CN115531521A

CN115531521A - Cell therapeutic agent, and preparation method and use thereof

Info

Publication number: CN115531521A
Application number: CN202211314449.6A
Authority: CN
Inventors: 肖海蓉; 刘冰; 徐勇
Original assignee: Shenzhen Boya Perception Medical Technology Co ltd
Current assignee: Shenzhen Boya Perception Medical Technology Co ltd
Priority date: 2022-10-26
Filing date: 2022-10-26
Publication date: 2022-12-30

Abstract

The present invention relates to a cell therapeutic agent, a preparation method and a use thereof. In one aspect, the cell composition of the invention comprises bone marrow enriched cells, granulocyte macrophage stimulating factor, supernatant concentrate, and optionally excipients; wherein the concentrated cells, granulocyte-macrophage stimulating factor and supernatant concentrate are mixed at a ratio of CD45 ⁺ The cell number is 4x10^6 cells: 10 to 15ng granulocyte macrophage stimulating factor: and (4) concentrating the supernatant by 75 to 125 mu L. Also relates to the preparation of said cellular compositionMethods, and uses of these cellular compositions in the manufacture of a medicament for the treatment of premature ovarian failure. The present invention comprises a combination therapeutic comprising bone marrow enriched cells, granulocyte macrophage stimulating factor, supernatant concentrate, exhibiting superior biological effects.

Description

Cell therapeutic agent, and preparation method and use thereof

Technical Field

The invention belongs to the field of biotechnology and biomedicine, and relates to a method for treating Premature Ovarian Failure (POF) by using a cell therapeutic agent.

Background

Premature Ovarian Failure (POF) refers to the phenomenon of amenorrhea and infertility in women before the age of 40 years due to ovarian failure. POF is a disease characterized by amenorrhea, infertility, estrogen deficiency, follicular reduction and gonadotropin elevation, accompanied by a range of low estrogen symptoms such as: hot flashes, profuse sweating, flushing of the face, low libido and the like seriously affect the physical and mental health of women. In addition, women with POF are at increased risk of osteoporosis, cardiovascular disease and senile dementia. POF is one of the important causes of infertility in women. POF has an incidence of about 1-3% in women of child bearing age and is on the rise and in the trend of youngness.

According to the guidelines of the European Society of Human Reproduction and Embryology (ESHRE), the diagnostic criteria for POF: FSH levels increased >40IU/L at least for 4 months with 4 weeks or more intervals. Premature ovarian failure is of unknown etiology, may be associated with genetic and autoimmune diseases, environmental factors, and iatrogenic and idiopathic conditions, and has no effective treatment. Hormone Replacement Therapy (HRT) is one of the most common treatments for POF, but the efficacy is not ideal and has been shown to increase the risk of venous thrombosis, breast cancer and ovarian cancer. POF can also cause climacteric symptoms such as hot flashes, hyperhidrosis, anxiety, depression, palpitation, insomnia, etc., in addition to symptoms such as scanty menstruation, amenorrhea, infertility, etc., and can accelerate female aging, thereby causing postmenopausal diseases such as osteoporosis, cardiovascular diseases, dementia, etc., and affecting the quality of life and lifespan of women.

POF has a complicated etiology, has not yet been completely elucidated, may be associated with autoimmune response, infection, genetic factors, chemotherapy, radiotherapy, surgery, etc., and endocrine dysfunction, and has no effective treatment method. Currently, the most common therapeutic method for POF is Hormone Replacement Therapy (HRT). Although the treatment has a certain relieving effect on the clinical symptoms of POF, HRT cannot fundamentally repair damaged ovaries and recover the 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 and ovarian cancer. Therefore, new therapeutic strategies are needed to restore ovarian function in POF patients.

Bone marrow (bone marrow) is the major hematopoietic organ of the human body and consists of hematopoietic cells, adipose tissue and stromal cells. Hematopoietic Stem Cells (HSCs) in bone marrow can differentiate into red blood cells, white blood cells and platelets in the blood circulation. Mesenchymal Stem Cells (MSCs) in bone marrow are a cell subset with multiple differentiation potentials for differentiating to form bone, cartilage, fat, nerves and myoblasts, and the MSCs play a supporting role on the HSCs by paracrine multiple growth factors, so that the stability of the bone marrow hematopoietic microenvironment is maintained.

Bone marrow condensed cells (BMACs) are a concentrate of nucleated cells obtained by centrifuging and separating bone marrow. Bone marrow enriched cells (BMAC) contain enriched Hematopoietic Stem Cells (HSCs) and Mesenchymal Stem Cells (MSCs), as well as a number of various cell growth factors. HSCs can differentiate into erythrocytes, leukocytes and platelets in the blood circulation. MSCs are a subset of cells with a variety of differentiation potential that differentiate to form bone, cartilage, fat, neural, and myoblast cells. BMACs contain various growth factors and cytokines such as Vascular Endothelial Growth Factor (VEGF), platelet-derived growth factor (PDGF), transforming growth factor beta (TGF-beta), 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).

The concentrated cells of bone marrow have antiinflammatory, immunoregulatory, angiogenesis promoting, and tissue regeneration and repair promoting effects. Preclinical and preliminary clinical studies confirm that BMAC improves ovarian function by improving ovarian microenvironment, promoting angiogenesis, promoting follicular development, increasing antral follicular numbers, and promoting ovulation, and is a potential therapeutic approach for POF patients.

However, the bone marrow aspiration liquid is separated by a density gradient centrifugation method in a manual operation mode in the prior art, and has the problems of complex operation, long time consumption, easy pollution, poor result repeatability and the like. One skilled in the art would expect a method of processing bone marrow aspirate to obtain bone marrow concentrate, i.e., bone marrow-enriched cells, that would be simple, time-consuming, less susceptible to contamination, and reproducible results, to be advantageous in one or more respects. This technical advance has been achieved in chinese patent application No. 2021116067849 by the present research team.

Granulocyte-macrophage colony stimulating factor (GM-CSF) is mainly produced by T cells and macrophages, can induce granulocyte precursor and macrophage precursor cells to grow in colony, and is called granulocyte-giant cell colony stimulating factor for short. The main biological effects of GM-CSF in vivo are to maintain the survival of the cells of the granulocytic and monocytic cell lines, promote growth, induce differentiation and enhance phagocytic function and bactericidal effect; inducing dendritic cell maturation and functional distribution. The granulocyte macrophage-stimulating factor used clinically is usually a recombinant human granulocyte macrophage-stimulating factor, is generally suitable for cancer chemotherapy and leukopenia caused by myelosuppression therapy, is also suitable for treating leucopenia of a bone marrow failure patient, can prevent potential infection complications caused by leukopenia, and can accelerate recovery of neutropenia caused by infection.

The existing methods for treating premature ovarian failure still need to be improved. Accordingly, it would be desirable to provide a method of treating premature ovarian failure, such as a method of treating premature ovarian failure using a myeloconcentrated cell therapeutic agent, for example, a method of treating premature ovarian failure using a combination therapeutic agent of myeloconcentrated cells and GM-CSF, wherein the combination therapeutic agent may further comprise a byproduct supernatant concentrate from the preparation of the concentrated cells.

Disclosure of Invention

An object of the present invention is to provide a method for preparing bone marrow-enriched cells, which is expected to have one or more beneficial effects of simple operation, short time consumption, low possibility of contamination, good result repeatability, etc.; alternatively, it is an object of the present invention to provide a novel method for treating premature ovarian failure by formulating bone marrow stromal cells and GM-CSF into a combination therapeutic agent. It has been surprisingly found that one or more of the above objects can be achieved by the present invention which uses a closed PXP cell autosegregation system to prepare concentrated cells of bone marrow, and/or to treat premature ovarian failure by formulating the obtained concentrated cells of bone marrow with GM-CSF into a combination therapeutic agent which may further comprise a byproduct supernatant concentrate from the preparation of the concentrated cells, and the present invention has been accomplished based on such findings.

