Umbilical cord blood concentrated cell preparation and use thereof in treating premature ovarian failure

Abstract

The present invention relates to a cord blood concentrated cell preparation and its use in treating premature ovarian failure. In one aspect, it relates to a cell composition made from umbilical cord blood concentrated cells, comprising concentrated cells, granulocyte macrophage stimulating factor, and an optional excipient. The ratio of the concentrated cells to the granulocyte macrophage stimulating factor is such that the concentrated cells are CD45+ / - / -. + The cell count is 5x10^6 cells: 10-15 ng of granulocyte macrophage stimulating factor; the excipient is physiological saline or 5% glucose solution; the concentration of granulocyte macrophage stimulating factor in the composition is 10-15 ng / ml. The present invention also relates to a method for preparing the composition and its pharmaceutical use. The cell preparation of the present invention exhibits excellent efficacy.

Description

Technical Field

[0001] The present invention belongs to the fields of biotechnology and biomedicine and relates to a method for treating premature ovarian failure (POF) using a cell therapy. Specifically, the present invention first relates to a cell therapy comprising rapidly isolating umbilical cord blood concentrated cells (cord blood concentrate) from umbilical cord blood, then formulating the concentrated cells with GM-CSF (granulocyte macrophage stimulating factor, also known as granulocyte / macrophage colony-stimulating factor, gmCSF) to form a cell therapy composition, and then using this umbilical cord blood concentrated cell composition to treat premature ovarian insufficiency and premature ovarian failure. The present method can effectively improve the efficiency of isolating umbilical cord blood concentrated cells from umbilical cord blood, providing a safe, efficient, and simple method for obtaining a umbilical cord blood concentrated cell preparation for treating premature ovarian failure and ovarian insufficiency, such as premature ovarian insufficiency. The present invention also provides a new and effective approach for treating premature ovarian failure by combining the prepared umbilical cord blood concentrated cells with GM-CSF. Background Art

[0002] Premature ovarian failure (POF) refers to ovarian failure leading to amenorrhea and infertility in women before the age of 40. POF is characterized by amenorrhea, infertility, estrogen deficiency, decreased ovarian follicle count, and elevated gonadotropin levels. It is accompanied by a series of low-estrogenic symptoms, such as hot flashes, excessive sweating, facial flushing, and low libido, seriously impacting women's physical and mental health. Furthermore, women with POF are at increased risk of osteoporosis, cardiovascular disease, and Alzheimer's disease. POF is a major cause of female infertility. The incidence of POF in women of childbearing age is approximately 1-3%, and is increasing and affecting younger women.

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

[0004] The etiology of POF is complex and has not yet been fully elucidated. It may be related to autoimmune responses, infections, genetic factors, the effects of treatments such as chemotherapy, radiotherapy, surgery, and endocrine dysfunction, and there is no effective treatment. Currently, the most commonly used treatment for POF is hormone replacement therapy (HRT). Although this therapy has a certain effect on alleviating the clinical symptoms of POF, HRT cannot fundamentally repair damaged ovaries and restore ovarian function. In addition, studies have shown that long-term HRT treatment increases the risk of heart disease and stroke, and may increase the risk of breast cancer and ovarian cancer. Therefore, new treatment strategies are needed to restore ovarian function in patients with POF.

[0005] Umbilical cord blood (UCB) is the blood that remains in the placenta and umbilical cord after fetal delivery, cord ligation, and cord severance. It is typically discarded. However, research over the past decade has revealed that UCB contains hematopoietic stem cells (HSCs), which can rebuild the human hematopoietic and immune systems. These cells can be used for HSC transplantation to treat over 80 diseases. Therefore, UCB has become an important source of HSCs, particularly for unrelated individuals, and a crucial human biological resource. Umbilical cord blood contains a large number of mesenchymal stem cells (MSCs). Stem cells are the seeds of life, capable of differentiating into various cell types in the human body and producing diverse fruits, including blood cells, nerve cells, and bone cells. With advances in technology, medical experts have developed methods to utilize UCB stem cells to treat diseases. Stem cells are a population of cells that possess self-renewal, high proliferation potential, and multiple differentiation potentials. These cells maintain their own characteristics and number through division and can further differentiate into various tissue cells, playing a vital role in tissue repair and other areas. Medical research over the past three decades has found that umbilical cord blood contains a very rich amount of hematopoietic stem cells, which can rebuild the human hematopoietic and immune systems and can be used for hematopoietic stem cell transplantation to treat blood system, immune system, as well as genetic metabolic and congenital diseases. Therefore, umbilical cord blood has become an important source of hematopoietic stem cells and has been widely used in clinical practice. It is a valuable human biological resource. Hematopoietic stem cells (HSCs) can differentiate into red blood cells, white blood cells and platelets in the blood circulation. Mesenchymal stem cells (MSCs) are a cell subpopulation with multiple differentiation potentials to form bone, cartilage, fat, nerve and myoblasts. MSCs support HSCs by paracrine secretion of multiple growth factors, maintaining the stability of the hematopoietic microenvironment.

[0006] Umbilical cord blood concentrate (UCBC) is a concentrate of nucleated cells obtained by centrifuging and separating umbilical cord blood. UCBC contains 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 circulating red blood cells, white blood cells, and platelets. MSCs are a subpopulation of cells with the potential to differentiate into bone, cartilage, fat, nerve, and myoblasts. UCBC contains 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).

[0007] UCBC has anti-inflammatory and immunomodulatory properties, promotes angiogenesis, and promotes tissue regeneration and repair. Preclinical and preliminary clinical studies have confirmed that UCBC improves ovarian function by improving the ovarian microenvironment, promoting angiogenesis, promoting follicle development, increasing the number of antral follicles, and promoting ovulation, making it a potential treatment for POF patients.

[0008] However, cord blood is traditionally separated manually using density gradient centrifugation, which presents significant challenges such as complex operation, time-consuming process, susceptibility to contamination, and poor reproducibility of results. Those skilled in the art desire a method for processing cord blood to obtain cord blood concentrates, or cord blood cell concentrates, that offers one or more advantages, such as simplicity, time efficiency, resistance to contamination, and high reproducibility. This technological advancement has been achieved in our research team's Chinese patent application No. 2021116067919.

[0009] Granulocyte-macrophage colony-stimulating factor (GM-CSF), produced primarily by T cells and macrophages, can induce the growth of granulocyte and macrophage precursor cells into colonies, hence its abbreviation, GM-CSF. GM-CSF's primary biological effects in vivo are to maintain the survival of granulocyte and monocyte lineage cells, promote their growth, induce differentiation, and enhance their phagocytic and bactericidal functions; it also induces the maturation and functional distribution of dendritic cells. Clinically used GM-CSF is typically recombinant human GM-CSF, which is typically indicated for treating leukopenia caused by cancer chemotherapy and myelosuppressive therapy. It is also used to treat low white blood cells in patients with bone marrow failure, prevent potential infectious complications associated with leukopenia, and accelerate recovery from neutropenia caused by infection.

[0010] Existing methods for treating premature ovarian failure still need to be improved. Therefore, those skilled in the art also expect to provide a method for treating premature ovarian failure, such as a method for treating premature ovarian failure using umbilical cord blood concentrated cell therapy. Summary of the Invention

[0011] One object of the present invention is to provide a method for preparing a concentrated umbilical cord blood cell preparation, which is expected to have one or more advantageous effects, including simplicity of operation, reduced time consumption, low susceptibility to contamination, and highly reproducible results. Alternatively, another object of the present invention is to provide a novel method for treating premature ovarian failure, achieved by formulating concentrated umbilical cord blood cells and GM-CSF into a combined therapeutic agent. The present invention has surprisingly discovered that one or more of these objectives can be achieved by preparing concentrated umbilical cord blood cells using a closed automated PXP cell separation system and / or by formulating the obtained concentrated umbilical cord blood cells with GM-CSF into a combined therapeutic agent for treating premature ovarian failure. The present invention has been completed based on these discoveries.

