Applications of peripheral blood concentrated cells and concentrated supernatant in the treatment of premature ovarian failure

Abstract

This invention relates to the use of a combination of peripheral blood concentrated cells and concentrated supernatant for the treatment of premature ovarian failure. In one aspect, the combination comprises peripheral blood concentrated cells, granulocyte-macrophage stimulating factors, a supernatant concentrate, and optional excipients; the peripheral blood concentrated cells and supernatant concentrate are prepared using an automated cell separation system and a tangential flow ultrafiltration system. The proportion of concentrated cells is such that the concentrated cells are CD45... + Cell count 4 x 10^6 cells: 10–15 ng granulocyte-macrophage stimulating factor: 75–125 μL supernatant concentrate. The invention also relates to a method for preparing the combination of the claims, comprising the step of mixing a specified amount of concentrated cells, granulocyte-macrophage stimulating factor, supernatant concentrate, and optional excipients to prepare a sterile formulation. The cell combination of the present invention exhibits excellent effects.

Description

Technical Field

[0001] This invention belongs to the fields of biotechnology and biomedicine, and relates to a method for treating premature ovarian failure (POF) using cell therapy agents. Specifically, this invention first relates to the rapid isolation and preparation of cell therapy agents from mobilized peripheral blood (MPB, also simply referred to as peripheral blood), followed by formulation of these agents with GM-CSF (granulocyte / macrophage colony-stimulating factor, gmCSF) to form a cell therapy agent composition. This mobilized peripheral blood cell composition is then used to treat premature ovarian insufficiency and POF. Using this method effectively improves the efficiency of isolating mobilized peripheral blood cells from mobilized peripheral blood, providing a safe, efficient, and simple method for obtaining mobilized peripheral blood cells for the treatment of premature ovarian failure and ovarian insufficiency, such as premature ovarian insufficiency. This invention also provides a new and effective approach for treating premature ovarian failure by combining the prepared peripheral blood concentrated cells, supernatant concentrate, and GM-CSF. Background Technology

[0002] Premature ovarian failure (POF) refers to the condition where ovarian function fails, leading to amenorrhea and infertility in women before the age of 40. POF is characterized by amenorrhea, infertility, estrogen deficiency, decreased follicle count, and elevated gonadotropins, accompanied by a range of low-estrogen symptoms such as hot flashes, night sweats, facial flushing, and decreased libido, severely impacting women's physical and mental health. Furthermore, women with POF have an increased risk of osteoporosis, cardiovascular disease, and Alzheimer's disease. POF is a significant cause of female infertility. The incidence of POF in women of reproductive age is approximately 1-3%, and it is showing an increasing trend and affecting younger women.

[0003] According to the guidelines of the European Society of Human Reproduction and Embryology (ESHRE), the diagnostic criteria for premature ovarian failure (POF) are: oligomenorrhea or amenorrhea for at least 4 months, and two FSH levels elevated >40 IU / L at an interval of more than 4 weeks. The cause of POF is unknown, but it may be related to genetic and autoimmune diseases, environmental factors, and iatrogenic and idiopathic conditions. There is currently no effective treatment. Hormone replacement therapy (HRT) is one of the most common treatments for POF, but its effectiveness is not ideal and it has been shown to increase the risk of venous thrombosis, breast cancer, and ovarian cancer. In addition to oligomenorrhea, amenorrhea, and infertility, POF can also cause menopausal symptoms such as hot flashes, night sweats, anxiety, depression, palpitations, and insomnia. It may accelerate female aging, leading to postmenopausal diseases such as osteoporosis, cardiovascular disease, and dementia, affecting women's quality of life and lifespan.

[0004] The etiology of POF is complex and not fully understood. It may be related to autoimmune responses, infections, genetic factors, the effects of treatments such as chemotherapy, radiotherapy, and surgery, as well as endocrine dysfunction. Currently, there is no effective treatment for POF. The most common treatment for POF is hormone replacement therapy (HRT). While this therapy can alleviate some of the clinical symptoms of POF, HRT cannot fundamentally repair damaged ovaries or restore ovarian function. Furthermore, studies have shown that long-term HRT treatment increases the risk of heart disease and stroke, and may increase the risk of breast and ovarian cancer. Therefore, new treatment strategies are needed to restore ovarian function in patients with POF.

[0005] Hematopoietic stem cells (HSCs) are a subset of cells that can differentiate into erythrocytes, leukocytes, and platelets in the bloodstream. Mesenchymal stem cells (MSCs) are a subset of cells with the potential to differentiate into bone, cartilage, fat, nerve, and myoblast cells. MSCs support HSCs by paracrine secretion of various growth factors, maintaining the stability of the bone marrow hematopoietic microenvironment. Currently, bone marrow transplantation is an effective treatment for hematological diseases. However, due to its high cost and scarcity of donors, bone marrow transplantation is gradually being replaced by peripheral blood hematopoietic stem cell transplantation. Peripheral blood hematopoietic stem cells (PBSCs) are more easily collected through the mobilization of colony-stimulating factors. Utilizing the hematopoietic and immune-enhancing functions of PBSCs, they are used to treat acute and chronic leukemia, multiple myeloma, and other hematological malignancies.

[0006] Peripheral blood contains very few hematopoietic stem cells (PBSCs) in normal peripheral blood. Mobilizing stem cells from the bone marrow into the peripheral blood via colony-stimulating factors (CSPF) to facilitate PBSC collection is one method of peripheral blood mobilization. PBSC mobilization is a crucial step in peripheral blood stem cell transplantation (PBSCT) and directly impacts its efficacy. Currently, there are three clinical regimens for stem cell mobilization: high-dose chemotherapy, hematopoietic growth factor (HGF) alone, and a combination of both.

[0007] Under normal circumstances, PBSCs are few in number, accounting for only about 0.1% of nucleated cells. Although PBSCs cannot be identified morphologically, they can be distinguished based on their immunophenotype. PBSCs are generally considered to include CD34+ cells in peripheral blood. After mobilization by colony-stimulating factors, peripheral blood stem cells typically peak on days 4-6. At this time, PBSCs are obtained through peripheral venous apheresis. Stem cells and granulocytes are separated from red blood cells and plasma components after centrifugation, and the red blood cells and plasma are reinfused into the donor.

[0008] Mobilized peripheral blood concentrates are concentrated nucleated cells obtained by centrifuging and separating mobilized peripheral blood. These concentrated cells contain enriched hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs), as well as a large number of various cell growth factors. HSCs can differentiate into erythrocytes, leukocytes, and platelets in the bloodstream. MSCs are a cell subpopulation with multiple differentiation potentials to form bone, cartilage, fat, nerve, and myoblast cells. Mobilized peripheral blood concentrates contain a variety of growth factors and cytokines, including vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), transforming growth factor β (TGF-β), hepatocyte growth factor (HGF), fibroblast growth factor (FGF), insulin-like growth factor I (IGF-I), bone morphogenetic proteins (BMP-2, BMP-7), and interleukins (IL-1, IL-6, IL-8).

[0009] Mobilizing peripheral blood concentrated cells possesses anti-inflammatory, immunomodulatory, angiogenesis-promoting, and tissue regeneration and repair-promoting effects. Preclinical and preliminary clinical studies have confirmed that mobilizing peripheral blood concentrated cells improves ovarian function by enhancing the ovarian microenvironment, promoting angiogenesis, promoting follicle development, increasing the number of antral follicles, and promoting ovulation, thus representing a potential treatment for POF patients.

[0010] However, the traditional method of separating mobilized peripheral blood using density gradient centrifugation is fraught with challenges, including complex procedures, long processing times, susceptibility to contamination, and poor reproducibility. Those skilled in the art have long desired a method for processing mobilized peripheral blood to obtain mobilized peripheral blood concentrates, i.e., mobilized peripheral blood concentrated cells, that offers advantages such as simplicity, shorter processing time, reduced susceptibility to contamination, and good reproducibility. This technological advancement has been achieved in our research team's Chinese patent application No. 2021116067853.

[0011] Granulocyte-macrophage colony-stimulating factor (GM-CSF) is mainly produced by T cells and macrophages. It can induce the colony growth of granulocyte precursor and macrophage precursor cells, hence the abbreviation granulocyte-macrophage colony-stimulating factor. The main biological functions of GM-CSF in vivo are to maintain the survival of granulocyte and monocyte cell lines, promote growth, induce differentiation, and enhance phagocytic function and bactericidal activity; it also induces dendritic cell maturation and functional distribution. The granulocyte-macrophage stimulating factor used clinically is usually recombinant human granulocyte-macrophage stimulating factor. It is generally used for leukopenia caused by cancer chemotherapy and myelosuppressive therapy, and is also used to treat leukopenia in patients with bone marrow failure. It can also prevent potential infectious complications in leukopenia and accelerate the recovery of neutropenia caused by infection.

[0012] Existing methods for treating premature ovarian failure still require improvement. Therefore, those skilled in the art also anticipate providing a method for treating premature ovarian failure, such as a method using a mobilized peripheral blood concentrated cell therapy agent, wherein the combined therapy agent of such a method may further include a concentrate of the supernatant obtained from the preparation of concentrated cells. Summary of the Invention

[0013] One object of the present invention is to provide a method for preparing peripheral blood, such as mobilized peripheral blood concentrated cells, which is expected to have one or more beneficial effects, such as simple operation, short time consumption, low susceptibility to contamination, and good reproducibility of results; or, another object of the present invention is to provide a novel method for treating premature ovarian failure, which is achieved by preparing a combination therapeutic agent using mobilized peripheral blood concentrated cells and GM-CSF. It has been unexpectedly discovered that the present invention can achieve one or more of the above-mentioned objectives by using a closed PXP automated cell separation system to prepare mobilized peripheral blood concentrated cells, and / or by preparing a combination therapeutic agent using the obtained mobilized peripheral blood concentrated cells and GM-CSF, wherein the combination therapeutic agent may further contain a concentrate of the supernatant of the byproducts obtained from the preparation of concentrated cells. The present invention is based on such discoveries.