To this end, the present invention provides, in a first aspect, a method for preparing bone marrow-enriched cells, comprising the steps of:

(1) Providing a biological sample bone marrow puncture fluid, and placing the biological sample bone marrow puncture fluid into a sterile bag containing an anticoagulant for later use;

(2) Taking off a protective cap on an input tube of the automatic cell separation system, connecting a syringe to a luer locking connector of the input tube, passing through a thrombus filter at a slow and stable speed, transferring an anticoagulated biological sample into a disposable sterile separation cup, and shaking the mixed sample along a horizontal shaft; the automatic cell separation system is a closed PXP separation system, which is composed of four components: a) a disposable sterile separation cup, b) a control module, c) a separation base for transmitting data, d) a DataTrak software processing system;

(3) Placing the disposable separating cup into a control module, displaying the state of the control module as '0' before centrifugation, weighing the separating cup/control module assembly, placing the separating cup/control module assembly into a programmable centrifuge after balancing, and setting parameters of the centrifuge according to the following procedures:

(4) Starting the centrifuge to centrifuge, and carrying out the following process:

4a) The P1 phase separates the cells in the biological sample into the lower, middle and upper three components in a disposable separation cup by centrifugation density stratification: red blood cell layer, cell concentrated layer, plasma layer;

4b) The P2 stage enables most of the red blood cells to enter the red blood cell recovery cabin;

4c) The P3 phase further stratifies the cells in the processing chamber, the P4 phase reduces centrifugal force to further remove red blood cells;

4d) The cell concentrated layer and the plasma are further layered in the stage P5, the centrifugal force is reduced until the stage P6, the cell concentrated layer is transferred to the recovery chamber through the conveying pipe, and the plasma is retained in the central chamber;

(5) After the centrifugation is finished, confirming that the window of the control module displays 'P', namely, the qualified state, taking out the separation cup from the control module, connecting an injector to an output pipe of the separation cup communicated with the recovery cabin, and collecting the obtained marrow concentrated cells;

optionally (c) is

(6) Placing the separation cup and control module on a separation base for data transmission and processing the data captured during centrifugation with a DataTrak software processing system;

and/or optionally, continuing the steps to prepare a (plasma) supernatant concentrate:

(7) Separating the supernatant (i.e. plasma layer) in the central chamber with an injector, centrifuging for 20min at 2000g to remove cell debris, and filtering with a sterile filter membrane of 0.22 μm;

(8) And (3) rinsing a pipeline of the tangential flow ultrafiltration system (Shibi pure KR2i type tangential flow ultrafiltration system) by using ultrapure water, installing a MidiKros filter of 100kD 100cm2, and performing ultrafiltration concentration on the filtrate obtained in the step (7) to 1/15 volume of the initial biological sample amount to obtain a supernatant concentrated solution.

The method according to the first aspect of the present invention, wherein the volume of the biological sample provided in step (1) is 20 to 200ml.

The method according to the first aspect of the present invention, wherein 1ml of the sample is additionally withdrawn in the step (1) for detection.

The method according to the first aspect of the present invention, wherein the anticoagulant used in step (1) is a sodium citrate solution.

The method according to the first aspect of the present invention, wherein the anticoagulant used in step (1) is a 3.2% sodium citrate solution.

The method according to the first aspect of the present invention, wherein the anticoagulant used in step (1) is a 3.2% sodium citrate solution supplemented with 0.5mg/ml histidine and 0.1mg/ml phosphatidylcholine.

The method according to the first aspect of the present invention, wherein the volume ratio of the anticoagulant to the biological sample used in step (1) is 1:12.

the method according to the first aspect of the present invention, wherein the anticoagulant used in step (1) is prepared by: adding sodium citrate, histidine and phosphatidylcholine into a proper amount of water, heating to 60 ℃, stirring to dissolve, adding water to full volume, filtering with a 0.22 mu m microporous filter membrane, and sterilizing at 121 ℃ under hot pressure.

The method according to the first aspect of the invention, further comprising the steps of: (6) The separation cup and control module were placed on the separation base to transmit the data and process the data captured during centrifugation with a DataTrak software processing system.

Further, the second aspect of the present invention provides a marrow condensed cell, which is prepared by a method comprising the steps of:

(2) Taking off a protective cap on an input tube of the automatic cell separation system, connecting a syringe to an input tube luer locking connector, passing through a thrombus filter at a slow and stable speed, transferring an anticoagulated biological sample into a disposable sterile separation cup, and shaking along a horizontal shaft to mix the sample; the automatic cell separation system is a closed PXP separation system, which is composed of four components: a) a disposable sterile separation cup, b) a control module, c) a separation base for transmitting data, d) a DataTrak software processing system;

(3) Placing the disposable separating cup into a control module, displaying the state of the control module as '0' before centrifugation, weighing the separating cup/control module assembly, placing the separating cup/control module assembly into a programmable centrifuge after balancing, and setting parameters of the centrifuge according to the following programs:

4d) The cell concentrated layer and the plasma are further layered in the period P5, the centrifugal force is reduced until the period P6, the cell concentrated layer is transferred to the recovery chamber through the conveying pipe, and the plasma is retained in the central chamber;

optionally (c) is

The bone marrow condensed cells according to the second aspect of the present invention, wherein the volume of the biological sample provided in step (1) is 20 to 200ml.

The bone marrow condensed cells according to the second aspect of the present invention, wherein 1ml of the sample is additionally extracted in the step (1) for examination.

The bone marrow condensed cells according to the second aspect of the present invention, wherein the anticoagulant used in the step (1) is a sodium citrate solution.

The bone marrow-enriched cells according to the second aspect of the present invention, wherein the anticoagulant used in the step (1) is a 3.2% sodium citrate solution.

The bone marrow-enriched cells according to the second aspect of the present invention, wherein the anticoagulant used in step (1) is a 3.2% sodium citrate solution supplemented with 0.5mg/ml histidine and 0.1mg/ml phosphatidylcholine.

The bone marrow-enriched cells according to the second aspect of the present invention, wherein the volume ratio of the anticoagulant to the biological sample used in the step (1) is 1:12.

the bone marrow-enriched cells according to the second aspect of the present invention, wherein the anticoagulant used in the step (1) is prepared by: adding sodium citrate, histidine and phosphatidylcholine into a proper amount of water, heating to 60 ℃, stirring to dissolve, adding water to full volume, filtering with a 0.22 mu m microporous filter membrane, and sterilizing at 121 ℃ under hot pressure.

The bone marrow condensed cells according to the second aspect of the present invention, further comprising the steps of: (6) The separation cup and control module were placed on the separation base to transmit the data and the data captured during centrifugation was processed with the DataTrak software processing system.

Further, the third aspect of the present invention provides a use of concentrated cells of bone marrow prepared by a method comprising the steps of:

(2) Taking off a protective cap on an input tube of the automatic cell separation system, connecting a syringe to an input tube luer locking connector, passing through a thrombus filter at a slow and stable speed, transferring an anticoagulated biological sample into a disposable sterile separation cup, and shaking along a horizontal shaft to mix the sample; the automatic cell separation system is a closed PXP separation system, which is composed of four components: a) a disposable sterile separator cup, b) a control module, c) a separation base for transmitting data, d) a DataTrak software processing system;

4a) The P1 phase separates the cells in the biological sample into the lower, middle and upper three components in a disposable separation cup by centrifugal density stratification: red blood cell layer, cell concentrated layer, plasma layer;

(5) After the centrifugation is finished, confirming that the window of the control module displays 'P', namely, the qualified state, taking out the separation cup from the control module, connecting an injector to an output pipe for communicating the separation cup with a recovery cabin, and collecting the obtained marrow concentrated cells;

optionally (c) is

and/or optionally, continuing the steps of:

(8) And (4) rinsing the pipeline of the tangential flow ultrafiltration system (Shibi pure KR2i type tangential flow ultrafiltration system) by using ultrapure water, installing a MidiKros filter of 100kD 100cm2, and performing ultrafiltration concentration on the filtrate obtained in the step (7) to 1/15 volume of the initial biological sample volume to obtain a supernatant concentrated solution.