[0012] To this end, the first aspect of the present invention provides a method for preparing an umbilical cord blood concentrated cell preparation, comprising the following steps:

[0013] (1) Provide umbilical cord blood as a biological sample and place it in a sterile bag containing an anticoagulant for later use;

[0014] (2) removing the protective cap on the input tube of the automated cell separation system, connecting the syringe to the Luer lock connector of the input tube, transferring the anticoagulated biological sample to a disposable sterile separation cup through the thrombus filter at a slow and steady rate, and shaking the sample along the horizontal axis to mix the sample; 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 transmitting data, and d) a DataTrak software processing system;

[0015] (3) Place the disposable separation cup into the control module. The control module status display should be "0" before centrifugation. Weigh the separation cup / control module assembly, balance it, and place it in the programmable centrifuge. Set the centrifuge parameters according to the following procedure:

[0016]

[0017]

[0018] (4) Start the centrifuge and centrifuge. The process is as follows:

[0019] 4a) In the P1 phase, cells in the biological sample are separated into three components: a red blood cell layer, a cell concentrate layer, and a plasma layer in a disposable separation cup by centrifugal density stratification.

[0020] 4b) P2 phase causes most red blood cells to enter the red blood cell recovery chamber;

[0021] 4c) P3 further separates the cells in the processing chamber, and P4 reduces the centrifugal force to further remove red blood cells;

[0022] 4d) P5 stage further separates the cell concentrate layer and plasma, and P6 stage reduces the centrifugal force, allowing the cell concentrate layer to be transferred to the recovery chamber through the transfer tube, while the plasma remains in the central chamber;

[0023] (5) After centrifugation is completed, confirm that the control module window displays "P", which means the qualified status, remove the separation cup from the control module, connect the syringe to the output tube connecting the separation cup and the recovery chamber, and collect the umbilical cord blood concentrated cell preparation.

[0024] 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 350 ml.

[0025] According to the method of the first aspect of the present invention, in step (1), 1 ml of sample is additionally drawn for detection.

[0026] According to the method of the first aspect of the present invention, the anticoagulant used in step (1) is sodium citrate solution.

[0027] According to the method of the first aspect of the present invention, the anticoagulant used in step (1) is 3.6% sodium citrate solution.

[0028] According to the method of the first aspect of the present invention, the anticoagulant used in step (1) is a 3.6% sodium citrate solution, to which 0.5 mg / ml histidine and 0.15 mg / ml phosphatidylcholine are supplemented.

[0029] 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.

[0030] According to the method of the first aspect of the present invention, the preparation method of 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 full amount, filtered through a 0.22 μm microporous filter membrane, and sterilized by autoclaving at 121°C.

[0031] The method according to the first aspect of the present invention further comprises the following steps: (6) placing the separation cup and the control module on a separation base to transmit data and process the data captured during the centrifugation process using a DataTrak software processing system.

[0032] Furthermore, a second aspect of the present invention provides an umbilical cord blood concentrated cell preparation, which is prepared by a method comprising the following steps:

[0033] (1) Provide umbilical cord blood as a biological sample and place it in a sterile bag containing an anticoagulant for later use;

[0034] (2) removing the protective cap on the input tube of the automated cell separation system, connecting the syringe to the Luer lock connector of the input tube, transferring the anticoagulated biological sample to a disposable sterile separation cup through the thrombus filter at a slow and steady rate, and shaking the sample along the horizontal axis to mix the sample; 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 transmitting data, and d) a DataTrak software processing system;

[0035] (3) Place the disposable separation cup into the control module. The control module status display should be "0" before centrifugation. Weigh the separation cup / control module assembly, balance it, and place it in the programmable centrifuge. Set the centrifuge parameters according to the following procedure:

[0036] 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 2.5 P4 9 9 50 1 P5 9 9 250 0.5 P6 9 9 50 1

[0037] (4) Start the centrifuge and centrifuge. The process is as follows:

[0038] 4a) In the P1 phase, cells in the biological sample are separated into three components: a red blood cell layer, a cell concentrate layer, and a plasma layer in a disposable separation cup by centrifugal density stratification.

[0039] 4b) P2 phase causes most red blood cells to enter the red blood cell recovery chamber;

[0040] 4c) P3 further separates the cells in the processing chamber, and P4 reduces the centrifugal force to further remove red blood cells;

[0041] 4d) P5 stage further separates the cell concentrate layer and plasma, and P6 stage reduces the centrifugal force, allowing the cell concentrate layer to be transferred to the recovery chamber through the transfer tube, while the plasma remains in the central chamber;

[0042] (5) After centrifugation is completed, confirm that the control module window displays "P", which means the qualified status, remove the separation cup from the control module, connect the syringe to the output tube connecting the separation cup and the recovery chamber, and collect the umbilical cord blood concentrated cell preparation.

[0043] According to the umbilical cord blood concentrated cell preparation of the second aspect of the present invention, the volume of the biological sample provided in step (1) is 20 to 350 ml.

[0044] According to the umbilical cord blood concentrated cell preparation of the second aspect of the present invention, in step (1), 1 ml of sample is additionally drawn for testing.

[0045] According to the umbilical cord blood concentrated cell preparation of the second aspect of the present invention, the anticoagulant used in step (1) is sodium citrate solution.

[0046] According to the umbilical cord blood concentrated cell preparation of the second aspect of the present invention, the anticoagulant used in step (1) is 3.6% sodium citrate solution.

[0047] According to the umbilical cord blood concentrated cell preparation of the second aspect of the present invention, the anticoagulant used in step (1) is a 3.6% sodium citrate solution, to which 0.5 mg / ml histidine and 0.15 mg / ml phosphatidylcholine are supplemented.

[0048] According to the umbilical cord blood concentrated cell preparation of the second aspect of the present invention, the volume ratio of the anticoagulant used in step (1) to the biological sample is 1:12.

[0049] According to the second aspect of the present invention, the umbilical cord blood concentrated cell preparation used in step (2) 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 amount, filtering through a 0.22 μm microporous filter membrane, and sterilizing by autoclaving at 121° C. to obtain the product.

[0050] The umbilical cord blood concentrated cell preparation according to the second aspect of the present invention further comprises the following steps: (6) placing the separation cup and the control module on the separation base to transmit data and process the data captured during the centrifugation process using the DataTrak software processing system.

[0051] Furthermore, a third aspect of the present invention provides use of a concentrated umbilical cord blood cell preparation in preparing a cell therapy agent for treating premature ovarian failure, wherein the concentrated umbilical cord blood cell preparation is prepared by a method comprising the following steps:

[0052] (1) Provide umbilical cord blood as a biological sample and place it in a sterile bag containing an anticoagulant for later use;

[0053] (2) removing the protective cap on the input tube of the automated cell separation system, connecting the syringe to the Luer lock connector of the input tube, transferring the anticoagulated biological sample to a disposable sterile separation cup through the thrombus filter at a slow and steady rate, and shaking the sample along the horizontal axis to mix the sample; 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 transmitting data, and d) a DataTrak software processing system;

[0054] (3) Place the disposable separation cup into the control module. The control module status display should be "0" before centrifugation. Weigh the separation cup / control module assembly, balance it, and place it in the programmable centrifuge. Set the centrifuge parameters according to the following procedure:

[0055] 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 2.5 P4 9 9 50 1 P5 9 9 250 0.5 P6 9 9 50 1

[0056] (4) Start the centrifuge and centrifuge. The process is as follows:

[0057] 4a) In the P1 phase, cells in the biological sample are separated into three components: a red blood cell layer, a cell concentrate layer, and a plasma layer in a disposable separation cup by centrifugal density stratification.