[0014] Therefore, the first aspect of the present invention provides a method for preparing peripheral blood, such as mobilized peripheral blood concentrated cells, comprising the following steps:

[0015] (1) Provide biological samples such as peripheral blood, for example, mobilize peripheral blood concentrated cells and place them in a sterile bag containing anticoagulant for later use;

[0016] (2) Remove the cap from the input tube of the automated cell separation system, connect the syringe to the Luer locking connector of the input tube, and transfer the anticoagulated biological sample to a disposable sterile separation cup by slowly and steadily passing it through the thrombus filter. Shake the mixed sample along the horizontal axis. The automated cell separation system is a closed PXP separation system, which consists of four components: a) a disposable sterile separation cup, b) a control module, c) a separation base for data transmission, and d) a DataTrak software processing system.

[0017] (3) Place the disposable separation cup into the control module. Before centrifugation, the control module status should be "0". Weigh the separation cup / control module assembly, balance it, and place it in a programmable centrifuge. Set the centrifuge parameters according to the following procedure:

[0018] Program number accelerate slow down Relative centrifugal force / RCF Time / min P1 9 9 2000 8.5 P2 9 9 50 2 P3 9 9 500 2 P4 9 9 50 1 P5 9 9 250 0.5 P6 9 9 50 1

[0019] (4) Start the centrifuge to perform centrifugation. The process is as follows:

[0020] 4a) During the P1 phase, cells in a biological sample are separated into three components—lower, middle, and upper—in a disposable separation cup by centrifugation density stratification: red blood cell layer, cell concentration layer, and plasma layer.

[0021] 4b) During the P2 phase, most red blood cells enter the red blood cell recycling chamber;

[0022] 4c) Phase P3 further separates the cells in the processing chamber into layers, and Phase P4 reduces centrifugation to further remove red blood cells;

[0023] 4d) During phase P5, the cell concentrate and plasma are further separated. During phase P6, the centrifugal force is reduced, allowing the cell concentrate to be transferred to the recovery chamber through the delivery tube, while the plasma is retained in the central chamber.

[0024] (5) After centrifugation, confirm that the control module window displays "P" indicating a qualified state. Remove the separation cup from the control module and connect the syringe to the output tube connecting the separation cup and the recovery chamber. Collect peripheral blood, for example, mobilize peripheral blood to concentrate cells.

[0025] Optional

[0026] (6) Place the separation cup and control module on the separation base to transmit data and process the data captured during centrifugation using the DataTrak software processing system;

[0027] And / or optionally, continue with the following steps to prepare a (plasma) supernatant concentrate:

[0028] (7) Use a syringe to separate the supernatant (i.e., plasma layer) in the central chamber, centrifuge at 2000g for 20min to remove cell debris, and then filter through a 0.22μm sterile filter membrane;

[0029] (8) Rinse the tubing of the tangential flow ultrafiltration system (Shibipure KR2i type tangential flow ultrafiltration system) with ultrapure water, install a 100kD 100cm2 MidiKros filter, and ultrafilter and concentrate the filtrate obtained in step (7) to 1 / 15 of the initial biological sample volume to obtain the supernatant concentrate.

[0030] According to the method of the first aspect of the present invention, the volume of the biological sample provided in step (1) is 20 to 200 ml.

[0031] According to the method of the first aspect of the present invention, in step (1), an additional 1 ml sample is drawn for testing.

[0032] According to the method of the first aspect of the present invention, the anticoagulant used in step (1) is a sodium citrate solution.

[0033] According to the method of the first aspect of the present invention, the anticoagulant used in step (1) is a 3.5% sodium citrate solution.

[0034] According to the method of the first aspect of the present invention, the anticoagulant used in step (1) is a 3.5% sodium citrate solution, wherein 0.5 mg / ml histidine and 0.12 mg / ml phosphatidylcholine are added.

[0035] According to the method of the first aspect of the present invention, the volume ratio of the anticoagulant used in step (1) to the biological sample is 1:12.

[0036] According to the method of the first aspect of the present invention, the anticoagulant used in step (1) is prepared by adding sodium citrate, histidine and phosphatidylcholine to an appropriate amount of water, heating to 60°C and stirring to dissolve, adding water to the total volume, filtering with a 0.22μm microporous membrane, and autoclaving at 121°C to obtain the product.

[0037] According to the method of the first aspect of the present invention, it further includes the following steps: (6) placing the separation cup and the control module on the separation base to transmit data and processing the data captured during centrifugation using the DataTrak software processing system.

[0038] Furthermore, a second aspect of the present invention provides peripheral blood, such as mobilized peripheral blood concentrated cells, which is prepared by a method comprising the following steps:

[0039] (1) Provide biological samples such as peripheral blood, mobilize peripheral blood, and place it in a sterile bag containing anticoagulant for later use;

[0040] (2) Remove the cap from the input tube of the automated cell separation system, connect the syringe to the Luer locking connector of the input tube, and transfer the anticoagulated biological sample to a disposable sterile separation cup by slowly and steadily passing it through the thrombus filter. Shake the mixed sample along the horizontal axis. The automated cell separation system is a closed PXP separation system, which consists of four components: a) a disposable sterile separation cup, b) a control module, c) a separation base for data transmission, and d) a DataTrak software processing system.

[0041] (3) Place the disposable separation cup into the control module. Before centrifugation, the control module status should be "0". Weigh the separation cup / control module assembly, balance it, and place it in a programmable centrifuge. Set the centrifuge parameters according to the following procedure:

[0042] Program number accelerate slow down Relative centrifugal force / RCF Time / min P1 9 9 2000 8.5 P2 9 9 50 2 P3 9 9 500 2 P4 9 9 50 1 P5 9 9 250 0.5 P6 9 9 50 1

[0043] (4) Start the centrifuge to perform centrifugation. The process is as follows:

[0044] 4a) During the P1 phase, cells in a biological sample are separated into three components—lower, middle, and upper—in a disposable separation cup by centrifugation density stratification: red blood cell layer, cell concentration layer, and plasma layer.

[0045] 4b) During the P2 phase, most red blood cells enter the red blood cell recycling chamber;

[0046] 4c) Phase P3 further separates the cells in the processing chamber into layers, and Phase P4 reduces centrifugation to further remove red blood cells;

[0047] 4d) During phase P5, the cell concentrate and plasma are further separated. During phase P6, the centrifugal force is reduced, allowing the cell concentrate to be transferred to the recovery chamber through the delivery tube, while the plasma is retained in the central chamber.

[0048] (5) After centrifugation, confirm that the control module window displays "P" indicating a qualified state. Remove the separation cup from the control module and connect the syringe to the output tube connecting the separation cup and the recovery chamber. Collect peripheral blood, for example, mobilize peripheral blood to concentrate cells.

[0049] Optional

[0050] (6) Place the separation cup and control module on the separation base to transmit data and process the data captured during centrifugation using the DataTrak software processing system;

[0051] And / or optionally, continue with the following steps to prepare a (plasma) supernatant concentrate:

[0052] (7) Use a syringe to separate the supernatant (i.e., plasma layer) in the central chamber, centrifuge at 2000g for 20min to remove cell debris, and then filter through a 0.22μm sterile filter membrane;

[0053] (8) Rinse the tubing of the tangential flow ultrafiltration system (Shibipure KR2i type tangential flow ultrafiltration system) with ultrapure water, install a 100kD 100cm2 MidiKros filter, and ultrafilter and concentrate the filtrate obtained in step (7) to 1 / 15 of the initial biological sample volume to obtain the supernatant concentrate.

[0054] According to the second aspect of the present invention, peripheral blood, for example, mobilized peripheral blood concentrated cells, wherein the volume of the biological sample provided in step (1) is 20 to 200 ml.

[0055] According to the second aspect of the present invention, peripheral blood, for example, mobilized peripheral blood concentrated cells, wherein in step (1), an additional 1 ml sample is drawn for testing.

[0056] According to a second aspect of the present invention, peripheral blood, for example, mobilizes peripheral blood concentrated cells, wherein the anticoagulant used in step (1) is a sodium citrate solution.

[0057] According to a second aspect of the invention, peripheral blood, for example, mobilized peripheral blood concentrated cells, wherein the anticoagulant used in step (1) is a 3.5% sodium citrate solution.

[0058] According to a second aspect of the invention, peripheral blood, for example, mobilized peripheral blood concentrated cells, wherein the anticoagulant used in step (1) is a 3.5% sodium citrate solution, wherein 0.5 mg / ml histidine and 0.12 mg / ml phosphatidylcholine are added.

[0059] According to the second aspect of the present invention, peripheral blood, for example, mobilized peripheral blood concentrated cells, wherein the volume ratio of the anticoagulant used in step (1) to the biological sample is 1:12.

[0060] According to the second aspect of the present invention, peripheral blood, for example, mobilizes peripheral blood concentrated cells, wherein the anticoagulant used in step (1) is prepared by adding sodium citrate, histidine and phosphatidylcholine to an appropriate amount of water, heating to 60°C and stirring to dissolve, adding water to the total volume, filtering through a 0.22 μm microporous membrane, and autoclaving at 121°C to obtain the product.