Use according to a third aspect of the invention, wherein the volume of the biological sample provided in step (1) is 20 to 200ml.

The use according to the third aspect of the present invention, wherein 1ml of the sample is additionally withdrawn in the step (1) for detection.

The use according to the third aspect of the present invention, wherein the anticoagulant used in step (1) is a sodium citrate solution.

The use according to the third aspect of the present invention, wherein the anticoagulant used in step (1) is a 3.2% sodium citrate solution.

The use according to the third aspect of the present invention, wherein the anticoagulant used in step (1) is a 3.2% sodium citrate solution supplemented with 0.5mg/ml histidine and 0.1mg/ml phosphatidylcholine.

Use according to the third aspect of the invention, wherein the volume ratio of anticoagulant to biological sample used in step (1) is 1:12.

the use according to the third aspect of the present invention, wherein the anticoagulant used in the step (1) is prepared by: adding sodium citrate, histidine and phosphatidylcholine into a proper amount of water, heating to 60 ℃, stirring for dissolving, adding water to full volume, filtering with a microporous filter membrane of 0.22 mu m, and carrying out hot-pressing sterilization at 121 ℃ to obtain the product.

The use according to the third aspect of the invention, further comprising the steps of: (6) The separation cup and control module were placed on the separation base to transmit the data and process the data captured during centrifugation with a DataTrak software processing system.

Further, a fourth aspect of the present invention provides a method for treating premature ovarian failure, the method comprising administering to a subject in need thereof a cell therapeutic comprising a therapeutically effective amount of bone marrow condensed cells prepared by a method comprising:

(2) Taking off a protective cap on an input tube of the automatic cell separation system, connecting a syringe to an input tube luer locking connector, passing through a thrombus filter at a slow and stable speed, transferring an anticoagulated biological sample into a disposable sterile separation cup, and shaking along a horizontal shaft to mix the sample; the automatic cell separation system is a closed PXP separation system, and consists of four components: a) a disposable sterile separator cup, b) a control module, c) a separation base for transmitting data, d) a DataTrak software processing system;

(4) Starting the centrifuge for centrifugation, and carrying out the following processes:

4c) Phase P3 further stratifies the cells in the processing chamber and phase P4 reduces centrifugal force to further remove red blood cells;

optionally (c) is

(7) Separating supernatant (plasma layer) in the central chamber with an injector, centrifuging at 2000g for 20min to remove cell debris, and filtering with 0.22 μm sterile filter membrane;

The method according to the fourth aspect of the present invention, wherein the volume of the biological sample provided in step (1) is 20 to 200ml.

The method according to the fourth aspect of the present invention, wherein 1ml of the sample is additionally withdrawn for detection in the step (1).

The method according to the fourth aspect of the present invention, wherein the anticoagulant used in step (1) is a sodium citrate solution.

The method according to the fourth aspect of the present invention, wherein the anticoagulant used in step (1) is a 3.2% sodium citrate solution.

The method according to the fourth aspect of the present invention, wherein the anticoagulant used in step (1) is a 3.2% sodium citrate solution supplemented with 0.5mg/ml histidine and 0.1mg/ml phosphatidylcholine.

The method according to the fourth aspect of the present invention, wherein the volume ratio of the anticoagulant to the biological sample used in step (1) is 1:12.

the method according to the fourth aspect of the present invention, wherein the anticoagulant used in the step (1) is prepared by: adding sodium citrate, histidine and phosphatidylcholine into a proper amount of water, heating to 60 ℃, stirring for dissolving, adding water to full volume, filtering with a microporous filter membrane of 0.22 mu m, and carrying out hot-pressing sterilization at 121 ℃ to obtain the product.

The method according to the fourth aspect of the present invention, further comprising the steps of: (6) The separation cup and control module were placed on the separation base to transmit the data and the data captured during centrifugation was processed with the DataTrak software processing system.

Further, the fifth aspect of the present invention provides a cell composition made of bone marrow condensed cells, which comprises the condensed cells, granulocyte macrophage stimulating factor and optionally an excipient.

The cell composition according to the fifth aspect of the present invention, wherein the ratio of the enriched cells to the granulocyte-macrophage stimulating factor is such that the enriched cells are CD45 ⁺ The cell number is 5x10^6 cells: 10 to 15ng granulocyte macrophage stimulating factor; for example, in a ratio of concentrating cells to CD45 ⁺ The cell number is 5x10^6 cells: 12.5ng granulocyte macrophage stimulating factor.

The cell composition according to the fifth aspect of the present invention, wherein the excipient is a physiological saline or a 5% glucose solution.

The cell composition according to the fifth aspect of the present invention, wherein the excipient is physiological saline, and the concentration of the granulocyte macrophage stimulating factor in the composition is 10 to 15ng/ml, for example, 12.5ng/ml.

The cell composition according to the fifth aspect of the present invention, wherein the granulocyte macrophage stimulating factor is a human granulocyte macrophage stimulating factor.

The cell composition according to the fifth aspect of the present invention, wherein the granulocyte macrophage-stimulating factor is a recombinant human granulocyte macrophage-stimulating factor.

The cell composition according to 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.

The cell composition according to the fifth aspect of the present invention, further comprising glutamine and sodium selenite.

The cell composition according to the fifth aspect of the present invention, further comprising glutamine and sodium selenite, wherein the weight ratio of the granulocyte macrophage stimulating factor to the glutamine and the sodium selenite in the composition is 12.5ng:0.1 to 0.5mg:5 to 20 micrograms.

The cell composition according to the fifth aspect of the present invention, further comprising glutamine and sodium selenite, wherein the weight ratio of the granulocyte macrophage stimulating factor to the glutamine and the sodium selenite in the composition is 12.5ng:0.2 to 0.3mg:10 to 15 mug.

The cell composition according to the fifth aspect of the present invention, further comprising glutamine and sodium selenite, wherein the weight ratio of the granulocyte macrophage stimulating factor to the glutamine and the sodium selenite in the composition is 12.5ng:0.2mg:15 mug.

A cell composition according to the fifth aspect of the invention, comprising: CD45 ⁺ Concentrated cells with the cell number of 4-6 x10^6, 10-15ng gmCSF, 0.1-0.5 mg glutamine, 5-20 μ g sodium selenite and a proper amount of normal saline to 1mL.

A cell composition according to the fifth aspect of the invention, comprising: CD45 ⁺ Concentrated cells with the cell number of 4 to 6x10^6 and 10 to 15ng of gmCSF, 0.2 to 0.3mg of glutamine, 10 to 15 mu g of sodium selenite and a proper amount of physiological saline to 1mL.

A cell composition according to the fifth aspect of the invention, comprising: CD45 ⁺ Concentrated cells with the cell number of 5x10^6, 12.5ng of gmCSF, 0.2 to 0.3mg of glutamine, 10 to 15 mu g of sodium selenite and a proper amount of physiological saline to 1mL.