[0058] 4b) P2 phase causes most red blood cells to enter the red blood cell recovery chamber;

[0059] 4c) P3 further separates the cells in the processing chamber, and P4 reduces the centrifugal force to further remove red blood cells;

[0060] 4d) P5 stage further separates the cell concentrate layer and plasma, and P6 stage reduces the centrifugal force, allowing the cell concentrate layer to be transferred to the recovery chamber through the transfer tube, while the plasma remains in the central chamber;

[0061] (5) After centrifugation is completed, confirm that the control module window displays "P", which means the qualified status, remove the separation cup from the control module, connect the syringe to the output tube connecting the separation cup and the recovery chamber, and collect the umbilical cord blood concentrated cell preparation.

[0062] According to the use of the third aspect of the present invention, the volume of the biological sample provided in step (1) is 20 to 350 ml.

[0063] According to the use of the third aspect of the present invention, in step (1), 1 ml of sample is additionally drawn for detection.

[0064] According to the use of the third aspect of the present invention, the anticoagulant used in step (1) is sodium citrate solution.

[0065] According to the use of the third aspect of the present invention, the anticoagulant used in step (1) is 3.6% sodium citrate solution.

[0066] According to the use of the third aspect of the present invention, the anticoagulant used in step (1) is a 3.6% sodium citrate solution, to which 0.5 mg / ml histidine and 0.15 mg / ml phosphatidylcholine are supplemented.

[0067] According to the use of the third aspect of the present invention, the volume ratio of the anticoagulant used in step (1) to the biological sample is 1:12.

[0068] According to the third 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 full amount, filtering with a 0.22 μm microporous filter membrane, and sterilizing by autoclaving at 121°C.

[0069] According to the third aspect of the present invention, the method further comprises the following steps: (6) placing the separation cup and the control module on the separation base to transmit data and process the data captured during the centrifugation process using the DataTrak software processing system.

[0070] Furthermore, a fourth aspect of the present invention provides a method for treating premature ovarian failure, comprising administering to a subject in need thereof a therapeutically effective amount of a concentrated umbilical cord blood cell preparation, wherein the concentrated umbilical cord blood cell preparation is prepared by a method comprising the following steps:

[0071] (1) Provide umbilical cord blood as a biological sample and place it in a sterile bag containing an anticoagulant for later use;

[0072] (2) removing the protective cap on the input tube of the automated cell separation system, connecting the syringe to the Luer lock connector of the input tube, transferring the anticoagulated biological sample to a disposable sterile separation cup through the thrombus filter at a slow and steady rate, and shaking the sample along the horizontal axis to mix the sample; 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 transmitting data, and d) a DataTrak software processing system;

[0073] (3) Place the disposable separation cup into the control module. The control module status display should be "0" before centrifugation. Weigh the separation cup / control module assembly, balance it, and place it in the programmable centrifuge. Set the centrifuge parameters according to the following procedure:

[0074] 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 2.5 P4 9 9 50 1 P5 9 9 250 0.5 P6 9 9 50 1

[0075] (4) Start the centrifuge and centrifuge. The process is as follows:

[0076] 4a) In the P1 phase, cells in the biological sample are separated into three components: a red blood cell layer, a cell concentrate layer, and a plasma layer in a disposable separation cup by centrifugal density stratification.

[0077] 4b) P2 phase causes most red blood cells to enter the red blood cell recovery chamber;

[0078] 4c) P3 further separates the cells in the processing chamber, and P4 reduces the centrifugal force to further remove red blood cells;

[0079] 4d) P5 stage further separates the cell concentrate layer and plasma, and P6 stage reduces the centrifugal force, allowing the cell concentrate layer to be transferred to the recovery chamber through the transfer tube, while the plasma remains in the central chamber;

[0080] (5) After centrifugation is completed, confirm that the control module window displays "P", which means the qualified status, remove the separation cup from the control module, connect the syringe to the output tube connecting the separation cup and the recovery chamber, and collect the umbilical cord blood concentrated cell preparation.

[0081] 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 350 ml.

[0082] According to the method of the fourth aspect of the present invention, in step (1), 1 ml of sample is additionally drawn for detection.

[0083] According to the method of the fourth aspect of the present invention, the anticoagulant used in step (1) is sodium citrate solution.

[0084] According to the method of the fourth aspect of the present invention, the anticoagulant used in step (1) is 3.6% sodium citrate solution.

[0085] According to the method of the fourth aspect of the present invention, the anticoagulant used in step (1) is a 3.6% sodium citrate solution, to which 0.5 mg / ml histidine and 0.15 mg / ml phosphatidylcholine are supplemented.

[0086] 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.

[0087] According to the method of the fourth aspect of the present invention, the preparation method of 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 full amount, filtered through a 0.22 μm microporous filter membrane, and sterilized by autoclaving at 121°C.

[0088] The method according to the fourth aspect of the present invention further comprises the following steps: (6) placing the separation cup and the control module on a separation base to transmit data and process the data captured during the centrifugation process using a DataTrak software processing system.

[0089] Furthermore, the fifth aspect of the present invention provides a cell composition made from concentrated cells of umbilical cord blood, comprising concentrated cells, granulocyte macrophage stimulating factor and optional excipients.

[0090] According to the cell composition of the fifth aspect of the present invention, the ratio of the concentrated cells to the granulocyte macrophage stimulating factor is: + Cell count: 5x10^6 cells: 10-15ng granulocyte macrophage stimulating factor; for example, the ratio is: concentrated cells with CD45 + Cell count: 5x10^6 cells: 12.5ng granulocyte macrophage stimulating factor.

[0091] According to the cell composition of the fifth aspect of the present invention, the excipient is physiological saline or 5% glucose solution.

[0092] According to the cell composition of the fifth aspect of the present invention, 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.

[0093] According to the cell composition of the fifth aspect of the present invention, the granulocyte macrophage stimulating factor is human granulocyte macrophage stimulating factor.

[0094] According to the cell composition of the fifth aspect of the present invention, the granulocyte macrophage stimulating factor is recombinant human granulocyte macrophage stimulating factor.

[0095] According to the cell composition of the fifth aspect of the present invention, the concentrated cells are as described in any embodiment of the second aspect of the present invention.

[0096] The cell composition according to the fifth aspect of the present invention further comprises glutamine and sodium selenite.

[0097] According to the fifth aspect of the present invention, the cell composition further comprises glutamine and sodium selenite, and 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.

[0098] According to the fifth aspect of the present invention, the cell composition further comprises glutamine and sodium selenite, and 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.

[0099] According to the fifth aspect of the present invention, the cell composition further comprises glutamine and sodium selenite, and the weight ratio of granulocyte macrophage stimulating factor to glutamine and sodium selenite in the composition is 12.5 ng:0.3 mg:10 μg.

[0100] The cell composition according to the fifth aspect of the present invention comprises: CD45 + Concentrated cells with a cell count of 4-6x10^6, 10-15ng of gmCSF, 0.1-0.5mg of glutamine, 5-20μg of sodium selenite, and an appropriate amount of normal saline to 1mL.

[0101] The cell composition according to the fifth aspect of the present invention comprises: CD45 + Concentrated cells with a cell count of 4-6x10^6, 10-15ng of gmCSF, 0.2-0.3mg of glutamine, 10-15μg of sodium selenite, and an appropriate amount of normal saline to 1mL.

[0102] The cell composition according to the fifth aspect of the present invention comprises: CD45 + Concentrated cells with a cell count of 5x10^6, 12.5ng of gmCSF, 0.2-0.3mg of glutamine, 10-15μg of sodium selenite, and normal saline to 1mL.

[0103] The cell composition according to the fifth aspect of the present invention comprises: CD45 + Concentrated cells with a cell count of 5x10^6, 12.5ng of gmCSF, 0.3mg of glutamine, 10μg of sodium selenite, and normal saline to 1mL.

[0104] Furthermore, the sixth aspect of the present invention provides use of the cell composition described in any one of the fifth aspects of the present invention in the preparation of a medicament for treating premature ovarian failure.

[0105] Furthermore, the seventh aspect of the present invention provides a method for preparing the cell composition described in any one of the fifth aspects of the present invention, which includes the steps of mixing a specified amount of concentrated cells, granulocyte macrophage stimulating factor, glutamine, sodium selenite, and optional excipients to prepare a sterile preparation.