[0061] According to the second aspect of the present invention, peripheral blood, such as mobilized peripheral blood concentrated cells, further includes the following steps: (6) placing the separation cup and control module on the separation base to transmit data and processing the data captured during centrifugation using the DataTrak software processing system.

[0062] Furthermore, a third aspect of the present invention provides the use of peripheral blood, such as mobilized peripheral blood concentrated cells, in the preparation of a cell therapy agent for treating premature ovarian failure, wherein the peripheral blood, such as mobilized peripheral blood concentrated cells, is prepared by a method comprising the following steps:

[0063] (1) Provide biological samples such as peripheral blood, mobilize peripheral blood, and place it in a sterile bag containing anticoagulant for later use;

[0064] (2) Remove the cap from the input tube of the automated cell separation system, connect the syringe to the Luer locking connector of the input tube, and transfer the anticoagulated biological sample to a disposable sterile separation cup by slowly and steadily passing it through the thrombus filter. Shake the mixed sample along the horizontal axis. The automated cell separation system is a closed PXP separation system, which consists of four components: a) a disposable sterile separation cup, b) a control module, c) a separation base for data transmission, and d) a DataTrak software processing system.

[0065] (3) Place the disposable separation cup into the control module. Before centrifugation, the control module status should be "0". Weigh the separation cup / control module assembly, balance it, and place it in a programmable centrifuge. Set the centrifuge parameters according to the following procedure:

[0066] Program number accelerate slow down Relative centrifugal force / RCF Time / min P1 9 9 2000 8.5 P2 9 9 50 2 P3 9 9 500 2 P4 9 9 50 1 P5 9 9 250 0.5 P6 9 9 50 1

[0067] (4) Start the centrifuge to perform centrifugation. The process is as follows:

[0068] 4a) During the P1 phase, cells in a biological sample are separated into three components—lower, middle, and upper—in a disposable separation cup by centrifugation density stratification: red blood cell layer, cell concentration layer, and plasma layer.

[0069] 4b) During the P2 phase, most red blood cells enter the red blood cell recycling chamber;

[0070] 4c) Phase P3 further separates the cells in the processing chamber into layers, and Phase P4 reduces centrifugation to further remove red blood cells;

[0071] 4d) During phase P5, the cell concentrate and plasma are further separated. During phase P6, the centrifugal force is reduced, allowing the cell concentrate to be transferred to the recovery chamber through the delivery tube, while the plasma is retained in the central chamber.

[0072] (5) After centrifugation, confirm that the control module window displays "P" indicating a qualified state. Remove the separation cup from the control module and connect the syringe to the output tube connecting the separation cup and the recovery chamber. Collect peripheral blood, for example, mobilize peripheral blood to concentrate cells.

[0073] Optional

[0074] (6) Place the separation cup and control module on the separation base to transmit data and process the data captured during centrifugation using the DataTrak software processing system;

[0075] And / or optionally, continue with the following steps to prepare a (plasma) supernatant concentrate:

[0076] (7) Use a syringe to separate the supernatant (i.e., plasma layer) in the central chamber, centrifuge at 2000g for 20min to remove cell debris, and then filter through a 0.22μm sterile filter membrane;

[0077] (8) Rinse the tubing of the tangential flow ultrafiltration system (Shibipure KR2i type tangential flow ultrafiltration system) with ultrapure water, install a 100kD 100cm2 MidiKros filter, and ultrafilter and concentrate the filtrate obtained in step (7) to 1 / 15 of the initial biological sample volume to obtain the supernatant concentrate.

[0078] According to the third aspect of the invention, the volume of the biological sample provided in step (1) is 20 to 200 ml.

[0079] According to the third aspect of the invention, in step (1), an additional 1 ml sample is drawn for testing.

[0080] According to the third aspect of the invention, the anticoagulant used in step (1) is a sodium citrate solution.

[0081] According to the third aspect of the invention, the anticoagulant used in step (1) is a 3.5% sodium citrate solution.

[0082] According to the third aspect of the invention, the anticoagulant used in step (1) is a 3.5% sodium citrate solution, wherein 0.5 mg / ml histidine and 0.12 mg / ml phosphatidylcholine are added.

[0083] According to the third aspect of the invention, the volume ratio of the anticoagulant used in step (1) to the biological sample is 1:12.

[0084] According to the third aspect of the present invention, the method for preparing the anticoagulant used in step (1) is as follows: sodium citrate, histidine and phosphatidylcholine are added to an appropriate amount of water, heated to 60°C and stirred to dissolve, water is added to the total volume, filtered through a 0.22 μm microporous membrane, and sterilized by autoclaving at 121°C to obtain the product.

[0085] According to the third aspect of the invention, it further includes the following steps: (6) placing the separation cup and control module on the separation base to transmit data and processing the data captured during centrifugation using the DataTrak software processing system.

[0086] Furthermore, a fourth aspect of the present invention provides a method for treating premature ovarian failure, the method comprising administering to a subject in need a cell therapy agent containing a therapeutically effective amount of peripheral blood, such as mobilized peripheral blood concentrates, prepared by a method comprising the following steps:

[0087] (1) Provide biological samples such as peripheral blood, mobilize peripheral blood, and place it in a sterile bag containing anticoagulant for later use;

[0088] (2) Remove the cap from the input tube of the automated cell separation system, connect the syringe to the Luer locking connector of the input tube, and transfer the anticoagulated biological sample to a disposable sterile separation cup by slowly and steadily passing it through the thrombus filter. Shake the mixed sample along the horizontal axis. The automated cell separation system is a closed PXP separation system, which consists of four components: a) a disposable sterile separation cup, b) a control module, c) a separation base for data transmission, and d) a DataTrak software processing system.

[0089] (3) Place the disposable separation cup into the control module. Before centrifugation, the control module status should be "0". Weigh the separation cup / control module assembly, balance it, and place it in a programmable centrifuge. Set the centrifuge parameters according to the following procedure:

[0090] Program number accelerate slow down Relative centrifugal force / RCF Time / min P1 9 9 2000 8.5 P2 9 9 50 2 P3 9 9 500 2 P4 9 9 50 1 P5 9 9 250 0.5 P6 9 9 50 1

[0091] (4) Start the centrifuge to perform centrifugation. The process is as follows:

[0092] 4a) During the P1 phase, cells in a biological sample are separated into three components—lower, middle, and upper—in a disposable separation cup by centrifugation density stratification: red blood cell layer, cell concentration layer, and plasma layer.

[0093] 4b) During the P2 phase, most red blood cells enter the red blood cell recycling chamber;

[0094] 4c) Phase P3 further separates the cells in the processing chamber into layers, and Phase P4 reduces centrifugation to further remove red blood cells;

[0095] 4d) During phase P5, the cell concentrate and plasma are further separated. During phase P6, the centrifugal force is reduced, allowing the cell concentrate to be transferred to the recovery chamber through the delivery tube, while the plasma is retained in the central chamber.

[0096] (5) After centrifugation, confirm that the control module window displays "P" indicating a qualified state. Remove the separation cup from the control module and connect the syringe to the output tube connecting the separation cup and the recovery chamber. Collect peripheral blood, for example, mobilize peripheral blood to concentrate cells.

[0097] Optional

[0098] (6) Place the separation cup and control module on the separation base to transmit data and process the data captured during centrifugation using the DataTrak software processing system;

[0099] And / or optionally, continue with the following steps to prepare a (plasma) supernatant concentrate:

[0100] (7) Use a syringe to separate the supernatant (i.e., plasma layer) in the central chamber, centrifuge at 2000g for 20min to remove cell debris, and then filter through a 0.22μm sterile filter membrane;

[0101] (8) Rinse the tubing of the tangential flow ultrafiltration system (Shibipure KR2i type tangential flow ultrafiltration system) with ultrapure water, install a 100kD 100cm2 MidiKros filter, and ultrafilter and concentrate the filtrate obtained in step (7) to 1 / 15 of the initial biological sample volume to obtain the supernatant concentrate.

[0102] According to the method of the fourth aspect of the present invention, the volume of the biological sample provided in step (1) is 20 to 200 ml.

[0103] According to the method of the fourth aspect of the present invention, in step (1), an additional 1 ml sample is drawn for testing.

[0104] According to the method of the fourth aspect of the present invention, the anticoagulant used in step (1) is a sodium citrate solution.

[0105] According to the method of the fourth aspect of the present invention, the anticoagulant used in step (1) is a 3.5% sodium citrate solution.

[0106] According to the method of the fourth aspect of the present invention, the anticoagulant used in step (1) is a 3.5% sodium citrate solution, wherein 0.5 mg / ml histidine and 0.12 mg / ml phosphatidylcholine are added.

[0107] According to the method of the fourth aspect of the present invention, the volume ratio of the anticoagulant used in step (1) to the biological sample is 1:12.

[0108] According to the method of the fourth aspect of the present invention, the anticoagulant used in step (1) is prepared by adding sodium citrate, histidine and phosphatidylcholine to an appropriate amount of water, heating to 60°C and stirring to dissolve, adding water to the total volume, filtering with a 0.22 μm microporous membrane, and autoclaving at 121°C to obtain the product.

[0109] According to the method of the fourth aspect of the present invention, it further includes the following steps: (6) placing the separation cup and the control module on the separation base to transmit data and processing the data captured during centrifugation using the DataTrak software processing system.

[0110] Furthermore, a fifth aspect of the present invention provides a cell composition made from mobilized peripheral blood concentrated cells, comprising concentrated cells, granulocyte-macrophage stimulating factors, and optional excipients.