A cell composition according to the fifth aspect of the invention, comprising: CD45 ⁺ The cell number is 5x10^6, the concentration of cells, 12.5ng of gmCSF, 0.2mg of glutamine, 15 microgram of sodium selenite and a proper amount of physiological saline to 1mL.

Further, the sixth aspect of the present invention provides a cell composition made of bone marrow condensed cells, which comprises the condensed cells, granulocyte macrophage stimulating factor, supernatant concentrate and optionally an excipient.

The cell composition according to the sixth aspect of the present invention, wherein the ratio of the enriched cells to the granulocyte-macrophage stimulating factor is that the enriched cells are expressed as CD45 ⁺ The cell number is 4x10^6 cells: 10 to 15ng granulocyte macrophage stimulating factor; for example, in a ratio of concentrating cells to CD45 ⁺ The cell number is 4x10^6 cells: 12.5ng granulocyte macrophage stimulating factor.

The cell composition according to the sixth aspect of the present invention, wherein the ratio of the concentrated cells to the supernatant concentrate is such that the concentrated cells are CD45 ⁺ Counting the number of cells to 4x10^6 cells: supernatant concentrated solution of 75 to 125 muL; for example, in a ratio of concentrating cells to CD45 ⁺ The cell number is 4x10^6 cells: 100 muL of supernatant concentrate.

The cell composition according to the sixth aspect of the present invention, wherein the excipient is a physiological saline or a 5% glucose solution.

The cell composition according to the sixth aspect of the present invention, wherein the excipient is physiological saline, and the concentration of the granulocyte macrophage stimulating factor in the composition is 10 to 15ng/ml, for example, 12.5ng/ml.

The cell composition according to the sixth aspect of the present invention, wherein the granulocyte macrophage stimulating factor is a human granulocyte macrophage stimulating factor.

The cell composition according to the sixth aspect of the present invention, wherein the granulocyte macrophage-stimulating factor is a recombinant human granulocyte macrophage-stimulating factor.

The cell composition according to the sixth aspect of the present invention, wherein the concentrated cells and the supernatant concentrate are prepared by the method according to any one of the embodiments of the first aspect of the present invention.

The cell composition according to the sixth aspect of the present invention, further comprising glutamine and sodium selenite.

The cell composition according to the sixth aspect of the present invention, further comprising glutamine and sodium selenite, wherein the weight ratio of the granulocyte macrophage stimulating factor to the glutamine and the sodium selenite in the composition is 12.5ng:0.1 to 0.5mg:5 to 20 micrograms.

The cell composition according to the sixth aspect of the present invention, further comprising glutamine and sodium selenite, wherein the weight ratio of the granulocyte macrophage stimulating factor to the glutamine and the sodium selenite in the composition is 12.5ng:0.2 to 0.3mg:10 to 15 mug.

The cell composition according to the sixth aspect of the present invention, further comprising glutamine and sodium selenite, wherein the weight ratio of the granulocyte macrophage stimulating factor to the glutamine and the sodium selenite in the composition is 12.5ng:0.2mg:15 mug.

A cell composition according to a sixth aspect of the invention, comprising: CD45 ⁺ The cell number is 3 to 5x10^6, the cell number is 10 to 15ng gmCSF, the supernatant concentrated solution is 75 to 125 muL, glutamine is 0.1 to 0.5mg, sodium selenite is 5 to 20 mug, and a proper amount of physiological saline is 1mL.

A cell composition according to a sixth aspect of the invention, comprising: CD45 ⁺ Concentrated cells with the cell number of 3-5x10 ^6, 10-15ng gmCSF, 80-120 muL supernatant concentrated solution, 0.2-0.3mg glutamine, 10-15 mug sodium selenite and a proper amount of physiological saline to 1mL.

A cell composition according to a sixth aspect of the invention, comprising: CD45 ⁺ 4x10^6 cells, 12.5ng of gmCSF, 100 muL of supernatant concentrated solution, 0.2 to 0.3mg of glutamine, 10 to 15 mug of sodium selenite and a proper amount of normal saline to 1mL.

A cell composition according to a sixth aspect of the invention, comprising: CD45 ⁺ 4x10^6 cells, 12.5ng of gmCSF, 100 muL of supernatant concentrated solution, 0.2mg of glutamine, 15 mug of sodium selenite and a proper amount of physiological saline to 1mL.

The unexpected discovery that the cell composition prepared by combining the supernatant concentrate with concentrated cells and gmCSF has excellent effect of treating premature ovarian failure, such as the expected effect achieved by using a lower dose of concentrated cells, is unexpected in the prior art.

Further, according to a seventh aspect of the present invention, there is provided a use of the cell composition according to any one of the fifth or sixth aspects of the present invention in the preparation of a medicament for treating premature ovarian failure.

Further, the eighth aspect of the present invention provides a method for preparing the cell composition according to any one of the sixth aspect of the present invention, which comprises the step of mixing the specified amounts of the concentrated cells, the granulocyte macrophage stimulating factor, the supernatant concentrate, glutamine, sodium selenite, and optionally an excipient to prepare a sterile preparation.

The phrase "CD 45" as described herein ⁺ Concentrated cells with a cell number of 5x10^6 "5x10 ^6 in" means 5 times to the power of 6 times 10, and the rest of similar expressions also have the same meaning.

The invention uses a PXP automatic cell rapid processing system and a closed system with automatic separation and concentration, and the method for safely, efficiently and simply obtaining the BMAC lays a foundation for the clinical application of the BMAC for treating POF patients. The invention provides a method for rapidly separating and obtaining marrow condensed cells, which separates and obtains the marrow condensed cells by using a closed automatic cell separation system from human marrow puncture fluid. The bone marrow condensed cells obtained by the invention can be used as an active ingredient for treating ovarian injury, and can promote angiogenesis and follicular development, thereby improving the ovarian function.

Previous studies have demonstrated that contaminating erythrocytes are associated with a decrease in stem/progenitor cell function, and that erythrocyte-contaminating cell concentrates are thought to reduce the effectiveness of cell therapy. To better exploit the potential of cell therapy, the industry is eagerly demanding new treatment systems that increase the purity of target cells and the removal rate of contaminating red blood cells.

The cell separation system used in the specific experiment of the invention is a PXP automatic separation system, and the manufacturer is ThermoGenesis Inc. in the United states. The innovative PXP system addresses many of the shortcomings of existing systems currently on the market. The PXP system enables clinicians to rapidly achieve very high stem and progenitor cell recoveries with little or no red blood cell contamination, typically less than 5% of the starting sample.

The PXP system is a rather efficient Point-of-Care (Point-of-Care) product for the clinical institution developing and using cell therapy technology to meet the need for rapid, efficient, sterile cell processing in the operating room environment. As a leading-edge automated rapid cell processing system, the PXP system does not require a cell separation medium or a precipitant, can process multiple samples simultaneously, has high recovery rates of MNC and CD34+ and CD45+ cells, enables clinicians to achieve efficient extraction of stem cells from biological samples (e.g., bone marrow) within 30 minutes in a hospital surgical center or clinic, and has a removal rate of red blood cells of over 90%. In addition, the PXP system is equipped with proprietary DataTrak software to track captured data to facilitate providing GMP flow control and reporting information to customers.

The present invention uses a PXP system for processing bone marrow extracts, can rapidly and automatically process bone marrow cells in real time, ensure the recovery rate of mononuclear cells (MNCs), can process multiple bone marrow cells simultaneously, and does not require a cell separation medium or a precipitant.

The PXP system is used for specific experiments, and the advantages of the PXP system include but are not limited to: the recovery rate of stable and excellent MNC (mononuclear cells) and CD34+ and CD45+ cells, the rapid processing of bone marrow samples within 30 minutes, the removal rate of more than 95 percent of red blood cells, an automatic closed sterile system, rapid and accurate data tracking and document recording, and sample processing data can be uploaded to a computer through DataTrak software, and production recording and reporting information meeting GMP requirements are provided.