[0106] As used herein, the phrase "CD45 +The 5x10^6 in "enriched cells with a cell count of 5x10^6" refers to 5 times 10 to the power of 6, and other similar expressions have the same meaning. In the various operating steps of the present invention described above, although the specific steps described therein differ in some details or language from the steps described in the preparation examples in the detailed implementation section below, those skilled in the art can fully summarize the above-mentioned method steps based on the detailed disclosure of the present invention.

[0107] Any embodiment of any aspect of the present invention may be combined with other embodiments, as long as they do not conflict. In addition, any technical feature in any embodiment of any aspect of the present invention may be applicable to the same technical feature in other embodiments, as long as they do not conflict. The present invention is further described below.

[0108] All documents cited herein are incorporated herein by reference in their entirety, and if the meanings expressed in these documents are inconsistent with those of the present invention, the present invention shall prevail. In addition, various terms and phrases used in the present invention have the general meanings known to those skilled in the art. Even so, the present invention still intends to provide a more detailed description and explanation of these terms and phrases herein. If the terms and phrases mentioned are inconsistent with the generally known meanings, the meanings expressed in the present invention shall prevail.

[0109] This invention utilizes the PXP automated rapid cell processing system, a closed system for automated separation and concentration, to safely, efficiently, and simply obtain concentrated umbilical cord blood cells. This method lays the foundation for the clinical application of concentrated umbilical cord blood cells in treating patients with POF. The invention provides a method for rapidly separating and obtaining concentrated umbilical cord blood cells from human umbilical cord blood using a closed, automated cell separation system. The resulting concentrated umbilical cord blood cells can be used as an active ingredient in treating ovarian damage, promoting angiogenesis and follicular development, thereby improving ovarian function.

[0110] Existing studies have confirmed that contaminating red blood cells are associated with decreased stem / progenitor cell function, and red blood cell contamination of cell concentrates is believed to reduce the efficacy of cell therapy. To further unleash the potential of cell therapy, the industry urgently needs new processing systems that can improve target cell purity and remove contaminating red blood cells.

[0111] The cell separation system used in the specific experiments of the present invention is The automated separation system, model 80065-01, is supplied by Shenzhen Boya Perception Medical Technology Co., Ltd. and manufactured by ThermoGenesis, Inc., USA. The innovative PXP system addresses many of the shortcomings of existing systems on the market. The PXP system enables clinicians to rapidly achieve high stem and progenitor cell recovery rates with virtually no red blood cell contamination, typically less than 5% of the starting sample.

[0112] The PXP system is a highly efficient point-of-care product designed for clinical institutions developing and using cell therapy technologies, meeting their needs for rapid, efficient, and sterile cell processing in the operating room environment. As a cutting-edge automated rapid cell processing system, the PXP system requires no cell separation media or precipitants and can process multiple samples simultaneously, achieving high recovery rates for MNCs, CD34+, and CD45+ cells. This system enables clinicians in hospital surgical centers or clinics to efficiently extract stem cells from biological samples (such as bone marrow and umbilical cord blood) within 30 minutes, achieving over 90% red blood cell removal. Furthermore, the PXP system is equipped with patented DataTrak software to track and capture data, providing customers with GMP process control and reporting information.

[0113] The PXP system has been registered as a medical device by the health ministries of many countries. This system has a wide range of clinical applications, primarily for cell therapy of orthopedic diseases. Some institutions have used the PXP system to efficiently and high-quality autologous bone marrow stem cell production in operating room environments, bringing orthopedic cell therapy to a new level and significantly enhancing the competitiveness of treatment centers. The present invention utilizes the PXP system for processing bone marrow extracts, enabling rapid, real-time, automated processing of umbilical cord blood cells, ensuring high recovery rates of mononuclear cells (MNCs), and enabling simultaneous processing of multiple umbilical cord blood units without the need for cell separation media or precipitants. Similar to the bone marrow stem cells described above, umbilical cord blood cells can also be used to treat premature ovarian failure.

[0114] The advantages of the PXP system used in the specific experiments of the present invention include, but are not limited to: stable and excellent MNC (monocyte) and CD34+, CD45+ cell recovery rates, rapid processing of umbilical cord blood samples within 30 minutes, red blood cell removal efficiency of more than 95%, an automated closed sterile system, rapid and accurate data tracking and documentation, and sample processing data can be uploaded to a computer via DataTrak software to provide production records and reporting information that meets GMP requirements.

[0115] Recent studies have shown that umbilical cord tissue and cord blood also contain mesenchymal stem cells (MSCs), which can be successfully isolated. Umbilical cord blood-derived stem cells are generally referred to as hematopoietic stem cells. MSCs derived from this source not only retain the biological properties of MSCs, but also exhibit more primitive characteristics and enhanced proliferation and differentiation capabilities. Their immune cell functional activity is low, significantly reducing the risk of triggering an immune response and causing graft-versus-host disease. The risk of infection and transmission of latent viruses and microorganisms is also relatively low. The collection process is simple and poses no risk to the mother or newborn. These factors make umbilical cord MSCs an ideal alternative to bone marrow MSCs.

[0116] Basic research has revealed that stem cells capable of differentiating into endothelial cells include endothelial progenitor cells (EPCs), bone marrow mononuclear cells (BMMNCs), and peripheral blood mononuclear cells (PBMNCs). However, these stem cells are limited in their tissue sources, and the quantities available for extraction and expansion are limited. Consequently, this therapy is currently still in the preclinical research stage.

[0117] Experiments have shown that human CD34 + These cells (CD34 is a marker of mature blood vessels) can alleviate CLI symptoms, improve function of affected limbs, and prevent amputation. However, since they are present in only about 5%-10% of the umbilical cord's Wharton's jelly, efficient production of these cells is crucial for their widespread application.

[0118] Premature ovarian failure (POF) is a common gynecological endocrine disease that affects women's health. The incidence rate is 1%-2% in women under 40 years of age and 0.1% in women under 30 years of age. The diagnostic criteria established by the Chinese Society of Obstetrics and Gynecology of the Chinese Medical Association are: amenorrhea for ≥4-6 months, follicle-stimulating hormone levels >40 IU / L with an interval of more than four weeks between two periods, and decreased estrogen levels and menopausal symptoms. Due to ovarian dysfunction and failure, POF causes a sharp decrease in hormone secretion, leading to amenorrhea or oligomenorrhea, often accompanied by symptoms such as hot flashes, night sweats, irritability, anxiety, osteoporosis, and temporary or permanent loss of fertility, threatening both physical and psychological health. The specific etiology of most cases of clinical POF remains unclear, but several factors are currently believed to be involved, including genetic, autoimmune, iatrogenic, and environmental factors. With the widespread use of chemotherapy in gynecological cancer treatment, the incidence of iatrogenic secondary POF is increasing. To address the aforementioned causes, current treatments for premature ovarian failure include hormone replacement therapy, immune regulation, ovarian tissue cryopreservation and transplantation, and embryo cryopreservation and transplantation. These clinical treatments can improve symptoms of premature ovarian failure and promote fertility, but they cannot fundamentally restore ovarian function and are associated with ethical concerns and adverse reactions. Therefore, finding a treatment that can effectively and safely restore ovarian function is a top priority for the academic community.

[0119] A 2019 study conducted in the United States showed that umbilical cord blood stem cells have demonstrated encouraging regenerative capacity in POF. In this treatment experiment, 18 female mice were divided equally into three groups: the first group was a control group (injected with phosphate-buffered saline alone), the second group was a chemotherapy group (administered a combination of busulfan and cyclophosphamide and phosphate-buffered saline), and the third group was an experimental group (in addition to the second group of chemotherapy drugs, umbilical cord blood stem cells were injected into both ovaries). The mice's weight and hormone levels were then assessed. After eight weeks of treatment, weight data for the mice showed that, compared to the significant weight loss after chemotherapy in the second group, the umbilical cord blood stem cell group had significantly increased their weight after stem cell infusion.

