrated using a gradient of ethanol solutions of varying concentrations (30%, 50%, 70%, 80%, and 90% ethanol, once for 15 min each, and 100% ethanol, twice for 20 min each). The cell samples were dropped onto silicon wafers, evaporated, and then gold-plated. The cells were then photographed using a scanning electron microscope (see [1] for details). Figure 4 ).

[0231] See Figure 3 and Figure 4 The frozen white blood cells obtained have a cell structure similar to that of living cells and are spherical in shape. It can be seen that the frozen white blood cells still have the basic structure of white blood cells and can still maintain the basic morphology of the cells.

[0232] Example 7. In vitro proliferation assay of frozen leukocytes

[0233] White blood cells that had not been rapidly frozen and frozen white blood cells (Example 1) were suspended in cell culture medium (the resuspension medium was 90% cell culture medium and 10% serum, by volume percentage) and seeded into 96-well plates at a cell density of 5×10 3 After culturing for 0.5 h, 24 h, 48 h, and 72 h in different experimental groups, 10 μL of CCK-8 solution was added to each well. After incubation for 3 h, the absorbance was measured at 450 nm using a microplate reader. Figure 5 According to the test results, the CCK8 value of frozen leukocytes did not change significantly after 72 hours of incubation in vitro, and it can be considered that the cells have lost their proliferation ability.

[0234] Example 8. Cytokine receptor expression test of frozen leukocytes

[0235] Take 1×10 6 Frozen leukocytes (Example 1) were resuspended in 500 μL of phosphate buffered saline (PBS) containing 1% (v / v) serum. Fluorescently labeled antibodies APC-CXCR2 (interleukin-8 receptor), APC-TLR4 (lipopolysaccharide receptor), PE-CD120a (interleukin-1 receptor), PE-gp130 (interleukin-1 receptor), PE-CD121a (tumor necrosis factor α receptor), and PE-CD14 (lipopolysaccharide receptor) were added, respectively. The cells were incubated on ice for 15-20 minutes. After the incubation, 10 mL of PBS (containing 1% (v / v) fetal bovine serum (FBS)) was added and centrifuged at 250 g for 5 minutes. The cells were washed once with PBS and analyzed by flow cytometry. Flow cytometric analysis results can be found at Figure 6 According to the test results, the above-mentioned cytokine receptors (interleukin-8 receptor, lipopolysaccharide receptor, interleukin-1 receptor, interleukin-1 receptor and tumor necrosis factor α receptor) were all clearly present on the frozen white blood cells.

[0236] Example 9. Frozen Leukocyte Cytokine Adsorption Test

[0237] Frozen leukocytes (Example 1) were incubated with FAM (fluorescein, maleimidofluorescein) fluorescently labeled interleukin-6. After 1 hour, the unadsorbed interleukin-6 was removed by centrifugation. After washing with phosphate buffer, the cells were photographed using a laser confocal microscope. The results can be found in Figure 7 According to the test results, frozen leukocytes have obvious fluorescent signals, which shows that frozen leukocytes can adsorb cytokines. Figure 7 From left to right in the middle, the bright field image, cell nucleus staining results, IL-6 staining results, and the overlay effect of cell nucleus and IL-6 counterstaining (overlay image) are shown.

[0238] Example 10. Cytokine secretion capacity of frozen leukocytes

[0239] The frozen leukocytes (Example 1) were collected at 1×10 5 Cells were seeded into 24-well plates and stimulated with lipopolysaccharide (50 ng / mL). After 8 hours, the cell culture medium was collected and centrifuged at 300 g for 10 minutes. The supernatant was collected and assayed for various cytokine levels (IL-6, IL-8, IL-1, and TNF-α) using enzyme-linked immunosorbent assay kits. A control group was treated with saline solution instead of lipopolysaccharide. Results can be found in the table below. Figure 8 According to the test analysis results, after being stimulated by lipopolysaccharide, the levels of various cytokines in the culture medium did not increase significantly, indicating that the frozen white blood cells had lost the ability to secrete cytokines.