[0111] According to a fifth aspect of the cell composition of the present invention, wherein the ratio of concentrated cells to granulocyte-macrophage stimulating factors is such that the concentrated cells contain CD45 + Cell count 5 x 10^6 cells: 10–15 ng granulocyte-macrophage stimulating factor; for example, the ratio is, concentrated cells with CD45 + Cell count: 5 x 10^6 cells; 12.5 ng granulocyte-macrophage stimulating factor.

[0112] According to the cell composition of the fifth aspect of the present invention, the excipient is physiological saline or 5% glucose solution.

[0113] According to a fifth aspect of the present invention, the cell composition wherein the excipient is physiological saline and the concentration of granulocyte-macrophage stimulating factor in the composition is 10-15 ng / ml, for example 12.5 ng / ml.

[0114] According to a fifth aspect of the present invention, the cell composition wherein the granulocyte-macrophage stimulating factor is a human granulocyte-macrophage stimulating factor.

[0115] According to a fifth aspect of the present invention, the cell composition wherein the granulocyte-macrophage stimulating factor is a recombinant human granulocyte-macrophage stimulating factor.

[0116] According to the cell composition of the fifth aspect of the present invention, wherein the concentrated cells are as described in any embodiment of the second aspect of the present invention.

[0117] The cell composition according to the fifth aspect of the invention further comprises glutamine and sodium selenite.

[0118] According to the cell composition of the fifth aspect of the present invention, it further comprises glutamine and sodium selenite, wherein the weight ratio of granulocyte-macrophage stimulating factor to glutamine and sodium selenite in the composition is 12.5 ng: 0.1-0.5 mg: 5-20 μg.

[0119] According to the cell composition of the fifth aspect of the present invention, the cell composition further comprises glutamine and sodium selenite, wherein the weight ratio of granulocyte-macrophage stimulating factor to glutamine and sodium selenite in the composition is 12.5 ng: 0.2-0.3 mg: 10-15 μg.

[0120] According to the cell composition of the fifth aspect of the present invention, it further comprises glutamine and sodium selenite, wherein the weight ratio of granulocyte-macrophage stimulating factor to glutamine and sodium selenite in the composition is 12.5 ng: 0.25 mg: 12.5 μg.

[0121] According to a fifth aspect of the present invention, a cell composition comprising: CD45 + Concentrated cells with a cell count of 4–6 x 10^6, 10–15 ng gmCSF, 0.1–0.5 mg glutamine, 5–20 μg sodium selenite, and appropriate amount of physiological saline to 1 mL.

[0122] According to a fifth aspect of the present invention, a cell composition comprising: CD45 + Concentrated cells with a cell count of 4–6 x 10^6, 10–15 ng gmCSF, 0.2–0.3 mg glutamine, 10–15 μg sodium selenite, and appropriate amount of physiological saline to 1 mL.

[0123] According to a fifth aspect of the present invention, a cell composition comprising: CD45 + Concentrated cells with a cell count of 5 x 10^6, 12.5 ng gmCSF, 0.2–0.3 mg glutamine, 10–15 μg sodium selenite, and appropriate amount of physiological saline to 1 mL.

[0124] According to a fifth aspect of the present invention, a cell composition comprising: CD45 + Concentrated cells with a cell count of 5 x 10^6, 12.5 ng gmCSF, 0.25 mg glutamine, 12.5 μg sodium selenite, and appropriate amount of physiological saline to 1 mL.

[0125] Furthermore, a sixth aspect of the present invention provides a cell composition made from mobilized peripheral blood concentrated cells, comprising concentrated cells, granulocyte-macrophage stimulating factors, supernatant concentrate, and optional excipients.

[0126] According to a sixth aspect of the cell composition of the present invention, wherein the ratio of concentrated cells to granulocyte-macrophage stimulating factors is such that the concentrated cells contain CD45 + Cell count: 4 x 10^6 cells; 10–15 ng granulocyte-macrophage stimulating factor; for example, the ratio is, concentrated cells with CD45 + Cell count: 4 x 10^6 cells; 12.5 ng granulocyte-macrophage stimulating factor.

[0127] According to a sixth aspect of the cell composition of the present invention, wherein the ratio of concentrated cells to supernatant concentrate is such that the concentrated cells are CD45 + Cell count: 4 x 10^6 cells: 75–125 μL supernatant concentrate; for example, the ratio is such that the concentrated cells are based on CD45.+ Cell count: 4 x 10^6 cells: 100 μL supernatant concentrate.

[0128] According to the cell composition of the sixth aspect of the present invention, the excipient is physiological saline or 5% glucose solution.

[0129] According to a sixth aspect of the present invention, the cell composition wherein the excipient is physiological saline and the concentration of granulocyte-macrophage stimulating factor in the composition is 10-15 ng / ml, for example 12.5 ng / ml.

[0130] According to a sixth aspect of the present invention, the cell composition wherein the granulocyte-macrophage stimulating factor is a human granulocyte-macrophage stimulating factor.

[0131] According to a sixth aspect of the present invention, the cell composition wherein the granulocyte-macrophage stimulating factor is a recombinant human granulocyte-macrophage stimulating factor.

[0132] According to the cell composition of the sixth aspect of the present invention, wherein the concentrated cells and the supernatant concentrate are prepared by the method described in any embodiment of the first aspect of the present invention.

[0133] The cell composition according to a sixth aspect of the invention further comprises glutamine and sodium selenite.

[0134] According to the sixth aspect of the present invention, the cell composition further comprises glutamine and sodium selenite, wherein the weight ratio of granulocyte-macrophage stimulating factor to glutamine and sodium selenite in the composition is 12.5 ng: 0.1-0.5 mg: 5-20 μg.

[0135] According to the sixth aspect of the present invention, the cell composition further comprises glutamine and sodium selenite, wherein the weight ratio of granulocyte-macrophage stimulating factor to glutamine and sodium selenite in the composition is 12.5 ng: 0.2-0.3 mg: 10-15 μg.

[0136] According to the sixth aspect of the present invention, the cell composition further comprises glutamine and sodium selenite, wherein the weight ratio of granulocyte-macrophage stimulating factor to glutamine and sodium selenite in the composition is 12.5 ng: 0.25 mg: 12.5 μg.

[0137] According to a sixth aspect of the present invention, a cell composition comprising: CD45 + Concentrated cells with a cell count of 3–5 x 10^6, 10–15 ng gmCSF, 75–125 μL supernatant concentrate, 0.1–0.5 mg glutamine, 5–20 μg sodium selenite, and appropriate amount of physiological saline to 1 mL.

[0138] According to a sixth aspect of the present invention, a cell composition comprising: CD45 + Concentrated cells with a cell count of 3–5 x 10^6, 10–15 ng gmCSF, 80–120 μL supernatant concentrate, 0.2–0.3 mg glutamine, 10–15 μg sodium selenite, and appropriate amount of physiological saline to 1 mL.

[0139] According to a sixth aspect of the present invention, a cell composition comprising: CD45 + Concentrated cells with a cell count of 4 x 10^6, 12.5 ng gmCSF, 100 μL supernatant concentrate, 0.2–0.3 mg glutamine, 10–15 μg sodium selenite, and appropriate amount of physiological saline to 1 mL.

[0140] According to a sixth aspect of the present invention, a cell composition comprising: CD45 + Concentrated cells with a cell count of 4 x 10^6, 12.5 ng gmCSF, 100 μL supernatant concentrate, 0.25 mg glutamine, 12.5 μg sodium selenite, and appropriate amount of physiological saline to 1 mL.

[0141] The present invention, after preparing concentrated cells using the PXP system, further processes the separated supernatant, i.e., the plasma portion, through a tangential flow ultrafiltration system to obtain a concentrated supernatant. The cell composition prepared by combining this concentrated supernatant with concentrated cells and gmCSF has excellent therapeutic effects on premature ovarian failure. For example, the desired effect can be achieved using a lower dose of concentrated cells. This unexpected discovery is not anticipated by the prior art.

[0142] Furthermore, a seventh aspect of the present invention provides the use of the cell composition described in any one of the fifth or sixth aspects of the present invention in the preparation of a medicament for treating premature ovarian failure.

[0143] Furthermore, the eighth aspect of the present invention provides a method for preparing the cell composition according to any one of the sixth aspects of the present invention, comprising the step of mixing a specified amount of concentrated cells, granulocyte-macrophage stimulating factor, supernatant concentrate, glutamine, sodium selenite, and optionally excipients to prepare a sterile formulation.

[0144] The phrase “CD45” mentioned in this article + The phrase "5x10^6 concentrated cells" refers to 5 multiplied by 10 to the power of 6, and other similar expressions have the same meaning.

[0145] Although the specific steps described in the various operational steps of the present invention may differ in some details or language from the steps described in the preparation examples in the detailed embodiments section below, those skilled in the art can fully summarize the above-described method steps based on the detailed disclosure of the entire present invention.

[0146] Any embodiment of any aspect of the present invention can be combined with other embodiments, as long as they do not contradict each other. Furthermore, any technical feature in any embodiment of any aspect of the present invention can be applied to the same technical feature in other embodiments, as long as they do not contradict each other. The present invention will now be further described.

[0147] All references cited in this invention are incorporated herein by reference in their entirety, and in the event of any inconsistency between the meanings expressed in these references and those expressed herein, the meanings expressed herein shall prevail. Furthermore, the various terms and phrases used in this invention have their general meanings known to those skilled in the art; however, this invention still seeks to provide a more detailed explanation and interpretation of these terms and phrases, and in the event of any inconsistency between the mentioned terms and their known meanings and those expressed herein, the meanings expressed herein shall prevail.