[0120] Starting from the fourth week, follicle-stimulating hormone (FSH) levels in the group receiving umbilical cord blood stem cells increased significantly. FSH is a hormone secreted by basophils in the anterior pituitary gland. Its primary function is to promote follicular maturation, thereby promoting the proliferation and differentiation of granulosa cells, leading to overall ovarian growth. Eighteen female mice were enrolled in the breeding experiment. The treatment protocol was repeated, but starting one week after surgery, one male mouse was placed in the same cage. The resulting pups were carefully inspected for abnormalities, and each animal in each group was counted. Over the three-month trial period, the mating rate was 100% in the umbilical cord blood stem cell-transfused and control groups, compared to only 25% in the chemotherapy group. A total of 26 pups were born in the umbilical cord blood stem cell-transfused group, compared to only 2 in the chemotherapy group. Although the total number of pups in the umbilical cord blood stem cell-transfused group was significantly lower than in the control group, the conception rate was comparable to that of the control group. This study found that umbilical cord blood stem cell-transfused mice had a positive therapeutic effect on chemotherapy-damaged ovaries. The mice gained weight, maintained good hormone secretion, and regained fertility.

[0121] Umbilical cord blood is the blood that remains in the placenta and umbilical cord after fetal delivery, cord ligation, and cord severance. It is typically discarded. However, research over the past decade has revealed that cord blood contains hematopoietic stem cells, which can rebuild the human hematopoietic and immune systems. These cells can be used for hematopoietic stem cell transplantation to treat over 80 diseases. Therefore, cord blood has become an important source of hematopoietic stem cells, particularly for unrelated individuals. It is also a crucial human biological resource. Umbilical cord blood contains a large number of stem cells, which are the seeds of life. They differentiate into various cells in the human body and produce a variety of fruits, including blood cells, nerve cells, and bone cells. With the advancement of technology, medical experts have developed methods to use stem cells from cord blood to treat diseases. Stem cells are a population of cells that possess self-renewal, high proliferation potential, and multiple differentiation potentials. These cells maintain their own characteristics and number through division and can further differentiate into various tissue cells, playing an active role in tissue repair and other areas. Medical research over the past three decades has revealed that umbilical cord blood contains a rich supply of hematopoietic stem cells (HSCs), which can rebuild the human hematopoietic and immune systems. These stem cells can be used for hematopoietic stem cell transplantation to treat blood and immune system diseases, as well as inherited metabolic and congenital diseases. Therefore, umbilical cord blood has become an important source of hematopoietic stem cells and is widely used in clinical practice, representing a valuable human biological resource.

[0122] The 21st century is the century of biology. Cell therapy, as a biotherapeutic approach, has opened up new avenues for treating human diseases. As of the end of May 2014, a total of 928 clinical studies involving umbilical cord blood had been registered with the U.S. National Institutes of Health (NIH). Diseases treated by umbilical cord blood research institutions include: autoimmune diseases (rheumatoid arthritis, lupus erythematosus, and multiple sclerosis); cardiovascular and cerebrovascular diseases (congenital heart disease, cardiac repair and recovery, and ischemic stroke); neurological diseases (cerebral palsy, hypoxic-ischemic encephalopathy, autism, traumatic brain injury, spinal cord injury, hearing loss, amyotrophic lateral sclerosis, and Alzheimer's disease); malignant diseases (breast cancer and kidney cancer); congenital diseases (Duchenne muscular dystrophy, Becker muscular dystrophy, and cystic fibrosis); and other diseases (type 1 diabetes, HIV / AIDS, cartilage repair, and critical limb ischemia).

[0123] So far, the research on CD34 + Cell separation usually adopts Ficoll separation method, hydroxyethyl starch separation method and gelatin natural sedimentation separation method, and then further purifies the obtained CD34 by immunomagnetic bead adsorption method (MACS). + cells to obtain CD34 + The above method is used to isolate and purify primary CD34 cells directly from human umbilical cord blood. + Given the limited supply of human umbilical cord blood, the CD34 + The number of cells is also extremely limited.

[0124] The umbilical cord blood cell extract market is an emerging one. After 30 to 40 years of research and development, various stem cell therapies and related technologies and equipment are gradually becoming commercialized. The emergence of umbilical cord blood cell concentrates, platelet-rich plasma, and stem cell derivatives is becoming a new trend in future healthcare. With the development of personalized medicine, the global clinical demand for cell therapy is becoming increasingly significant, and the trend of liberalizing the clinical application of cell therapy is also becoming more and more obvious. As the clinical application of cell therapy gradually expands, medical institutions will increasingly demand the next generation of cell processing and preparation solutions based on automated technologies, and these technology platforms will have a significant impact on the success of cell therapy.

[0125] The present invention achieves satisfactory results by using the PXP system for cell separation.

[0126] GM-CSF can be human granulocyte macrophage stimulating factor or recombinant granulocyte macrophage stimulating factor, which has been included in multiple versions of the Chinese Pharmacopoeia and has many brands of products approved for clinical use. In the present invention, unless otherwise specified, the GM-CSF used in the experiment is a commercially available recombinant human granulocyte macrophage stimulating factor for injection (S19991012, specification 750000 IU / 75 μg, 10 IU / ng). When preparing the composition, if necessary, it can be diluted in advance with 0.9% sodium chloride injection to an appropriate concentration. GM-CSF (Granulocyte-macrophage Colony Stimulating Factor, granulocyte-macrophage colony stimulating factor, or granulocyte macrophage stimulating factor) acts on hematopoietic progenitor cells to promote their proliferation and differentiation. Its important role is to stimulate the maturation of granulocytes, mononuclear macrophages, promote the release of mature cells into the peripheral blood, and promote the various functions of macrophages and eosinophils. GM-CSF is a clinically used drug for leukopenia or granulocytopenia caused by various reasons. It stimulates bone marrow hematopoiesis, stimulates the proliferation of granulocytes, monocytes, and T cells, and promotes the maturation of monocytes and granulocytes. GM-CSF also overcomes the bone marrow toxicity caused by radiotherapy and chemotherapy, shortens the duration of neutropenia during cancer chemotherapy, and improves patient tolerance to chemotherapy. GM-CSF enhances the function of monocytes, granulocytes, eosinophils, and macrophages, thereby improving the body's anti-tumor and anti-infection immunity.

[0127] The present invention combines concentrated umbilical cord blood cells separated by the PXP system with GM-CSF and uses a premature ovarian failure model for verification, achieving positive effects. DETAILED DESCRIPTION

[0128] The present invention can be further described by the following examples. However, the scope of the present invention is not limited to the following examples. Those skilled in the art will appreciate that, without departing from the spirit and scope of the present invention, various changes and modifications may be made to the present invention. The present invention provides general and / or specific descriptions of the materials and test methods used in the test. Although many materials and operating methods employed for achieving the purpose of the present invention are well known in the art, the present invention is still described in as much detail as possible herein.

[0129] In the present invention, unless otherwise specified, the cell separation system used in the specific experiments is The automated separation system, also referred to herein as the PXP system, automated PXP cell separation system, automated PXP separation system, or PXP separation system, is model 80065-01. The supplier is Shenzhen Boya Perception Medical Technology Co., Ltd., and the manufacturer is ThermoGenesis, Inc., USA. In this invention, the term "umbilical cord blood myeloid concentrate" may also be referred to as "umbilical cord blood concentrated cell preparation." Unless otherwise specified, the two have the same meaning.