The bone marrow concentrate is widely applied to clinics of related disciplines such as orthopedics, severe lower limb ischemia and the like because of being rich in a large amount of stem cells and growth factors. The bone marrow concentrate is a concentrate of nucleated cells obtained by centrifuging and separating bone marrow, and is rich in mesenchymal stem cells. As is well known, the worldwide market for bone marrow concentrates can be divided into bone surgery, wound healing, chronic pain, peripheral vascular disease, skin lesions and others. Due to the exacerbation of global aging and the rising incidence of osteoarthritis, the application of orthopaedic diseases will dominate the whole bone marrow concentrate market in the future. With the explosive development of the industry, the advancement of technology and the increase in disposable income, the dermatological field is expected to grow at the highest composite annual growth rate in the future (6.0%). The regenerative capacity of fibroblasts and keratinocytes in human skin has been exploited for the development of cell therapy, while scientists are studying the plasticity of bone marrow-derived dermal cells in skin regeneration, which will accelerate the application of cell therapy in the dermatological field. Bone marrow concentrate is considered the "platinum standard" for bone substitute materials. It has been found that cells, precursor cells, growth factors, platelets, and the like in bone marrow concentrate play an important role in bone regeneration. For example, endothelial cells in bone marrow concentrates play an important role in angiogenesis; a large amount of growth factors contained in the bone marrow concentrate can act on osteoblasts and osteoclasts, regulate the bone reconstruction process and promote the generation of new bones; platelets in bone marrow concentrate provide a faster and more efficient environment for bone regeneration of mesenchymal stem cells.

According to the survey results of the united nations and the world health organization, more than 4 hundred million people worldwide suffer from arthritis, and the development of the orthopedics field is promoted by such a huge background of patient groups. Bone marrow-derived stem cell therapy is considered a promising advanced therapy that can shorten the wound healing time of orthopedic disorders from 4 to 6 months required for surgical treatment to 5 to 6 weeks. The reduction in healing time is one of the factors driving the development of the bone marrow concentrate market. In recent years, the progress of the treatment and preparation technology of the bone marrow concentrate provides effective choices for hospitals and clinics to improve the clinical effect of orthopedic treatment.

The bone marrow concentrate market is an emerging market, and various stem cell therapies and related technical equipment are gradually commercialized after 30-40 years of research and development. The advent of bone marrow concentrates, platelet rich plasma and stem cell derivatives is becoming a new trend for future medical treatments. With the development of personalized medicine, the global clinical needs of cell therapy become more and more significant, and the release trend of clinical application of cell therapy becomes more and more obvious. With the gradual coverage of clinical applications of cell therapy, the demand of medical institutions for new generation cell processing and preparation solutions based on automation technology is increasing, and these technology platforms will also have important impact on the success of cell therapy.

The present invention achieves satisfactory results by cell separation using the PXP system.

GM-CSF can be human granulocyte macrophage stimulating factor or recombinant granulocyte macrophage stimulating factor, which has been loaded into various versions of the Chinese pharmacopoeia and has been approved for clinical use by various brands. In the present invention, GM-CSF used in the test is a commercially available recombinant human granulocyte macrophage stimulating factor for injection (S19991012, specification 750000IU/75 μ g,10 IU/ng), and the composition can be prepared by diluting with 0.9% sodium chloride injection to a suitable concentration in advance if necessary, if not otherwise specified. GM-CSF (Granulocyte-macrophage Colony Stimulating Factor, or Granulocyte-macrophage Stimulating Factor) acts on hematopoietic progenitor cells to promote proliferation and differentiation thereof, and has the important functions of Stimulating the maturation of Granulocyte and monocyte macrophages, promoting the release of mature cells to peripheral blood, and promoting multiple functions of macrophages and acid-phagocytic cells. GM-CSF is a clinically used drug for leukopenia or granulocytopenia caused by various reasons, which can stimulate hematopoietic function of bone marrow, stimulate proliferation of granulocytes, monocytes, T cells, and promote maturation of monocytes and granulocytes. In addition, GM-CSF can overcome the bone marrow toxicity caused by radiotherapy and chemotherapy, shorten the neutrophilic granulocyte reduction time in tumor chemotherapy, and make the patient easily tolerate the chemotherapy. GM-CSF can enhance the functions of monocyte, granulocyte, eosinophil and macrophage, and further improve the anti-tumor and anti-infection immunity of the organism.

The invention combines the marrow concentrated cells obtained by PXP system separation with GM-CSF, and uses the premature ovarian failure model for verification, thereby obtaining positive effects.

Detailed Description

The present invention will be further described by the following examples, however, the scope of the present invention is not limited to the following examples. It will be understood by those skilled in the art that various changes and modifications may be made to the invention without departing from the spirit and scope of the invention. The present invention has been described generally and/or specifically with respect to materials used in testing and testing methods. Although many materials and methods of operation are known in the art for the purpose of carrying out the invention, the invention is nevertheless described herein in as detail as possible.

In the invention, if not otherwise stated, the cell separation system used in the specific experiment is a PXP automatic separation system, which may also be referred to as a PXP system, a PXP cell automatic separation system, a PXP separation system, etc. in the invention. The model number of the PXP system used in the experiment is 80065-01, the supplier is Shenzhen Boya perception medical technology Limited, and the producer is ThermoGenesis in America. In the present invention, the term "bone marrow enriched cells" may also be referred to as "bone marrow enriched cell preparation", and both have the same meaning, unless otherwise specified.

Example 1: rapid isolation preparation of bone marrow enriched cells (BMAC)

(1) Providing a biological sample bone marrow puncture solution (a sample with the volume of 20 to 200ml can be processed), putting the biological sample bone marrow puncture solution into an aseptic bag containing an anticoagulant for later use, and extracting 1ml of sample for detection; the anticoagulant is 3.2% sodium citrate solution, wherein 0.5mg/ml histidine and 0.1mg/ml phosphatidylcholine are additionally added, and the volume ratio of the anticoagulant to the biological sample is 1:12; the preparation method of the anticoagulant comprises the following steps: adding sodium citrate, histidine and phosphatidylcholine into a proper amount of water, heating to 60 ℃, stirring for dissolving, adding water to full volume, filtering with a microporous filter membrane of 0.22 mu m, and carrying out hot-pressing sterilization at 121 ℃ to obtain the compound sodium citrate;

(6) The separation cup and control module were placed on the separation base to transmit the data and process the data captured during centrifugation with a DataTrak software processing system.

The following steps were continued to prepare (plasma) supernatant concentrate:

In the present invention, as not otherwise specified, the tangential flow ultrafiltration system used is a Shibi pure KR2i type tangential flow ultrafiltration system; of course other brands of tangential flow ultrafiltration systems may be used.

In this example 1, 10 collected biological samples of human bone marrow aspirate (obtained by methods known in the art, for example, the biological samples of the present invention were obtained by cell separation and supernatant concentrate preparation according to the methods described in Song Jianhua literature (Song Jianhua, et al, J.Aus. J.Osseur., 2008,14 (8): 467), and 10 bone marrow-enriched cells (BMAC) and 10 supernatant concentrates (each labeled as No. 1-No. 10) were obtained.