[0148] This invention utilizes the PXP automated cell rapid processing system, employing a closed system for automated separation and concentration, to safely, efficiently, and simply obtain BMAC (biologically active macromolecular cells), laying the foundation for the clinical application of BMAC in treating POF patients. The invention provides a method for rapidly separating and obtaining peripheral blood, such as mobilized peripheral blood concentrated cells, using a closed automated cell separation system. The peripheral blood, such as mobilized peripheral blood, obtained by this invention, can be used as an active ingredient in the treatment of ovarian damage, promoting angiogenesis and follicle development, thereby improving ovarian function.

[0149] Existing research has confirmed that contaminated red blood cells are associated with a decline in stem cell / progenitor cell function, and red blood cell contamination of cell concentrates is believed to reduce the effectiveness of cell therapy. To better realize the potential of cell therapy, the industry urgently needs new processing systems that can improve target cell purity and the removal rate of contaminated red blood cells.

[0150] The cell separation system used in the specific experiments of this invention is: The automated separation system, model number 80065-01, is supplied by Shenzhen Boya Sensing Medical Technology Co., Ltd., and manufactured by ThermoGenesis, Inc. (USA). The innovative PXP system addresses many shortcomings of existing systems on the market. The PXP system enables clinicians to rapidly obtain high rates of stem cell and progenitor cell recovery, typically less than 5% of the starting sample, with virtually no red blood cell contamination.

[0151] The PXP system is designed for clinical institutions developing and using cell therapy technologies, providing rapid, efficient, and sterile cell processing in an operating room environment. It is a highly efficient point-of-care (POC) system. As a cutting-edge automated rapid cell processing system, the PXP system eliminates the need for cell separation media or precipitants, can process multiple samples simultaneously, and boasts high recovery rates for MNC, CD34+, and CD45+ cells. It enables clinicians to efficiently extract stem cells from biological samples (such as peripheral blood, including mobilized peripheral blood) within 30 minutes in hospital surgical centers or clinics, with a red blood cell removal rate exceeding 90%. Furthermore, the PXP system is equipped with patented DataTrak software to track captured data, facilitating GMP process control and reporting information for clients.

[0152] The PXP system has already obtained medical device product registration from the health ministries of multiple countries. This system has a wide range of clinical applications, primarily in cell therapy for orthopedic diseases. Some institutions have used the PXP system to achieve highly efficient and high-quality autologous bone marrow stem cell preparation in operating room environments, bringing orthopedic cell therapy to a new level and significantly enhancing the competitiveness of treatment centers. This invention utilizes the PXP system to process peripheral blood, such as mobilized peripheral blood. It can process peripheral blood cells, such as mobilized peripheral blood cells, in real-time, rapidly, and automatically, ensuring the recovery rate of mononuclear cells (MNCs). It can process multiple peripheral blood units, such as mobilized peripheral blood units, simultaneously without requiring cell separation media or precipitants. Similar to bone marrow stem cells, peripheral blood cells can also be used to treat premature ovarian failure.

[0153] The advantages of the PXP system used in the specific experiments of this invention include, but are not limited to: stable and excellent MNC (monocyte) and CD34+, CD45+ cell recovery rates; rapid processing of peripheral blood, such as mobilized peripheral blood samples, within 30 minutes; red blood cell removal rate of over 95%; automated closed aseptic system; rapid and accurate data tracking and documentation; and sample processing data can be uploaded to a computer via DataTrak software, providing production records and reporting information that meet GMP requirements.

[0154] Studies have shown that under normal circumstances, there are only a very small number of hematopoietic stem cells in the peripheral blood circulation. Stress, endotoxins, strenuous exercise, chemotherapy, and cytokines can significantly increase CD45 levels. +The mobilization of hematopoietic stem cells, including CD34+ cells, is clinically most effective, with the latter two being the most efficient. This discovery makes peripheral blood an important source of stem cells for hematopoietic stem cell transplantation (HSCT). Peripheral blood mobilization (i.e., peripheral hematopoietic stem cell mobilization) is a method to release hematopoietic stem cells from the bone marrow into the peripheral blood, significantly increasing the proportion of stem / progenitor cells in the peripheral blood. In normal individuals, stem / progenitor cells account for approximately 1% of the total number of nucleated cells in peripheral blood. After effective mobilization, hematopoietic stem cells from the bone marrow are released into the peripheral blood, significantly increasing the proportion of stem / progenitor cells. Peripheral blood mobilization methods are well-known and mature techniques in the field, and mobilized peripheral blood can be obtained through conventional techniques.

[0155] The present invention achieved satisfactory results by using the PXP system for cell separation.

[0156] GM-CSF can be human granulocyte-macrophage colony-stimulating factor or recombinant granulocyte-macrophage colony-stimulating factor. It is included in multiple versions of the Chinese Pharmacopoeia and numerous brands of products have been approved for clinical use. In this invention, unless otherwise specified, the GM-CSF used in the experiment is commercially available recombinant human granulocyte-macrophage colony-stimulating factor for injection (S19991012, specification 750000 IU / 75 μg, 10 IU / ng). If necessary, it can be pre-diluted with 0.9% sodium chloride injection to a suitable concentration when preparing the composition. GM-CSF (Granulocyte-macrophage Colony Stimulating Factor) acts on hematopoietic progenitor cells, promoting their proliferation and differentiation. Its important function is to stimulate the maturation of granulocytes, monocytes, and macrophages, promote the release of mature cells into the peripheral blood, and promote various functions of macrophages and eosinophils. GM-CSF is a clinically used drug for leukopenia or granulocytopenia caused by various reasons. It can stimulate the hematopoietic function of bone marrow, stimulate the proliferation of granulocytes, monocytes, and T cells, and promote the maturation of monocytes and granulocytes. In addition, GM-CSF can overcome the bone marrow toxicity caused by radiotherapy and chemotherapy, shorten the neutropenia time during tumor chemotherapy, and make patients more tolerant to chemotherapy. GM-CSF can enhance the function of monocytes, granulocytes, eosinophils, and macrophages, thereby improving the body's anti-tumor and anti-infective immunity.

[0157] This invention combines mobilized peripheral blood concentrated cells obtained through PXP system separation with GM-CSF and uses a premature ovarian failure model for verification, achieving positive results. Detailed Implementation

[0158] The invention can be further described through the following embodiments; however, the scope of the invention is not limited to the embodiments described below. Those skilled in the art will understand that various changes and modifications can be made to the invention without departing from its spirit and scope. The invention provides a general and / or specific description of the materials and methods used in the experiments. Although many materials and methods of operation used to achieve the objectives of the invention are well known in the art, the invention is still described herein in as much detail as possible.

[0159] In this invention, unless otherwise specified, the cell separation system used in the specific experiments is as follows: The automated separation system, which in this invention may also be referred to as the PXP system, PXP cell automated separation system, PXP automated separation system, PXP separation system, etc., is model 80065-01. The supplier is Shenzhen Boya Sensing Medical Technology Co., Ltd., and the manufacturer is ThermoGenesis, Inc. (USA). In this invention, the term "peripheral blood concentrated cells" may also be referred to as "peripheral blood concentrated cell preparation," and unless otherwise specified in the context, the two have the same meaning.

[0160] Example 1: Rapid isolation and preparation of concentrated cells from mobilized peripheral blood

[0161] (1) Provide a biological sample of mobilized peripheral blood (which can process samples of 20-200 ml volume), place it in a sterile bag containing anticoagulant for later use, and extract another 1 ml sample for testing; the anticoagulant is a 3.5% sodium citrate solution, supplemented with 0.5 mg / ml histidine and 0.12 mg / ml phosphatidylcholine, and the volume ratio of anticoagulant to biological sample is 1:12; the anticoagulant is prepared by adding sodium citrate, histidine and phosphatidylcholine to an appropriate amount of water, heating to 60°C and stirring to dissolve, adding water to the total volume, filtering through a 0.22 μm microporous membrane, and autoclaving at 121°C to obtain the final product;

[0162] (2) Remove the cap from the input tube of the automated cell separation system, connect the syringe to the Luer locking connector of the input tube, and transfer the anticoagulated biological sample to a disposable sterile separation cup by slowly and steadily passing it through the thrombus filter. Shake the mixed sample along the horizontal axis. The automated cell separation system is a closed PXP separation system, which consists of four components: a) a disposable sterile separation cup, b) a control module, c) a separation base for data transmission, and d) a DataTrak software processing system.

[0163] (3) Place the disposable separation cup into the control module. Before centrifugation, the control module status should be "0". Weigh the separation cup / control module assembly, balance it, and place it in a programmable centrifuge. Set the centrifuge parameters according to the following procedure:

[0164] Program number accelerate slow down Relative centrifugal force / RCF Time / min P1 9 9 2000 8.5 P2 9 9 50 2 P3 9 9 500 2 P4 9 9 50 1 P5 9 9 250 0.5 P6 9 9 50 1

[0165] (4) Start the centrifuge to perform centrifugation. The process is as follows:

[0166] 4a) During the P1 phase, cells in a biological sample are separated into three components—lower, middle, and upper—in a disposable separation cup by centrifugation density stratification: red blood cell layer, cell concentration layer, and plasma layer.

[0167] 4b) During the P2 phase, most red blood cells enter the red blood cell recycling chamber;

[0168] 4c) Phase P3 further separates the cells in the processing chamber into layers, and Phase P4 reduces centrifugation to further remove red blood cells;

[0169] 4d) During phase P5, the cell concentrate and plasma are further separated. During phase P6, the centrifugal force is reduced, allowing the cell concentrate to be transferred to the recovery chamber through the delivery tube, while the plasma is retained in the central chamber.