[0130] Example 1: Rapid separation and preparation of umbilical cord blood concentrated cells (UCBC)

[0131] (1) Provide a biological sample of umbilical cord blood (a sample volume of 20 to 350 ml can be processed), place it in a sterile bag containing an anticoagulant for standby use, and draw 1 ml of sample for testing; the anticoagulant is a 3.6% sodium citrate solution, to which 0.5 mg / ml histidine and 0.15 mg / ml phosphatidylcholine are added, and the volume ratio of the anticoagulant to the biological sample is 1:12; the preparation method of the anticoagulant is: add sodium citrate, histidine and phosphatidylcholine to an appropriate amount of water, heat to 60°C and stir to dissolve, add water to the full amount, filter with a 0.22 μm microporous filter membrane, and sterilize by autoclaving at 121°C;

[0132] (2) removing the protective cap on the input tube of the automated cell separation system, connecting the syringe to the Luer lock connector of the input tube, transferring the anticoagulated biological sample to a disposable sterile separation cup through the thrombus filter at a slow and steady rate, and shaking the sample along the horizontal axis to mix the sample; 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 transmitting data, and d) a DataTrak software processing system;

[0133] (3) Place the disposable separation cup into the control module. The control module status display should be "0" before centrifugation. Weigh the separation cup / control module assembly, balance it, and place it in the programmable centrifuge. Set the centrifuge parameters according to the following procedure:

[0134] 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 2.5 P4 9 9 50 1 P5 9 9 250 0.5 P6 9 9 50 1

[0135] (4) Start the centrifuge and centrifuge. The process is as follows:

[0136] 4a) In the P1 phase, cells in the biological sample are separated into three components: a red blood cell layer, a cell concentrate layer, and a plasma layer in a disposable separation cup by centrifugal density stratification.

[0137] 4b) P2 phase causes most red blood cells to enter the red blood cell recovery chamber;

[0138] 4c) P3 further separates the cells in the processing chamber, and P4 reduces the centrifugal force to further remove red blood cells;

[0139] 4d) P5 stage further separates the cell concentrate layer and plasma, and P6 stage reduces the centrifugal force, allowing the cell concentrate layer to be transferred to the recovery chamber through the transfer tube, while the plasma remains in the central chamber;

[0140] (5) After centrifugation is completed, confirm that the control module window displays "P", indicating a qualified status. Remove the separation cup from the control module, connect the syringe to the output tube connecting the separation cup and the recovery chamber, and collect the concentrated umbilical cord blood cells.

[0141] (6) Place the separation cup and control module on the separation base to transmit data and process the data captured during the centrifugation process using the DataTrak software processing system.

[0142] In this Example 1, 10 human umbilical cord blood biological samples were collected and subjected to cell separation and preparation to obtain 10 umbilical cord blood concentrated cells, which were labeled No. 1 to No. 10, respectively.

[0143] Experimental Example 1: Analysis of MNC Recovery Rate in Umbilical Cord Blood Concentrated Cells

[0144] Using the method of Example 1, 10 collected umbilical cord blood samples (pre-separation volume ranged from 172 to 193 ml) were subjected to cell separation. Subsequently, referring to the method described in Chinese Patent Application No. 2021116067919, cell analysis was performed on each separated fraction. The results for the 10 samples showed: an average input volume of 182.4 ml of umbilical cord blood, an average output volume of 23.6 ml of concentrated umbilical cord blood, an average red blood cell (RBC) removal rate of 98.5%, an average mononuclear cell (MNC) recovery rate of 96.2%, and an average increase in MNC concentration of 7.73 times. For example, the results for one umbilical cord blood sample (No. 1) showed: pre-separation volume = 187.6 ml, final volume = 23.3 ml, RBC removal rate = 98.1%, MNC recovery rate = 96.8%, and MNC concentration factor = 8.05.

[0145] The results showed that the PXP system can be used to enrich MNCs in umbilical cord blood while removing most red blood cells in a simple, time-saving, less susceptible to contamination, and highly reproducible manner.

[0146] Experimental Example 2: Cell Viability in Cord Blood and Cord Blood Concentrated Cell Samples

[0147] The 10 samples involved in Experimental Example 1 were investigated. Cell viability is the most intuitive indicator of whether cells have biological functions. Within 24-36 hours of sample collection (T<36 hours), the cell viability of umbilical cord blood and umbilical cord blood concentrated cells was analyzed using an FC500 flow cytometer and 7-AAD staining method. The cell viability of the 10 umbilical cord blood samples before PXP treatment was 87.32±3.62%, and the cell viability of the 10 umbilical cord blood samples after PXP treatment was 98.19±1.76%. For example, the result of a certain umbilical cord blood sample (No. 1) was: the cell viability of the umbilical cord blood was 88.13%, and the cell viability of the umbilical cord blood concentrated cells was 99.16%. It shows that the cell viability in the umbilical cord blood concentrated cell sample is significantly higher than that of the umbilical cord blood cell.

[0148] Experimental Example 3: Counting CD45+ and CD34+ cells in umbilical cord blood and umbilical cord blood concentrated cell samples

[0149] The 10 samples involved in Experimental Example 1 were examined. Using the 7-AAD staining method on an FC500 flow cytometer, the number of CD45+ and CD34+ cells and cell viability in all umbilical cord blood and concentrated cell samples before and after treatment were analyzed. The results were as follows:

[0150] In terms of the number of CD45+ live cells, the umbilical cord blood sample = (12.24±2.16)x10^6 / mL, and the umbilical cord blood cell concentrate sample = (91.13±4.74)x10^6 / mL, an increase of 7.4 times;

[0151] In terms of the number of CD34+ live cells, the umbilical cord blood sample = (117.6±12.4)x10^3 / mL, and the umbilical cord blood concentrated cell sample = (802.4±22.7)x10^3 / mL, an increase of 6.8 times.

[0152] Test Example 4: Sterility Testing of Umbilical Cord Blood and Umbilical Cord Blood Concentrated Cell Samples

[0153] The 10 samples involved in Experimental Example 1 were examined for sterility using Gram staining. Smears of umbilical cord blood and umbilical cord blood concentrated cell samples were prepared, fixed with methanol, and stained for testing. The results showed that Gram staining analysis of the smears from all 10 umbilical cord blood samples revealed no microorganisms, and Gram staining analysis of the smears from all 10 umbilical cord blood concentrated cell samples revealed no microorganisms.

[0154] The process of preparing umbilical cord blood concentrated cells using the PXP system in the present invention is characterized by being rapid, closed, and sterile throughout.

[0155] Experimental Example 5: Cell-level effectiveness study

[0156] The umbilical cord blood concentrated cells prepared by the PXP system of the present invention are an injectable cell preparation containing multiple stem cell components, including hematopoietic stem cells (HSCs), mesenchymal stem cells (MSCs), endothelial progenitor cells (EPCs), and multiple cytokines, such as vascular endothelial growth factor (VEGF), stromal cell-derived factor (SDF-1), and endostatin, which promote angiogenesis and endothelial cell migration.

[0157] This test example investigated the 10 samples involved in Test Example 1. The stem cell biological efficacy of umbilical cord blood concentrated cells was evaluated by CFU colony-forming ability, and ELISA was used to quantitatively detect the cytokines rich in umbilical cord blood concentrated cells.

[0158] 5.1 Stem Cell Biological Efficacy—CFU Colony Formation Assay

[0159] Umbilical cord blood stem cell biological potency (Potency Assays) use in vitro CFU colony formation assays to identify and analyze the colony-forming ability of progenitor / stem cells and characterize the stemness of cells in a mixture of umbilical cord blood concentrate cells. The potency of various stem cells in umbilical cord blood and umbilical cord blood concentrate samples was analyzed using CFU-H (hematopoietic progenitor / stem cells) and CFU-F (stromal progenitor cells). The results were as follows:

[0160] In terms of CFU-H (hematopoietic progenitor / stem cell) counts, the umbilical cord blood sample was (24.7±3.3)x10^3 / mL, and the umbilical cord blood concentrated cell sample was (183.2±17.5)x10^3 / mL, a 7.4-fold increase.

[0161] In terms of CFU-F (stromal progenitor cells), the umbilical cord blood sample = (44.8±7.1)x10^3 / mL, and the umbilical cord blood concentrated cell sample = (316.3±32.6)x10^3 / mL, an increase of 7.1 times.

[0162] The results showed that the PXP system can effectively enrich umbilical cord blood stem cells while maintaining the biological efficacy of the stem cells.