Test example 1: analysis of MNC recovery in Bone Marrow Aspirate (BMA) and bone marrow concentrate (BMAC)

Using the method of example 1, 10 collected human bone marrow aspirate fluid (volume before dissection ranged from 79 to 97ml) was subjected to cell isolation; then, cell detection was performed on each fraction by the method described in chinese patent application No. 2021116067849, and results of 10 samples: the mean value of the input bone marrow puncture liquid is 88.26ml, the mean value of the output BMAC is 12.74ml, the mean value of the Red Blood Cell (RBC) removal rate is 97.9 percent, the mean value of the single nuclear cell (MNC) recovery rate is 97.2 percent, and the MNC concentration is averagely improved by 6.93 times. For example, the result of a certain amount of the puncturing solution (No. 2): pre-separation volume =91.34ml, final volume =12.97ml, erythrocyte removal rate =98.3%, MNC recovery rate =97.6%, MNC concentration multiple =7.04.

The results show that the PXP kinetic separation system can be used for enriching MNC in bone marrow and removing most of red blood cells simultaneously in a mode of simple operation, short time consumption, difficult pollution and good result repeatability.

Test example 2: cell viability in BMA and BMAC samples

The 10 samples of test example 1 were examined. Cell viability is the most intuitive indicator of whether a cell has a biological function. Bone marrow samples were collected over 24-36 hours (T <36 hours) and analyzed for cell viability in BMA, BMAC using a FC500 flow cytometer using 7-AAD staining.

Cell viability =88.17 ± 3.14% for 10 bone marrow aspirate fluid samples (BMA) before PXP treatment, and 97.42 ± 1.24% for 10 bone marrow concentrated cell samples (BMAC) after PXP treatment, for example, results of a certain aspirate fluid sample (No. 2): BMA cell viability =89.64%, BMAC cell viability =98.27%. Cell viability rates in BMAC samples were shown to be significantly higher than BMA cell viability rates.

Test example 3: CD45+, CD34+ cell counts in BMA and BMAC samples

The 10 samples involved in test example 1 were examined. CD45+ and CD34+ cell numbers were analyzed on an FC500 flow cytometer using 7-AAD staining for all pre-treatment and post-treatment bone marrow samples, and the results:

in terms of the number of CD45+ living cells, a bone marrow puncture fluid sample (BMA) = (15.08 +/-2.84) x10^6/mL, a bone marrow concentrated cell sample (BMAC) = (106.41 +/-6.94) x10^6/mL is increased by 7.1 times;

in terms of the number of CD34+ living cells, a bone marrow aspirate fluid sample (BMA) = (127.42 +/-10.36) x10^3/mL, and a bone marrow concentrated cell sample (BMAC) = (882.63 +/-16.41) x10^3/mL are increased by 6.9 times.

Test example 4: sterility testing of bone marrow samples

The 10 cell concentrate samples and the 10 supernatant concentrate samples referred to in test example 1 were examined. Sterility testing was performed using gram stain, smears of BMA, BMAC samples were prepared, fixed with methanol, tested after staining, results: no microorganisms were found in any of the gram stain analysis sample smears for 10 BMA samples, no microorganisms were found in any of the gram stain analysis sample smears for 10 BMAC samples, and no microorganisms were found in any of the gram stain analysis sample smears for 10 supernatant concentrate samples.

The process for preparing the BMAC by using the PXP system has the characteristics of rapidness, sealing and whole-process sterility.

Test example 5: cellular level of efficacy study

The BMAC prepared using the PXP system of the present invention is an injection of a cell preparation, which contains 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), endostatin (Entostatin), etc., and promotes neovascularization and endothelial cell migration.

This test example was conducted on 10 samples related to test example 1, and the biological efficacy of stem cells of BMAC was evaluated by CFU colony-forming ability, and cytokines abundant in BMAC were quantitatively detected by ELISA.

5.1 Stem cell biological potency-CFU colony formation assay

Bone marrow stem cell biological Potency (patent Assays), characterization of the colony forming ability of progenitor/stem cells by in vitro CFU colony formation assay, characterizes cell sternness in BMAC mixed cells. The efficacy of various stem cells in BMA and BMAC samples was analyzed using CFU-H (hematopoietic progenitor/stem cells), CFU-F (stromal progenitor cells), and the results:

CFU-H (hematopoietic progenitor/stem cell), bone marrow aspirate sample (BMA) = (34.3 + -4.1) x10^3/mL, bone marrow enriched cell sample (BMAC) = (213.6 + -19.3) x10^3/mL, 6.2-fold increase;

CFU-F (stromal progenitor cells), bone marrow aspirate sample (BMA) = (41.7 + -5.3) x10^3/mL, bone marrow enriched cell sample (BMAC) = (302.4 + -28.1) x10^3/mL, 7.3-fold increase.

The results indicate that the PXP system is able to efficiently enrich bone marrow stem cells while maintaining the biological potency of the bone marrow stem cells.

5.2 quantitative analysis of cytokines

The BMAC injection prepared by the PXP system contains a plurality of cytokines. Enzyme linked immunosorbent assay (ELISA) quantitative assays assay transformed growth factor-beta (TGF-beta), vascular Endothelial Growth Factor (VEGF), and Hepatocyte Growth Factor (HGF) levels in BMA and BMAC samples, results:

TGF-beta of BMA and BMAC is 34.1 +/-3.1 pg/ml and 218.3 +/-17.2 pg/ml respectively,

VEGF for BMA and BMAC were 17.4 + -2.6 pg/ml and 151.6 + -13.4 pg/ml, respectively,

HGF for BMA and BMAC were 194.3. + -. 24.2pg/ml and 1312.4. + -. 44.7pg/ml, respectively.

The results show that TGF-beta, VEGF and HGF in BMAC are obviously higher than TGF-beta, VEGF and HGF levels in BMA (p < 0.01), which indicates that PXP system can effectively concentrate and enrich cell growth factors.

Test example 6: effectiveness of BMAC in treating Premature Ovarian Failure (POF)

In Chinese patent application No. 2021116067849, an effectiveness study on the treatment of Premature Ovarian Failure (POF) was carried out by using BMAC concentrated solution. In this test example, the BMAC concentrate prepared above was used in combination with GM-CSF (also referred to herein as gmCSF) to study the effectiveness of POF.

(1) Establishing POF mouse model

And C57BL/6 mice of 8 weeks old female are injected with 50mg/kg/day Cyclophosphamide (CTX) in the abdominal cavity, and are continuously injected with 15 days in the abdominal cavity at the same time every day to establish a Premature Ovarian Failure (POF) mouse model. The control group was not treated at all. After the POF modeling is finished, BMAC cell transplantation treatment is carried out, and model animals are randomly grouped.

(2) The reserve function of the ovary is evaluated by the indexes of hormone level, follicle number, fertility test and the like.

A. Hormone levels

Animal grouping:

control group (n = 20),

POF model group (n = 20),

BMAC treatment group (n = 20),

BMAC + gmCSF treatment group (n = 20),

BMAC + supernatant concentrate + gmCSF treatment group (n = 20).

BMAC treatment group mice were administered 200 μ l of BMAC concentrated cell composition (of 200 μ l) by tail vein injection for each animal on day 1 after POF modeling, respectivelyThe BMAC concentrated cell composition was a sample of No.2 concentrate according to example 1, and was diluted with sterile physiological saline to prepare CD45 ⁺ A solution with a cell count concentration of 4x10^6 cells/200 μ l);

in the BMAC + gmCSF treatment group mice, 200 mul of BMAC concentrated solution gmCSF composition is respectively administered to the tail vein injection of each animal on the 1 st day after POF modeling;

the BMAC + supernatant concentrate + gmCSF treatment group mice are respectively injected with 200 mul of BMAC + supernatant concentrate + gmCSF composition in tail vein of each animal on the 1 st day after POF modeling;

the POF model group is injected with physiological saline with equal volume; the control group was not treated by injection;

after cell transplantation, each group was given diet and water as normal.