[0170] (5) After centrifugation, confirm that the control module window displays "P" indicating a qualified state. Remove the separation cup from the control module and connect the syringe to the output tube connecting the separation cup and the recovery chamber to collect the mobilized peripheral blood concentrated cells.

[0171] (6) Place the separation cup and control module on the separation base to transmit data and process the data captured during centrifugation using the DataTrak software processing system.

[0172] Continue with the following steps to prepare a (plasma) supernatant concentrate:

[0173] (7) Use a syringe to separate the supernatant (i.e., plasma layer) in the central chamber, centrifuge at 2000g for 20min to remove cell debris, and then filter through a 0.22μm sterile filter membrane;

[0174] (8) Rinse the tubing of the tangential flow ultrafiltration system (Shibipure KR2i type tangential flow ultrafiltration system) with ultrapure water, install a 100kD 100cm2 MidiKros filter, and ultrafilter and concentrate the filtrate obtained in step (7) to 1 / 15 of the initial biological sample volume to obtain the supernatant concentrate.

[0175] In this invention, unless otherwise stated, the tangential flow ultrafiltration system used is the Specle KR2i tangential flow ultrafiltration system; of course, other brands of tangential flow ultrafiltration systems can also be used.

[0176] In this embodiment 1, 10 biological samples of human mobilized peripheral blood (mobilized peripheral blood can be obtained by methods known in the art, for example, the biological sample of this invention is the peripheral blood stem cell mobilization performed by subcutaneous injection of 7.5 μg / kg of recombinant human granulocyte colony-stimulating factor injection (rhG-CSF) for 5 consecutive days, and peripheral blood collected on the 6th day) were subjected to cell separation and preparation of supernatant concentrate to obtain 10 mobilized peripheral blood concentrated cells and 10 supernatant concentrates, which were labeled No.1 to No.10 respectively.

[0177] Experimental Example 1: Analysis of MNC recovery rate in mobilized peripheral blood and concentrated cells from mobilized peripheral blood

[0178] Using the method of Example 1, cell separation was performed on 10 collected human mobilized peripheral blood samples (volumes ranging from 157 to 164 ml before separation). Then, referring to the method described in Chinese Patent Application No. 2021116067853, cell analysis was performed on each separation fraction. The results for the 10 samples were as follows: the average input peripheral blood volume was 161.2 ml, the average output peripheral blood concentrated cells volume was 17.7 ml, the average red blood cell (RBC) removal rate was 98.1%, the average mononuclear cell (MNC) recovery rate was 97.3%, and the average MNC concentration increased by 9.11 times. For example, the results for one peripheral blood sample (No. 1) were: initial volume = 159.4 ml, final volume = 17.8 ml, red blood cell removal rate = 98.3%, MNC recovery rate = 97.2%, and MNC concentration factor = 8.96.

[0179] The results showed that the PXP dynamic separation system can enrich MNCs in mobilized peripheral blood in a simple, time-saving, non-contamination-prone, and reproducible manner, while removing most of the red blood cells.

[0180] Experimental Example 2: Cell viability in mobilized peripheral blood and concentrated cell samples from mobilized peripheral blood

[0181] Ten samples from Experiment 1 were examined. Cell viability is the most direct indicator of whether cells possess biological function. Peripheral blood samples were collected within 24-36 hours (T<36 hours). Cell viability in mobilized peripheral blood and its concentrated cells was analyzed using an FC500 flow cytometer and 7-AAD staining. The cell viability of the ten peripheral blood samples before PXP treatment was 85.62±3.23%, and the cell viability of the ten peripheral blood samples after PXP treatment was 97.87±2.42%. For example, the results for one peripheral blood sample (No. 1) showed a cell viability of 86.07% for peripheral blood and 97.53% for concentrated peripheral blood cells. This indicates that the cell viability in the concentrated peripheral blood cell sample was significantly higher than that of the peripheral blood cells.

[0182] Experimental Example 3: CD45+ and CD34+ cell counts in mobilized peripheral blood and concentrated peripheral blood cell samples

[0183] Ten samples from Experiment 1 were examined. Using an FC500 flow cytometer, 7-AAD staining was applied to analyze the number and viability of CD45+ and CD34+ cells in all pre- and post-treatment mobilized peripheral blood and concentrated cell samples. Results:

[0184] Regarding the number of CD45+ live cells, the number of mobilized peripheral blood samples was (14.16±2.44)x10^6 / mL, and the number of mobilized peripheral blood concentrated cells was (97.53±6.36)x10^6 / mL, an increase of 6.9 times;

[0185] Regarding the number of CD34+ live cells, the mobilized peripheral blood sample was (92.3±7.9)x10^3 / mL, and the mobilized peripheral blood concentrated cell sample was (674.2±25.1)x10^3 / mL, an increase of 7.3 times.

[0186] Experimental Example 4: Aseptic Testing of Mobilized Peripheral Blood Samples

[0187] Ten cell concentrate samples and ten supernatant concentrate samples from Experiment Example 1 were investigated. Gram staining was used for sterility testing. Smears of mobilized peripheral blood and mobilized peripheral blood concentrated cell samples were prepared, fixed with methanol, stained, and then tested. Results: No microorganisms were found in the Gram staining smears of the ten mobilized peripheral blood samples, the ten mobilized peripheral blood concentrated cell samples, and the ten supernatant concentrate samples.

[0188] The process of preparing mobilized peripheral blood concentrated cells using the PXP system in this invention is characterized by being rapid, closed, and completely sterile.

[0189] Experimental Example 5: Cellular Level Efficacy Study

[0190] The mobile peripheral blood concentrated cells prepared using the PXP system in this invention are an injectable cell preparation containing various stem cell components, including hematopoietic stem cells (HSCs), mesenchymal stem cells (MSCs), endothelial progenitor cells (EPCs), and various cytokines such as vascular endothelial growth factor (VEGF), stromal cell-derived factor (SDF-1), and entostatin, which promote angiogenesis and endothelial cell migration.

[0191] This study investigated 10 samples from Experiment 1. The stem cell biological efficacy of mobilized peripheral blood concentrates was evaluated by CFU colony-forming ability, and the cytokines abundant in mobilized peripheral blood concentrates were quantitatively detected by ELISA.

[0192] 5.1 Stem Cell Biological Efficacy—CFU Colony Formation Experiment

[0193] The biological potency of mobilized peripheral blood concentrated cells was assessed using in vitro CFU colony-forming assays to identify and analyze the colony-forming ability of progenitor / stem cells and characterize the stem cell nature of mixed cells from mobilized peripheral blood concentrated cells. The potency of various stem cells in mobilized peripheral blood and mobilized peripheral blood concentrated cell samples was analyzed using CFU-H (hematopoietic progenitor / stem cells) and CFU-F (stromal progenitor cells). Results:

[0194] Regarding CFU-H (hematopoietic progenitor / stem cell), the number of mobilized peripheral blood samples was (23.3±4.1)x10^3 / mL, and the number of mobilized peripheral blood concentrated cell samples was (196.4±21.6)x10^3 / mL, an increase of 8.4 times;

[0195] Regarding CFU-F (stromal progenitor cells), the mobilized peripheral blood sample was (42.6±5.3)x10^3 / mL, and the mobilized peripheral blood concentrated cell sample was (347.6±30.3)x10^3 / mL, an increase of 8.2 times.

[0196] The results showed that the PXP system can effectively enrich stem cells while maintaining their biological efficacy.

[0197] 5.2 Quantitative analysis of cytokines

[0198] The mobilized peripheral blood concentrated cell injection prepared by the PXP system contains multiple cytokines. Enzyme-linked immunosorbent assay (ELISA) was used to quantitatively detect and analyze the levels of transforming growth factor-β (TGF-β), vascular endothelial growth factor (VEGF), and hepatocyte growth factor (HGF) in mobilized peripheral blood and mobilized peripheral blood concentrated cell samples. Results:

[0199] The TGF-β levels of mobilized peripheral blood and mobilized peripheral blood concentrate cells were 27.2±4.1 pg / ml and 212.3±12.4 pg / ml, respectively.

[0200] The VEGF levels of mobilized peripheral blood and mobilized peripheral blood concentrated cells were 19.3±3.6 pg / ml and 167.3±14.6 pg / ml, respectively.

[0201] The HGF levels of mobilized peripheral blood and mobilized peripheral blood concentrate cells were 195.2±18.4 pg / ml and 1084.3±33.7 pg / ml, respectively.

[0202] The results showed that the levels of TGF-β, VEGF, and HGF in BMAC were significantly higher than those in BMA (p<0.01), indicating that the PXP system can effectively concentrate and enrich cell growth factors.

[0203] Case Study 6: Efficacy of Mobilizing Peripheral Blood Concentrated Cells in the Treatment of Premature Ovarian Failure (POF)

[0204] Chinese Patent Application No. 2021116067853 investigated the efficacy of peripheral blood concentrated cells in treating premature ovarian failure (POF). This study uses peripheral blood concentrated cells prepared as described above in conjunction with GM-CSF (also referred to as gmCSF) to investigate the efficacy of POF treatment.

[0205] (1) Establishing a POF mouse model

[0206] Eight-week-old female C57BL / 6 mice were intraperitoneally injected with 50 mg / kg / day of cyclophosphamide (CTX) for 15 consecutive days at the same time each day to establish a mouse model of premature ovarian failure (POF). The control group received no treatment. After POF modeling was completed, mobilized peripheral blood concentrated cells were transplanted for treatment, and the model animals were randomly assigned to different groups.