[0163] 5.2. Quantitative Analysis of Cytokines

[0164] The cord blood concentrate injection prepared by the PXP system contains a variety of cytokines. Enzyme-linked immunosorbent assay (ELISA) quantitatively analyzed the levels of transforming growth factor-β (TGF-β), vascular endothelial growth factor (VEGF), and hepatocyte growth factor (HGF) in cord blood and cord blood concentrate samples. The results were as follows:

[0165] The TGF-β levels of umbilical cord blood and umbilical cord blood concentrated cells were 25.6±3.3 pg / ml and 203.5±15.7 pg / ml, respectively.

[0166] The VEGF levels of umbilical cord blood and umbilical cord blood concentrated cells were 19.8±4.7pg / ml and 172.4±17.2pg / ml, respectively.

[0167] The HGF levels of umbilical cord blood and umbilical cord blood concentrated cells were 171.6±21.3 pg / ml and 1124.8±38.3 pg / ml, respectively.

[0168] The results showed that the levels of TGF-β, VEGF and HGF in umbilical cord blood concentrated cells were significantly higher than those in umbilical cord blood (p<0.01), indicating that the PXP system can effectively concentrate and enrich cell growth factors.

[0169] Experimental Example 6: Effectiveness of Umbilical Cord Blood Concentrated Cells in Treating Premature Ovarian Failure (POF)

[0170] Chinese Patent Application No. 2021116067919 used umbilical cord blood concentrated cells to study the effectiveness of treating premature ovarian failure (POF). This experimental example used the umbilical cord blood concentrated cells prepared above in combination with GM-CSF (also referred to herein as gmCSF) to study the effectiveness of POF.

[0171] (1) Establishment of POF mouse model

[0172] 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 premature ovarian failure (POF) mouse model. A control group received no treatment. Following POF model establishment, umbilical cord blood concentrated cell transplantation was performed, and the model animals were randomly divided into groups.

[0173] (2) Assess ovarian reserve function through indicators such as hormone levels, follicle count, and fertility tests.

[0174] A. Hormone levels

[0175] Animal Grouping:

[0176] Control group (n=20),

[0177] POF model group (n=20),

[0178] Umbilical cord blood concentrated cell therapy group (n=20),

[0179] Umbilical cord blood concentrated cells + gmCSF treatment group (n=20).

[0180] On the first day after POF was established, mice in the cord blood concentrated cell treatment group were injected with 200 μl of the cord blood concentrated cell composition (the 200 μl cord blood concentrated cell composition was the No. 1 cord blood concentrated cell sample in Example 1, diluted with sterile saline to prepare a CD45 + The cell count concentration is 4x10^6 cells / 200μl solution);

[0181] For the mice in the umbilical cord blood concentrated cell + gmCSF treatment group, 200 μl of the umbilical cord blood concentrated cell gmCSF combination was injected into the tail vein of each animal on the first day after POF establishment;

[0182] The POF model group was injected with an equal volume of normal saline; the control group was not injected;

[0183] After cell transplantation, each group was given normal diet and drinking water.

[0184] Note: The umbilical cord blood concentrated cell gmCSF composition (hereinafter referred to as the umbilical cord blood cell gmCSF composition) administered to the above umbilical cord blood concentrated cell + gmCSF treatment group contains, per 200 μL: an appropriate amount of the umbilical cord blood concentrated cell sample No. 1 obtained in Example 1, + The cell number is calculated to be 1x10^6, 2.5ng of gmCSF is added, and the volume is adjusted to sterile physiological saline to obtain a composition; the composition is stored at a temperature of 2-4°C after preparation and is injected within 4 hours. gmCSF is a commercially available lyophilized powder injection.

[0185] Fourteen and 28 days after transplantation of the umbilical cord blood concentrate, 10 mice were collected from each group, and orbital blood was collected. Serum was separated and stored at −20°C. Estradiol (E2) and follicle-stimulating hormone (FSH) levels were analyzed by enzyme-linked immunosorbent assay (ELISA) (referring to the method described in 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, Issue 2, 2018). The results are shown in the table below.

[0186]

[0187]

[0188] The results showed that compared with the POF model group, the E2 level in the serum of mice in the umbilical cord blood concentrated cell group increased and the FSH level decreased at 28 days, both of which were significantly different (P<0.05). In addition, it has been found that by combining with GM-CSF, the amount of umbilical cord blood concentrated cells can be significantly reduced while achieving basically the same effect.

[0189] B. Follicle counting in mouse ovarian tissue

[0190] 28 days after the transplantation of the umbilical cord blood concentrated cell group, 10 mice were taken from each group and sacrificed. The left ovarian tissue of the mice was fixed with 4% paraformaldehyde, dehydrated with alcohol, transparentized with xylene, embedded in paraffin, and serially sectioned with a thickness of 5 μm. The sections were stained with HE and observed under a microscope.

[0191] 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, the number of follicles at all levels in the umbilical cord blood concentrate group recovered to varying degrees, with increased granulosa cell growth and decreased apoptosis. The morphology of ovarian epithelial cells remained stable, with a significant increase in the number of primary, secondary, and mature follicles and a significant decrease in the number of atretic follicles. Follicle counts at all levels in the umbilical cord blood concentrate group 28 days after transplantation were significantly different from those in the POF group. Specific results are shown in the table below.

[0192]

[0193] Compared with the POF model group, **p<0.01.

[0194] C. Observation of mouse fertility

[0195] On day 28 after transplantation of the cord blood concentrate group, male and female mice were co-housed in a 2:1 ratio. The fertility rates of the mice were measured, and the litter sizes were compared to investigate the effect of the cord blood concentrate group on restoring ovarian function in the mice. The results showed a significant difference between the cord blood concentrate group and the POF group. The results of the litter size comparison are shown in the table below.

[0196] Group control group POF model group Umbilical cord blood cell concentrate group Umbilical cord blood concentrated cells + gmCSF treatment group litter size 13-15 1-2 8 to 10 8 to 9

[0197] According to the above results, the transplantation of umbilical cord blood concentrated cell group can significantly improve the reserve function of the damaged ovaries of POF mice, increase the number of follicles, increase estrogen and progesterone, and restore the fertility of mice, providing an experimental basis for the application of umbilical cord blood concentrated cell group in the clinical treatment of POF.

[0198] Test Example 7: Umbilical cord blood concentrated cell + gmCSF composition

[0199] The cord blood cell concentrate + gmCSF composition used in Experimental Example 6 was administered by injection as soon as possible after preparation. The inventors discovered that the biological activity of GM-CSF decreased after the liquid composition was stored at 4°C for 12 and 24 hours. This decreased biological activity was significantly alleviated by adding trace amounts of glutamine and sodium selenite to the liquid composition. Specific experiments are as follows.

[0200] Formula a: Using the five cord blood concentrated cells No. 1 to No. 5 obtained in Example 1, five liquid compositions were prepared according to the following formulas and were recorded as Composition a No. 1 to Composition a No. 5: containing CD45 + 5x10^6 umbilical cord blood concentrate, 12.5ng (i.e. 125IU) of gmCSF, and sterile saline to 1mL;

[0201] Formulation b: Prepare a liquid composition according to the above formulation a without adding umbilical cord blood concentrated cells, denoted as composition b;

[0202] Formulation c: Using the five cord blood cell concentrates No. 1 to No. 5 obtained in Example 1, five liquid compositions were prepared according to Formulation a, except that glutamine (to a final concentration of 0.3 mg / ml) and sodium selenite (to a final concentration of 10 μg / ml) were added. These compositions were designated Composition c No. 1 to Composition c No. 5.

[0203] Formulation d: Using the five cord blood cell concentrates No. 1 to No. 5 obtained in Example 1, five liquid compositions were prepared according to Formulation a above, but with the addition of glutamine (to a final concentration of 0.3 mg / ml). These compositions were designated as Composition dNo. 1 to Composition dNo. 5.