[0240] Example 11. Investigation of the in vitro immunosuppressive activity of frozen leukocytes

[0241] Macrophages were cultured at a rate of 1×10 5 Each well was inoculated with 1×10 cells / well in a 24-well plate. After 12 h, lipopolysaccharide (50 ng / mL) was added to each well for stimulation. After 2 h, the preparations of each group (normal saline as the control group, 1×10 frozen leukocytes in Example 1) were added. 5 The cells were incubated for 2 h, 4 h, 6 h, and 22 h, and the cell culture medium was collected and centrifuged at 300 g for 10 min. The supernatant was collected and the levels of various cytokines (IL-6, IL-1, and TNF-α) were detected using enzyme-linked immunosorbent assay kits.

[0242] The results can be found in Figure 9 According to the test analysis results, the amount of cytokine secretion by macrophages was significantly reduced in the presence of frozen white blood cells. It is speculated that this may be due to the lipopolysaccharide receptors on the surface of frozen white blood cells adsorbing some free lipopolysaccharide, reducing the stimulatory effect of free lipopolysaccharide on macrophages.

[0243] Example 12. In vitro immunosuppressive activity of various types of frozen leukocytes

[0244] Mononuclear macrophage J774A.1 cells were seeded into 24-well plates at a cell density of 1×10 5 Each well was stimulated by adding lipopolysaccharide (50 ng / ml) prepared with blank culture medium. After 2 hours, physiological saline, 1×10 frozen leukocytes in Example 1, and 1×10 5 1×10 cells / well, frozen mononuclear cells in Example 2 5 / well, frozen lymphocytes in Example 3 1×10 5 / well, and 1×10 frozen neutrophils in Example 4 5 The cells were incubated for 8 h, and the cell culture supernatant was collected and centrifuged at 300 g for 10 min. The supernatant was collected and the levels of various cytokines were detected using an enzyme-linked immunosorbent assay kit. Figure 10 Compared with single-type frozen leukocytes (frozen monocytes, frozen lymphocytes, and frozen neutrophils), the frozen leukocyte group had the strongest inhibitory effect on cytokine secretion. Compared with the normal saline group, the secretion of interleukin-6 decreased by 83.7%, the secretion of interleukin-1 decreased by 48.1%, and the secretion of tumor necrosis factor-α decreased by 80.0%.

[0245] Example 13. In vivo anti-inflammatory efficacy of frozen leukocytes

[0246] Pneumonia model (severe pneumonia model): After fasting for 24 hours, mice were intratracheally instilled with lipopolysaccharide (10 mg / kg) to establish a severe pneumonia model. 4 hours after model establishment, normal saline (control group), 1×10 6 Frozen white blood cells (Example 1), 1×10 6 Normal blood white blood cells (labeled as the "white blood cell" group). The results can be found in Figure 11 The survival period of mice injected with frozen leukocytes was significantly prolonged, with the median survival period doubled compared with the saline group (saline control group: 3 days; frozen leukocytes: 6 days). However, the survival period of mice injected with unfrozen normal leukocytes was significantly shortened compared with the saline group (saline control group: 3 days; normal leukocytes: 2 days), indicating that frozen leukocytes have a significantly enhanced anti-inflammatory effect in vivo.

[0247] The technical features of the above-mentioned embodiments and examples can be combined in any appropriate manner. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments and examples are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0248] The above embodiments and examples only express several embodiments of the present application, which are convenient for understanding the technical solutions of the present application in detail, but they cannot be understood as limiting the scope of protection of the present application. It should be pointed out that for ordinary technicians in this field, without departing from the concept of the present application, several variations and improvements can be made, which all fall within the scope of protection of the present application. In addition, it should be understood that after reading the above teaching content of the present application, those skilled in the art can make various changes or modifications to the present application, and the equivalent forms obtained also fall within the scope of protection of the present application. It should also be understood that the technical solutions obtained by those skilled in the art through logical analysis, reasoning or limited experiments on the basis of the technical solutions provided in the present application are all within the scope of protection of the claims attached to the present application. Therefore, the scope of protection of the patent of this application shall be based on the content of the attached claims, and the description and drawings can be used to interpret the content of the claims.