[0207] (2) Ovarian reserve function is assessed by indicators such as hormone levels, number of follicles and fertility tests.

[0208] A. Hormone levels

[0209] Animal groups: control group (n=20), POF model group (n=20), mobilized peripheral blood concentrated cells treatment group (n=20), mobilized peripheral blood concentrated cells + gmCSF treatment group (n=20), mobilized peripheral blood concentrated cells + supernatant concentrate + gmCSF treatment group (n=20).

[0210] Mice in the peripheral blood cell mobilization and treatment group were administered 200 μl of a peripheral blood cell mobilization and concentration composition via tail vein injection on day 1 after POF modeling. (This 200 μl of concentrated cell composition was the No. 1 concentrated cell sample mentioned in Example 1, diluted with sterile physiological saline to prepare a CD45-rich solution.) + (A solution with a cell count concentration of 4 x 10^6 cells / 200 μl);

[0211] Mice in the mobilization of peripheral blood concentrated cells + gmCSF treatment group were given 200 μl of the mobilization of peripheral blood concentrated cells gmCSF composition via tail vein injection on day 1 after POF modeling.

[0212] Mice in the POF-treated group were given 200 μl of the POF-treated group via tail vein injection on day 1 after POF modeling.

[0213] The POF model group was injected with an equal volume of physiological saline; the control group received no injection treatment.

[0214] After cell transplantation, all groups were given the same normal diet and water.

[0215] Note: The peripheral blood cell concentrate + gmCSF treatment group mentioned above received a peripheral blood cell concentrate gmCSF composition (which can be simply referred to as the peripheral blood cell gmCSF composition), each 200 μL of which contained: an appropriate amount of the No. 1 mobilized peripheral blood cell concentrate sample obtained in Example 1, in CD45... + The composition is obtained by diluting 2.5 ng of gmCSF (with a cell count of 1 x 10^6) with sterile physiological saline to a final volume.

[0216] The above-mentioned treatment group received a combination of mobilized peripheral blood concentrated cells + supernatant concentrate + gmCSF, which contained, per 200 μL: an appropriate amount of concentrated cell sample No. 1 obtained in Example 1, in CD45... + The composition was obtained by adding 2.5 ng of gmCSF (cell count of 0.8 x 10^6), 20 μL of the No. 1 supernatant concentrate obtained in Example 1, and sterile physiological saline to a final volume.

[0217] The above composition is stored at a temperature of 2-4°C after preparation and administered by injection within 4 hours. gmCSF is a commercially available lyophilized powder for injection.

[0218] Fourteen and twenty-eight days after peripheral blood-concentrated cell transplantation, ten mice from each group were harvested, and blood was collected from the orbital sinus. Serum was separated and stored at -20°C. Enzyme-linked immunosorbent assay (ELISA) was used to analyze the levels of estradiol (E2) and follicle-stimulating hormone (FSH) (specific methods were performed according to Xiang Li's paper, Xiang Li, et al., Human placental mesenchymal stem cell transplantation improves ovarian function by reducing the expression of superoxide dismutase 1 and uncoupling protein-2, Chinese Journal of Reproduction and Contraception, 2018, No. 2). The results are shown in the table below.

[0219]

[0220] The results showed that, compared with the POF model group, the serum E2 level of mice in the mobilized peripheral blood concentrated cells group increased and the FSH level decreased on day 28, with significant differences (P<0.05). In addition, it was found that by using it in combination with GM-CSF and / or supernatant concentrate, the amount of peripheral blood concentrated cells required can be significantly reduced and the same effect can be obtained.

[0221] B. Follicle count in mouse ovarian tissue

[0222] Twenty-eight days after peripheral blood concentrated cell transplantation, 10 mice from each group were sacrificed, and the left ovarian tissue of the mice was fixed in 4% paraformaldehyde. The fixed tissue was then dehydrated with alcohol, cleared with xylene, embedded in paraffin, serially sectioned, and sectioned to a thickness of 5 μm. The sections were stained with hematoxylin and eosin (HE) and observed under a microscope.

[0223] The results showed that compared with the control group, the number of primary, secondary, and mature follicles in the POF model group was significantly reduced, while the number of atretic follicles was significantly increased. Twenty-eight days after treatment with mobilized peripheral blood concentrated cells, the number of follicles at all levels recovered to varying degrees, granulosa cell growth increased, apoptosis decreased, ovarian epithelial cell morphology stabilized, and the number of primary, secondary, and mature follicles significantly increased, while the number of atretic follicles significantly decreased. The follicle counts at all levels 28 days after transplantation of mobilized peripheral blood concentrated cells showed significant differences compared with the POF group; specific results are shown in the table below.

[0224]

[0225] Compared with the POF model group, **p<0.01.

[0226] C. Observation of mouse fertility

[0227] On day 28 after peripheral blood concentrated cell transplantation, male and female mice were housed together at a ratio of 2:1. The fertility rate of the mice was recorded, and the number of litters was compared to observe the restorative effect of peripheral blood concentrated cell transplantation on ovarian function in mice. The results showed a significant difference between the peripheral blood concentrated cell group and the POF group. The comparison of litter sizes is shown in the table below.

[0228]

[0229] As can be seen from the above results, transplantation of mobilized peripheral blood concentrated cells can significantly improve the ovarian reserve function of POF mice, increase the number of follicles, increase estrogen and progesterone levels, and restore fertility in mice, providing experimental evidence for the application of mobilized peripheral blood concentrated cells in the clinical treatment of POF.

[0230] Experimental Example 7: Mobilization of peripheral blood concentrated cells + gmCSF composition

[0231] In the above Experiment Example 6, the mobilized peripheral blood concentrated cells + gmCSF composition was injected as soon as possible after preparation. The inventors found that the biological activity of GM-CSF decreased after the liquid composition was placed at 4°C for 12 hours and 24 hours. This decreasing biological activity was significantly alleviated by adding trace amounts of glutamine and sodium selenite to the liquid composition. The specific experiments are as follows.

[0232] Formula a: Using the five types of mobilized peripheral blood concentrated cells No. 1 to No. 5 obtained in Example 1, five liquid compositions were prepared according to the following formula, denoted as Composition a No. 1 to Composition a No. 5: containing CD45 + Peripheral blood concentrate with a cell count of 5x10^6, 12.5 ng (i.e. 125 IU) of gmCSF, and an appropriate amount of sterile physiological saline to 1 mL;

[0233] Formula a1: Using the five types of mobilized peripheral blood concentrated cells (No. 1 to No. 5) and five types of supernatant concentrates obtained in Example 1, five liquid compositions were prepared according to the following formula, denoted as Composition a1 No. 1 to Composition a1 No. 5: Containing CD45 + Mobilized peripheral blood concentrated cells with a cell count of 4 x 10^6, 12.5 ng (i.e. 125 IU) of gmCSF, 100 μL of supernatant concentrate, and an appropriate amount of sterile physiological saline to 1 mL.

[0234] Formula b: Prepare a liquid composition according to Formula a above but without adding peripheral blood concentrated cells, and denot it as Composition b;

[0235] Formula b1: Formula a1 above is prepared by using No.1 supernatant concentrate but without adding mobilized peripheral blood concentrated cells to form a liquid composition, which is referred to as composition b1.

[0236] Formula c: Using the five types of peripheral blood concentrated cells No.1 to No.5 obtained in Example 1, glutamine (to a final concentration of 0.25 mg / ml) and sodium selenite (to a final concentration of 12.5 μg / ml) were added according to Formula a above to prepare five liquid compositions, which are respectively designated as Composition c No.1 to Composition c No.5;

[0237] Formula c1: Using the five types of mobilized peripheral blood concentrated cells (No.1 to No.5) and five types of supernatant concentrates obtained in Example 1, and following the above formula a1 but with the addition of glutamine (to a final concentration of 0.25 mg / ml) and sodium selenite (to a final concentration of 12.5 μg / ml), five liquid compositions were prepared, which are respectively designated as Composition c1 No.1 to Composition c1 No.5;

[0238] Formula d: Using the five types of peripheral blood concentrated cells No.1 to No.5 obtained in Example 1, glutamine (to a final concentration of 0.25 mg / ml) was added according to Formula a above to prepare five liquid compositions, which are respectively designated as Composition d No.1 to Composition d No.5;

[0239] Formula d1: Using five types of mobilized peripheral blood concentrated cells (No.1 to No.5) and five types of supernatant concentrates obtained in Example 1, glutamine (to a final concentration of 0.25 mg / ml) was added according to Formula a1 above to prepare five liquid compositions, which are respectively designated as Composition d1 No.1 to Composition d1 No.5;

[0240] Formula e: Using the five types of peripheral blood concentrated cells No.1 to No.5 obtained in Example 1, sodium selenite (to a final concentration of 12.5 μg / ml) was added according to Formula a above to prepare five liquid compositions, which are respectively designated as Composition e No.1 to Composition e No.5;

[0241] Formula e1: Using five types of mobilized peripheral blood concentrated cells (No.1 to No.5) and five types of supernatant concentrates obtained in Example 1, and adding sodium selenite (to a final concentration of 12.5 μg / ml) according to Formula a1 above, five liquid compositions were prepared, which are respectively designated as Composition e1 No.1 to Composition e1 No.5.