[0204] Formulation e: Using the five cord blood cell concentrates No. 1 to No. 5 obtained in Example 1, five liquid compositions were prepared according to Formulation a above, except that sodium selenite (to a final concentration of 10 μg / ml) was added. These compositions were designated Composition e No. 1 to Composition e No. 5.

[0205] The preparation methods of the above-mentioned various compositions are conventional methods well known to those skilled in the art. For example, under aseptic operating conditions, a predetermined amount of lyophilized powdered gmCSF, optionally glutamine, and optionally sodium selenite are quantitatively dissolved in sterile saline to a predetermined volume. In addition, the umbilical cord blood concentrated cells are diluted with sterile saline to a concentration of CD45 + To obtain the appropriate concentration of the cell counter, dilute the two solutions with sterile saline to the specified concentration according to the formula ratio, and package them in glass bottles.

[0206] Each of the five compositions described above was placed at 4°C, and samples were collected at 0 h, 12 h, and 24 h. The biological activity (IU / ml) of each composition at the specified time was determined according to the "3526 Recombinant Human Granulocyte Macrophage Stimulating Factor Biological Activity Assay" in Appendix 4 of the 2015 Chinese Pharmacopoeia. For a given composition, the residual percentage of gmCSF biological activity at that time point was calculated by dividing the biological activity at 12 h or 24 h by the biological activity at 0 h, and then multiplying the result by 100%.

[0207] result:

[0208] The biological activities of all the compositions of formula a to formula e at 0 h were within the range of 121.7 to 127.4 IU / ml. For example, the biological activity of composition a No. 1 at 0 h was 124.3 IU / ml;

[0209] The residual percentage of the composition of formula b after 12 hours is 98.6%.

[0210] The residual percentages of all the compositions of formula c after 12 hours were in the range of 94-97%. For example, the residual percentage of composition c No. 1 after 12 hours was 95.3%.

[0211] The residual percentages of all the compositions of formula a, formula d, and formula e at 12 h were all in the range of 81 to 85%. For example, the residual percentage of composition a No. 1 at 12 h was 83.4%;

[0212] The residual percentage of the composition of formula b after 24 hours is 95.3%.

[0213] The 24h residual percentages of all compositions of formula c were in the range of 88-91%. For example, the 24h residual percentage of composition c No. 1 was 90.1%.

[0214] The residual percentages of all the compositions of formula a, formula d, and formula e after 24 hours were all within the range of 63-69%. For example, the residual percentage of composition a No. 1 after 24 hours was 67.2%.

[0215] These results indicate that the biological activity of gmCSF decreases rapidly in the combination containing cells, and this phenomenon of biological activity decrease can be significantly overcome when trace amounts of glutamine and sodium selenite are added to the composition.

[0216] In addition, the number of CD45+ viable cells was measured according to the method of Experimental Example 3 above, and the results were:

[0217] The number of CD45+ viable cells of all the compositions of formula a and formula c to formula e at 0h was in the range of 476 to 513 x 10^4 / ml. For example, the number of CD45+ viable cells of composition a No. 1 at 0h was 486.3 x 10^4 / ml.

[0218] The number of CD45+ viable cells in all compositions of formula a and formula c to formula e at 24 hours was within the range of 121 to 187 x 10^4 / ml. For example, the number of CD45+ viable cells in composition a No. 1 at 24 hours was 156.2 x 10^4 / ml;

[0219] These results demonstrate no significant differences in the number of viable CD45+ cells across the various compositions at different time points, suggesting that glutamine and sodium selenite do not affect the biological activity of the cells. Therefore, while the cord blood concentrated cell + gmCSF composition of Formula a exhibits excellent biological efficacy in treating premature ovarian failure, the addition of small amounts of glutamine and sodium selenite significantly improves the stability of the gmCSF biological activity within the composition, while maintaining no significant differences in the number of viable cells within the composition. This improvement in gmCSF biological activity stability is highly significant for therapeutic applications.

[0220] In addition, since gmCSF is cheap and readily available, by combining it with umbilical cord blood concentrated cells, which are less available, the amount of cells used can be significantly reduced while still achieving excellent biological effects in treating premature ovarian failure.

[0221] 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 embodied 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 illustrative and non-restrictive, and the scope of the invention is defined by the appended claims rather than the foregoing description, and it is intended that all variations that come within the meaning and range of equivalents of the claims be embraced therein.

Claims

1. A cell composition made from concentrated umbilical cord blood cells, comprising: concentrated cells with CD45 + Cell count (4~6)×10 6 cells, 10-15 ng of human granulocyte macrophage stimulating factor, 0.1-0.5 mg of glutamine, 5-20 μg of sodium selenite, and an appropriate amount of normal saline to 1 mL; the concentrated cells are prepared according to a method comprising the following steps: (1) Providing a biological sample of umbilical cord blood, which is placed in a sterile bag containing an anticoagulant for standby use; the anticoagulant is a 3.6% sodium citrate solution supplemented with 0.5 mg / ml histidine and 0.15 mg / ml phosphatidylcholine; (2) Remove the protective cap from the input tube of the automated cell separation system, connect the syringe to the Luer lock connector of the input tube, transfer the anticoagulated biological sample through the thrombus filter at a slow and steady rate into a disposable sterile separation cup, and shake along the horizontal axis to mix the sample; the automated cell separation system is a closed PXP separation system, which consists of four components: a) disposable sterile separation cup, b) control module, c) separation base for transmitting data, d) DataTrak software processing system; (3) Place the disposable separation cup into the control module. The control module status display should be "0" before centrifugation. Weigh and balance the separation cup / control module assembly, then place it in the programmable centrifuge. Set the centrifuge parameters as follows: ； (4) Start the centrifuge and centrifuge. The process is as follows: 4a) In the P1 phase, cells in the biological sample are separated into three components: a red blood cell layer, a cell concentrate layer, and a plasma layer in a disposable separation cup by centrifugal density stratification. 4b) P2 phase causes most red blood cells to enter the red blood cell recovery chamber; 4c) P3 further separates the cells in the processing chamber, and P4 reduces the centrifugal force to further remove red blood cells; 4d) During the P5 stage, the cell concentrate layer and plasma are further separated. In the P6 stage, the centrifugal force is reduced, and the cell concentrate layer is transferred to the recovery chamber through the transfer tube, while the plasma remains in the central chamber. (5) After centrifugation is completed, confirm that the control module window displays "P", which means the qualified status, remove the separation cup from the control module, connect the syringe to the output tube connecting the separation cup and the recovery chamber, and collect the concentrated cells of the umbilical cord blood.

2. The cell composition according to claim 1, comprising: concentrated cells with CD45 + Cell count (4~6)×10 6 cells, human granulocyte macrophage stimulating factor 10-15ng, glutamine 0.2-0.3mg, sodium selenite 10-15µg, and normal saline q.s to 1mL.

3. The cell composition according to claim 1, comprising: concentrated cells with CD45 + The cell count was 5×10 6 cells, human granulocyte macrophage stimulating factor 12.5ng, glutamine 0.2-0.3mg, sodium selenite 10-15µg, and normal saline q.s to 1mL.

4. The cell composition according to claim 1, comprising: concentrated cells with CD45 + The cell count was 5×10 6 cells, human granulocyte macrophage stimulating factor 12.5ng, glutamine 0.3mg, sodium selenite 10µg, and normal saline q.s to 1mL. The cell composition according to claim 1 , wherein the granulocyte macrophage stimulating factor is recombinant human granulocyte macrophage stimulating factor.

6. The cell composition according to claim 1, wherein The volume of the biological sample provided in step (1) is 20-350 ml.

7. The cell composition according to claim 1, wherein In step (1), another 1 ml sample was taken for testing.

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

12.

9. The cell composition 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 total amount, filtering through a 0.22µm microporous membrane, and sterilizing by autoclaving at 121°C.

10. Use of the cell composition according to any one of claims 1 to 9 in the preparation of a medicament for treating premature ovarian failure.

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