Claims

1. A leukocyte product, characterized in that The non-viable leukocytes include one or more non-viable leukocytes from the blood, wherein the non-viable leukocytes have the basic structure of leukocytes and carry cytokine receptors, but the non-viable leukocytes have substantially no proliferation ability; The cytokine receptor is capable of binding to cytokines that can be bound by normal blood leukocytes.

2. The leukocyte product according to claim 1, characterized in that The cytokines to which normal blood leukocytes can bind include cytokines to which normal blood leukocytes can bind in inflammatory responses; Optionally, the cytokines to which the normal blood leukocytes can bind include one or more of interleukin 1, interleukin 6, interleukin 8, interleukin 12, interleukin 17, tumor necrosis factor α, interferon-α, interferon-β, interferon-γ and monocyte chemoattractant protein-1; Optionally, the cytokines to which normal blood leukocytes can bind include cytokines to which normal blood leukocytes can bind in severe inflammation; Optionally, the cytokines that the normal blood leukocytes can bind to participate in the formation of a cytokine storm in the severe inflammation.

3. The leukocyte product according to claim 1 or 2, characterized in that Meet one or more of the following characteristics: The non-viable leukocytes include one or more of monocytes, neutrophils and lymphocytes; The normal blood leukocytes include one or more of monocytes, neutrophils and lymphocytes; The non-viable leukocytes and the normal blood leukocytes together include one or more of monocytes, neutrophils and lymphocytes.

4. The leukocyte product according to any one of claims 1 to 3, characterized in that The non-viable leukocytes include dead leukocytes; Optionally, the non-viable leukocytes include dead cells of one or more leukocytes among monocytes, neutrophils and lymphocytes.

5. The leukocyte product according to any one of claims 1 to 3, characterized in that The leukocyte product is a cell suspension, a cryoprotectant, or a cell mud; Optionally, the leukocyte product is a pharmaceutical preparation.

6. A frozen leukocyte, characterized in that: The non-viable leukocytes are those included in the leukocyte product according to any one of claims 1 to 5; the frozen leukocytes are obtained by subjecting normal blood leukocytes isolated from the body to rapid freezing, wherein the rapid freezing temperature is between -50°C and -196°C; Optionally, the frozen leukocytes include one or more of frozen monocytes, frozen neutrophils and frozen lymphocytes.

7. A method for preparing a leukocyte product or frozen leukocytes, characterized in that: The steps include: Extract target leukocytes from isolated blood to prepare a leukocyte suspension; The leukocyte suspension is subjected to rapid freezing treatment to obtain the leukocyte product or the frozen leukocytes, wherein the temperature for the rapid freezing treatment is -50°C to -196°C.