Note: a BMAC concentrate gmCSF composition (which may be abbreviated as BMAC-gmCSF composition) administered in the above BMAC + gmCSF treatment group, which includes per 200 μ L: an appropriate amount of sample No.2 concentrate obtained in example 1 was CD45 ⁺ The cell count is 1x10^6, 2.5ng of gmCSF and the volume is fixed by sterile physiological saline to obtain the composition;

the BMAC + supernatant concentrate + gmCSF composition administered in the BMAC + supernatant concentrate + gmCSF treatment group includes, per 200 μ L: a suitable amount of CD45 was used as a sample of concentrate No.2 obtained in example 1 ⁺ The cell count is 0.8x10^6, 2.5ng gmCSF, no.2 supernatant concentrate obtained in example 1 is 20 mu L, and the volume is fixed by sterile physiological saline, so that the composition is obtained;

the composition is stored at the temperature of 2-4 ℃ after preparation and is injected and administered within 4 hours, and the gmCSF is a commercially available freeze-dried powder injection.

After 14 days and 28 days after BMAC transplantation, 10 mice were collected from each group, blood was collected from the orbit, and serum was separated and stored at-20 ℃. Enzyme-linked immunosorbent assay (ELISA) was performed to analyze the levels of estradiol (E2) and Follicle Stimulating Hormone (FSH) (the specific method is described in the beauty article (beauty, et al, transplantation of human placental mesenchymal stem cells to improve ovarian function by reducing the expression of superoxide dismutase 1 and uncoupling protein-2, journal of chinese reproduction and contraception, 2018, stage 02), and the results are shown in the table below.

The results show that: compared with the POF model group, the serum E2 level of the mice in the BMAC group is increased at 28d, the FSH level is reduced, and the serum levels are all significantly different (P < 0.05); in addition, it has been found that by using in combination with GM-CSF and/or supernatant concentrate, the amount of BMAC can be significantly reduced and substantially the same effect can be obtained.

B. Follicle count of mouse ovarian tissue

28 days after BMAC transplantation, 10 mice are taken from each group respectively and killed, the left ovary tissue of the mice is taken and fixed in 4% paraformaldehyde, the fixed tissue is dehydrated by series alcohol, xylene is transparent, paraffin is embedded, the tissues are continuously sliced, the slice thickness is 5 mu m, HE staining is carried out, and observation is carried out under a microscope.

The results show that: compared with a control group, the number of primary follicles, secondary follicles and mature follicles of the mice in the POF model group is obviously reduced, and the number of atretic follicles is obviously increased; after 28 days of BMAC treatment, the number of all levels of follicles is recovered to different degrees, the growth of granulosa cells is increased, the apoptosis is reduced, the form of ovarian epithelial cells is stable, the number of primary follicles, secondary follicles and mature follicles is obviously increased, and the number of atretic follicles is obviously reduced. The follicle counts at each stage 28 days after BMAC transplantation have significant differences compared with the POF group, and specific results are shown in the following table.

C. Observation of mouse fertility

On day 28 post-BMAC transplantation, male and female mice were treated as 2: the mice are bred in a cage with a proportion of 1, the fertility rate of the mice is counted, the litter size of the mice is compared, the repairing effect of BMAC transplantation on the ovarian function of the mice is observed, and the result shows that the BMAC group and the POF group have obvious difference. The litter size comparison of the mice is shown in the following table.

According to the results, the BMAC transplantation treatment can obviously improve the reserve function of the damaged ovaries of the POF mice, increase the number of follicles, increase the estrogen and the progestogen, recover the fertility of the mice and provide experimental basis for the BMAC applied to the clinical treatment of the POF.

Test example 7: BMAC-gmCSF compositions

The BMAC-gmCSF composition used in test example 6 above was administered as soon as possible after preparation for injection, and the present inventors found that the biological activity of GM-CSF showed a tendency to decrease after the composition in a liquid state was left at 4 ℃ for 12 hours and 24 hours, and that this tendency of decrease in biological activity could be significantly alleviated by adding trace amounts of glutamine and sodium selenite to the liquid composition, as specifically tested below.

Formula a: 5 kinds of liquid compositions were prepared according to the following formulation using 5 kinds of BMACs of Nos. 1 to 5 obtained in example 1, and these compositions were denoted as compositions aNo.1 to aNo.5, respectively: comprising CD45 ⁺ BMAC with the cell number of 5x10^6, gmCSF with the cell number of 12.5ng (namely 125 IU) and proper amount of sterile physiological saline to 1mL;

formulation a1: 5 kinds of compositions in a liquid state were prepared as compositions a1No.1 to a1No.5, respectively, using 5 kinds of BMACs and 5 kinds of supernatant concentrates obtained in example 1 according to the following formulation: comprising CD45 ⁺ BMAC with the cell number of 4x10^6, gmCSF with the cell number of 12.5ng (namely 125 IU), supernatant concentrated solution of 100 mu L and a proper amount of sterile physiological saline of 1mL;

and (b) a formula: preparing a composition in a liquid state according to the formula a but without adding BMAC, and marking as a composition b;

formulation b1: a composition in a liquid state was prepared as described in the above formulation a1 using the concentrate of No.1 supernatant without addition of BMAC and was designated as composition b1;

and (c) formula: using 5 BMACs of No.1 to No.5 obtained in example 1, according to the formula a, glutamine (to a final concentration of 0.2 mg/ml) and sodium selenite (to a final concentration of 15 mu g/ml) are further added to prepare 5 compositions in a liquid state, which are respectively marked as composition cNo.1 to composition cNo.5;

the formula c1: 5 liquid compositions, namely, compositions c1No.1 to c1No.5, were prepared by using 5 BMACs and 5 supernatant concentrates of No.1 to No.5 obtained in example 1 according to the above formulation a1 and further adding glutamine (to a final concentration of 0.2 mg/ml) and sodium selenite (to a final concentration of 15 μ g/ml);

and (3) formula d: 5 kinds of liquid compositions (designated as compositions dNO.1 to dNO.5, respectively) were prepared by using 5 kinds of BMACs of Nos. 1 to 5 obtained in example 1, according to the above formulation a except that glutamine was added (to a final concentration of 0.2 mg/ml);

formulation d1: 5 kinds of compositions in a liquid state, designated as compositions d1No.1 to d1No.5, were prepared by using 5 kinds of BMACs of Nos. 1 to 5 obtained in example 1 and 5 kinds of supernatant concentrates and adding glutamine (to a final concentration of 0.2 mg/ml) in the same manner as in the above formulation a 1; and (e) formula: using the 5 BMACs of No. 1-No. 5 obtained in the embodiment 1, preparing 5 liquid compositions according to the formula a and adding sodium selenite (to a final concentration of 15 mu g/ml), and respectively marking as compositions eNO.1-eNO.5;

formulation e1: 5 kinds of compositions in a liquid state, designated as compositions e1No.1 to e1No.5, were prepared by using 5 kinds of BMACs and 5 kinds of supernatant concentrates obtained in example 1 according to the above formulation a1 and further adding sodium selenite (to a final concentration of 15 μ g/ml).

The formulation of the above-mentioned various compositions is a conventional method well known to those skilled in the art, for example, under aseptic conditions, a prescribed amount of gmCSF in the form of lyophilized powder and optionally glutamine and optionally sodium selenite is quantitatively dissolved to a prescribed volume with sterile physiological saline, and BMAC is diluted with sterile physiological saline to CD45 ⁺ Diluting the two solutions to specified concentration with sterile normal saline according to formula proportion, and packaging with glass bottle.