[0242] The preparation methods of the above-mentioned compositions are conventional methods well known to those skilled in the art. For example, under aseptic conditions, a specified amount of lyophilized powder gmCSF, optional glutamine, and optional sodium selenite are quantitatively dissolved in sterile physiological saline to a specified volume. Additionally, peripheral blood concentrated cells are diluted with sterile physiological saline to a CD45 concentration. + To determine the appropriate concentration for the cell counter, dilute the two solutions with sterile physiological saline according to the formula ratio to the specified concentration, and dispense them into glass bottles.

[0243] Each of the 10 formulations was placed at 4°C, and samples were taken at 0h, 12h, and 24h. The biological activity (IU / ml) of each composition at the specified time was determined according to the "Assay Method for Biological Activity of Recombinant Human Granulocyte-Macrophage Stimulating Factor 3526" in Appendix IV of the 2015 edition of the Chinese Pharmacopoeia. For a given composition, the percentage obtained by dividing its biological activity at 12h or 24h by its biological activity at 0h and then multiplying by 100% is the residual percentage of gmCSF biological activity of that composition at that time point.

[0244] result:

[0245] The biological activity of all compositions from formulations a to e and from formulations a1 to e1 at 0 h was in the range of 123.5 to 133.2 IU / ml. For example, the biological activity of composition aNo.1 at 0 h was 131.6 IU / ml.

[0246] The residual percentages of formulations b and b1 after 12 hours were 98.1% and 97.8%, respectively.

[0247] The residual percentage of all compositions in formulations c and c1 after 12 hours was in the range of 97% to 103%. For example, the residual percentage of composition cNo.1 after 12 hours was 102.3%.

[0248] The residual percentage of all compositions of formulations a and a1, formulations d and d1, and formulations e and e1 after 12 hours is in the range of 79% to 85%. For example, the residual percentage of composition aNo.1 after 12 hours is 82.1%.

[0249] The residual percentages of formulations b and b1 after 24 hours were 93.5% and 94.6%, respectively.

[0250] The residual percentage of all compositions in formulations c and c1 after 24 hours is in the range of 90-95%. For example, the residual percentage of composition cNo.1 after 24 hours is 93.1%.

[0251] The residual percentage of all compositions of formulations a and a1, formulations d and d1, and formulations e and e1 after 24 hours is in the range of 63% to 71%. For example, the residual percentage of composition aNo.1 after 24 hours is 69.1%.

[0252] These results indicate that the biological activity of gmCSF decreases rapidly in cell-containing compositions, and this decrease in biological activity can be significantly overcome by adding trace amounts of glutamine and sodium selenite to the composition.

[0253] In addition, the number of CD45+ viable cells was determined according to the method in Experiment Example 3 above, and the results were as follows:

[0254] All compositions of formulations a, c, d, and e had CD45+ viable cell counts in the range of 496–537 x 10^4 / ml at 0 h. For example, composition a No. 1 had a CD45+ viable cell count of 526.1 x 10^4 / ml at 0 h.

[0255] All compositions of formulations a1, c1, d1, and e1 had CD45+ viable cell counts in the range of 384–419 x 10^4 / ml at 0 h. For example, composition a1 No. 1 had a CD45+ viable cell count of 403.4 x 10^4 / ml at 0 h.

[0256] The CD45+ viable cell counts of all compositions of formulations a, c, d, and e were in the range of 157–233 x 10^4 / ml after 24 hours. For example, the CD45+ viable cell count of composition a No. 1 was 214.6 x 10^4 / ml after 24 hours.

[0257] All compositions of formulations a1, c1, d1, and e1 had a CD45+ viable cell count in the range of 142–177 x 10^4 / ml over 24 hours. For example, composition a1 No.1 had a CD45+ viable cell count of 149.3 x 10^4 / ml over 24 hours.

[0258] These results indicate that there was no significant difference in the number of CD45+ viable cells at different time points among the various compositions, suggesting that glutamine and sodium selenite will not affect the biological activity of the cells. Therefore, although the peripheral blood concentrated cells + gmCSF composition of formulation a and the peripheral blood concentrated cells + supernatant concentrate + gmCSF composition of formulation a1 exhibit excellent biological effects in treating premature ovarian failure, the addition of small amounts of glutamine and sodium selenite significantly improved the stability of the biological activity of gmCSF in the compositions, and there was no significant difference in the number of viable cells in the compositions. This improvement in the stability of gmCSF biological activity is extremely significant for therapeutic applications.

[0259] In addition, since gmCSF is inexpensive and readily available, it can be combined with peripheral blood concentrated cells, which are less readily available, to significantly reduce the amount of cells used while still achieving excellent biological effects in the treatment of premature ovarian failure.

[0260] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

Claims

1. A method for preparing a composition, comprising the step of mixing materials in the following proportions to prepare a sterile formulation composition: mobilizing peripheral blood concentrated cells in an amount of CD45... + Cell count (3~5)×10 6 10-15 ng granulocyte-macrophage stimulating factor, 75-125 µL supernatant concentrate, 0.1-0.5 mg glutamine, 5-20 µg sodium selenite, and appropriate amount of physiological saline to 1 mL; The mobilized peripheral blood concentrate and supernatant concentrate are prepared according to the following method: (1) providing a biological sample of mobilized peripheral blood, which is placed in a sterile bag containing an anticoagulant, the anticoagulant being 3.5% sodium citrate solution supplemented with 0.5 mg / ml histidine and 0.12 mg / ml phosphatidylcholine; (2) Remove the cap from the input tube of the automated cell isolation system and attach a syringe to the luer lock fitting on the input tube to transfer the anticoagulated biological sample through the thrombus filter into the sterile disposable isolation cup at a slow and steady rate, mixing the sample by rocking along the horizontal axis; the automated cell isolation system is a closed PXP isolation system consisting of four components: a) a disposable separation cup, b) a control module, c) a separation base for transmitting data, and d) a DataTrak software processing system; (3) placing the disposable separation cup into the control module, the control module showing "0" before centrifugation, weighing the separation cup / control module assembly, and placing it in a programmable centrifuge and setting the parameters of the centrifuge according to the following procedure: ； (4) starting the centrifuge for centrifugation, the process being as follows: 4a) P1 phase makes the cells in the peripheral blood stratify into three components, red blood cell layer, cell concentrate layer, and plasma layer, by centrifugal density in the disposable separation cup; 4b) P2 phase makes the red blood cells enter the red blood cell recovery cabin; 4c) P3 phase further stratifies the cells in the processing chamber, and P4 phase reduces the centrifugal force to further remove the red blood cells; 4d) P5 phase further stratifies the cell concentrate layer and the plasma, and P6 phase reduces the centrifugal force to make the cell concentrate layer transfer to the recovery cabin through the delivery tube, and the plasma remains in the central cabin; (5) after centrifugation, confirming that the control module window shows "P" (qualified state), removing the separation cup from the control module, connecting a syringe to the output tube of the separation cup connected to the recovery cabin to collect the mobilized peripheral blood concentrate cells; (6) placing the separation cup and the control module on the separation base to transmit data and processing the captured data in the centrifugation process with the DataTrak software processing system; (7) taking the supernatant, i.e. the plasma layer, in the central cabin with a syringe, centrifuging at 2000g for 20 min to remove cell debris, and filtering through a 0.22 µm sterile filter membrane; (8) Rinse the piping of the tangential flow ultrafiltration system with ultrapure water, and install the 100kD 100cm... 2 The filtrate obtained in step (7) was ultrafiltered and concentrated to 1 / 15 of the initial peripheral blood volume using a MidiKros filter to obtain a supernatant concentrate.

2. The method according to claim 1, wherein the tangential flow ultrafiltration system is a tangential flow ultrafiltration system of the type Amicon® KR2i.

3. The method according to claim 1, wherein the volume of the biological sample provided in step (1) is 20-200 ml.

4. The method according to claim 1, wherein the ratio of the anticoagulant to the volume of the biological sample used in step (1) is 1:

12.

5. The method according to claim 1, wherein the anticoagulant used in step (1) is prepared by adding sodium citrate, histidine, and phosphatidylcholine to an appropriate amount of water, heating to 60°C and stirring to dissolve, adding water to the full amount, filtering through a 0.22 µm microporous filter membrane, and autoclaving at 121°C to obtain the anticoagulant.

6. The method according to claim 1, said ratio of concentrated cells to granulocyte macrophage colony stimulating factor is, concentrated cells to CD45 + cells counted 4 x 10 6 cells: 12.5 ng granulocyte macrophage colony stimulating factor.

7. The method according to claim 1, wherein the ratio of concentrated cells to supernatant concentrate is 4 x 10 + cells: 100 μL supernatant concentrate. 6 cells: 100 μL supernatant concentrate.

8. The method according to claim 1, wherein the concentration of the granulocyte macrophage colony-stimulating factor in the composition is 12.5 ng / ml.

9. The method according to claim 1, wherein the granulocyte macrophage colony-stimulating factor is human granulocyte macrophage colony-stimulating factor.

10. The method according to claim 1, wherein the granulocyte macrophage colony-stimulating factor is recombinant human granulocyte macrophage colony-stimulating factor.

11. The method according to claim 1, wherein the weight ratio of granulocyte macrophage colony-stimulating factor to glutamine and sodium selenite in the composition is 12.5 ng: 0.2-0.3 mg: 10-15 μg.

12. The method according to claim 1, wherein the weight ratio of granulocyte macrophage colony-stimulating factor to glutamine and sodium selenite in the composition is 12.5 ng: 0.25 mg: 12.5 μg.

13. Use of a composition prepared according to the method of any one of claims 1-12 for the manufacture of a medicament for the treatment of premature ovarian failure.

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