8. The method for preparing a leukocyte product or frozen leukocytes according to claim 7, characterized in that: Meet one or more of the following characteristics: The isolated blood is whole blood; The isolated blood is subjected to anticoagulation treatment; The step of extracting target leukocytes from the ex vivo blood to prepare a leukocyte suspension is achieved using Method A, Method B, or Method C; wherein Method A comprises the following steps: diluting the ex vivo blood, centrifuging, collecting blood cell sediment, rupturing the red blood cells with a red blood cell lysis solution, washing and centrifuging, collecting centrifugal sediment A, and resuspending the centrifugal sediment A to prepare the leukocyte suspension; Method B comprises the following steps: separating one or more target types of leukocytes from the ex vivo blood with a cell separation solution and preparing a leukocyte suspension in a desired ratio; optionally, Method B comprises the following steps: diluting the ex vivo blood, treating it with a cell separation solution, and horizontally centrifuging to form multiple liquid phase layers, the multiple liquid phase layers comprising an upper layer containing monocytes, a middle layer containing lymphocytes, and a bottom layer containing neutrophils and red blood cells, extracting at least one of the upper layer, the middle layer, and the bottom layer to obtain at least one target extraction layer, washing and centrifuging the at least one target extraction layer to obtain centrifugal sediments of the corresponding target extraction layers, and resuspending the centrifugal sediments of each target extraction layer in a desired ratio. The method comprises the steps of: extracting the blood from the blood sample and performing a leukocyte suspension resuspending the blood sample into the leukocyte suspension; wherein, when one of the target extraction layers is the upper layer, at least the upper layer is extracted, and then the extracted upper layer is washed and centrifuged to collect a centrifugal precipitate B1. In this case, the centrifugal precipitates of each target extraction layer include the centrifugal precipitate B1; when one of the target extraction layers is the middle layer, at least the middle layer is extracted, and then the extracted middle layer is washed and centrifuged to collect a centrifugal precipitate B2. In this case, the centrifugal precipitates of each target extraction layer include the centrifugal precipitate B2; when one of the target extraction layers is the bottom layer, at least the bottom layer is extracted, and then the extracted bottom layer is subjected to erythrocyte lysis treatment and centrifugation using an erythrocyte lysis solution to collect a centrifugal precipitate B31. The centrifugal precipitate B31 is washed and centrifuged to obtain a centrifugal precipitate B32. In this case, the centrifugal precipitates of each target extraction layer include the centrifugal precipitate B3; the method C comprises the following steps: subjecting the isolated blood to a blood component separator to separate the leukocyte components therein, obtaining one or more leukocytes of the target type, and preparing a leukocyte suspension according to a desired ratio; The leukocyte suspension comprises a resuspension and the extracted target leukocytes; wherein, the resuspension is composed of, by volume percentage, 0% to 90% cell culture medium, 0% to 90% serum, and 0% to 30% dimethyl sulfoxide, and the resuspension comprises at least one of the cell culture medium and the serum; The rapid freezing process is achieved by using at least one of a freezing medium and a freezing device, wherein the freezing medium includes at least one of liquid nitrogen, liquid carbon dioxide, and liquefied air, and the freezing device includes an ultra-low temperature refrigerator; the ultra-low temperature refrigerator can provide at least one temperature between -50°C and -196°C; After the rapid freezing treatment and before the leukocyte product or the frozen leukocytes are obtained, the method further comprises thawing the frozen product obtained by the rapid freezing treatment, wherein the thawing temperature is 4° C. to 37° C.; The leukocyte product or frozen leukocytes is prepared by mixing prepared frozen leukocytes of different types in a desired ratio.

9. The method for preparing a leukocyte product or frozen leukocytes according to claim 8, characterized in that: Meet one or more of the following characteristics: In the step of diluting the isolated blood, the diluent is phosphate buffer or physiological saline, and the amount of the diluent is 3 to 20 times the volume of the isolated blood; The centrifugal precipitate of each target extraction layer is a mixture of centrifugal precipitates of multiple target extraction layers or a centrifugal precipitate of one target extraction layer; After the rapid freezing treatment and before the leukocyte product or the frozen leukocytes are obtained, the method further comprises thawing and centrifuging the frozen product obtained by the rapid freezing treatment, with or without a washing step after thawing and before centrifugation; Each washing step independently adopts the following washing method: the washing solution is phosphate buffer or physiological saline, the amount of the washing solution is 3 to 20 times the volume of the washed object, and the number of washing times is 1 to 5 times; Each centrifugation step independently adopts the following centrifugation mode: centrifugation speed is 200g~800g, and centrifugation time is 2min~10min; The temperature for performing the rethawing is 37°C; The prepared leukocyte product is the leukocyte product according to any one of claims 1 to 5; The prepared frozen leukocytes are the frozen leukocytes according to claim 6.

10. Use of the leukocyte product according to any one of claims 1 to 5, or the frozen leukocytes according to claim 6, or the leukocyte product or frozen leukocytes prepared by the preparation method according to any one of claims 7 to 9 in the preparation of a drug for inhibiting inflammation; Optionally, the drug for suppressing inflammation is a drug for suppressing severe inflammation; Optionally, the severe inflammation is severe pneumonia; Optionally, the drug that suppresses inflammation is used to control cytokine storm.

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