Placing each composition of the above 10 formulas at 4 deg.C, sampling at 0h, 12h, and 24h, respectively, and determining biological activity (IU/ml) of each composition at a specified time according to "method for measuring biological activity of recombinant human granulocyte macrophage-stimulating factor 3526" in the four parts appendix of Chinese pharmacopoeia 2015 edition; for a composition, the percentage obtained by dividing the biological activity of 12h or 24h by the biological activity of 0h multiplied by 100% is the residual percentage of the biological activity of the composition at the time point gmCSF.

As a result:

the biological activity of all the compositions from the formula a to the formula e and from the formula a1 to the formula e1 in 0h ranges from 121.3 to 129.6IU/ml, for example, the biological activity of the composition aNo.1 in 0h is 126.4IU/ml;

the 12h residual percentages of the compositions of formulation b and formulation b1 were 97.4% and 96.8% respectively,

the 12h residual percentage of the total composition of formulation c and formulation c1 is in the range of 97 to 102% for example 98.6% for 12h residual percentage of composition cNo.1,

the 12h residual percentage of all the compositions of formula a and formula a1, formula d and formula d1, and formula e1 are in the range of 81 to 88%, for example, the residual percentage of composition aNo.1 in 12h is 85.3%;

the 24h residual percentages of the compositions of formulation b and formulation b1 were 94.7% and 95.4% respectively,

the 24h residual percentage of the total composition of the formulation c and the formulation c1 is in the range of 91 to 95%, for example the 24h residual percentage of the composition cNo.1 is 93.6%,

the residual percentages of all the compositions of the formula a and the formula a1, the formula d and the formula d1, and the formula e1 in 24 hours are in a range of 64 to 73 percent, for example, the residual percentage of the composition aNo.1 in 24 hours is 70.4 percent.

These results indicate that the biological activity of gmCSF in the cell-containing composition decreased more rapidly, and this decrease in biological activity was significantly overcome when minor amounts of glutamine and sodium selenite were added to the composition.

In addition, the number of CD45+ live cells was measured as in test example 3 above, and the results were:

the CD45+ viable cell count of all the compositions of the formula a, the formula c, the formula d and the formula e in 0h is in the range of 472 to 524 x10^4/ml, for example, the CD45+ viable cell count of the composition aNo.1 in 0h is 511.7 x10^4/ml,

the CD45+ living cell number of all the compositions of the formula a1, the formula c1, the formula d1 and the formula e1 at 0h is 387 to 416 x10^4/ml, for example, the CD45+ living cell number of the composition a1No.1 at 0h is 394.5 x10^4/ml,

the CD45+ viable cell number of all the compositions of the formula a, the formula c, the formula d and the formula e in 24h is within the range of 144 to 212 x10^4/ml, for example, the CD45+ viable cell number of the composition aNo.1 in 24h is 196.7 x10^4/ml;

the CD45+ viable cell count of all the compositions of the formula a1, the formula c1, the formula d1 and the formula e1 in 24h is in the range of 121 to 158 x10^4/ml, for example, the CD45+ viable cell count of the composition a1No.1 in 24h is 142.2 x10^4/ml.

These results indicate that there was no significant difference in the number of CD45+ viable cells at different time points for each composition, and thus it can be expected that glutamine and sodium selenite would not affect the biological activity of the cells. Therefore, although the BMAC + gmCSF composition of formulation a and the BMAC + supernatant concentrate + gmCSF composition of formulation a1 can exhibit excellent biological effects for treating premature ovarian failure, the stability of gmCSF biological activity in the composition can be significantly improved when a small amount of glutamine and sodium selenite is supplemented, and there is no significant difference in living cells in the composition, and such improvement of stability of gmCSF biological activity would be of great significance for therapeutic application.

In addition, since gmCSF is inexpensive and readily available, the amount of cells used can be significantly reduced by combining with BMAC that is poorly available but still can obtain an excellent biological effect for treating premature ovarian failure.

It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.

Claims

1. A cellular composition comprising bone marrow stromal cells, granulocyte macrophage stimulating factor, supernatant concentrate, glutamine, sodium selenite, and optionally an excipient; wherein the content of the first and second substances,

the proportion of the concentrated cells to the granulocyte-macrophage stimulating factor is that the concentrated cells are CD45 ⁺ Counting the number of cells to 4x10^6 cells: 10 to 15ng granulocyte macrophage stimulating factor;

the ratio of the concentrated cells to the supernatant concentrate is that the concentrated cells are CD45 ⁺ The cell number is 4x10^6 cells: supernatant concentrated solution of 75 to 125 muL;

the weight ratio of the granulocyte-macrophage stimulating factor to the glutamine and the sodium selenite in the composition is 12.5ng:0.1 to 0.5mg:5 to 20 microgram;

the marrow condensed cells and the supernatant concentrated solution are prepared by the method comprising the following steps:

(7) Separating the supernatant in the central chamber, i.e. the plasma layer, by using an injector, centrifuging for 20min at 2000g to remove cell debris, and filtering by using a sterile filter membrane of 0.22 mu m;

(8) And (4) flushing a pipeline of the tangential flow ultrafiltration system by using ultrapure water, installing a MidiKros filter of 100kD 100cm2, and performing ultrafiltration concentration on the filtrate obtained in the step (7) to 1/15 volume of the initial biological sample volume to obtain a supernatant concentrated solution.

2. The cellular composition of claim 1, wherein the ratio of the enriched cells to granulocyte macrophage stimulating factor is such that the enriched cells are CD45 ⁺ The cell number is 4x10^6 cells: 12.5ng granulocyte macrophage stimulating factor; the ratio of the concentrated cells to the supernatant concentrate is that the concentrated cells are CD45 ⁺ Counting the number of cells to 4x10^6 cells: 100 muL of supernatant concentrated solution; and/or the weight ratio of granulocyte macrophage stimulating factor to glutamine and sodium selenite in the composition is 12.5ng:0.2 to 0.3mg:10 to 15 mu g.

3. The cell composition according to claim 1, wherein the excipient is physiological saline or a 5% glucose solution, and the concentration of the granulocyte macrophage stimulating factor in the composition is 10 to 15ng/ml.

4. The cellular composition of claim 1, the granulocyte macrophage-stimulating factor is a human granulocyte macrophage-stimulating factor.

5. The cellular composition of claim 1, comprising: CD45 ⁺ Concentrated cells with the cell number of 3-5x10 ^6, granulocyte macrophage stimulating factors of 10-15ng, supernatant concentrated solution of 75-125 muL, glutamine of 0.1-0.5mg, sodium selenite of 5-20 muL and proper amount of physiological saline to 1mL; alternatively, it comprises: CD45 ⁺ Concentrated cells with the cell number of 3 to 5x10^6, granulocyte macrophage stimulating factor of 10 to 15ng, supernatant concentrated solution of 80 to 120 mu L, glutamine of 0.2 to 0.3mg, sodium selenite of 10 to 15 mu g and proper amount to 1mL of physiological saline.

6. The cellular composition of claim 1, comprising: CD45 ⁺ 4x10^6 cells, 12.5ng granulocyte-macrophage stimulating factor, 100 muL supernatant concentrate, 0.2 to 0.3mg glutamine, 10 to 15 mug sodium selenite and a proper amount of normal saline to 1mL.

7. The cellular composition of claim 1, comprising: CD45 ⁺ 4x10^6 cells, 12.5ng granulocyte-macrophage stimulating factors, 100 muL supernatant concentrate, 0.2mg glutamine, 15 mug sodium selenite and proper amount of normal saline to 1mL.

8. The cell composition of claim 1, wherein the anticoagulant used in step (1) is a 3.2% sodium citrate solution supplemented with 0.5mg/ml histidine and 0.1mg/ml phosphatidylcholine.

9. A method for preparing a cell composition, which comprises the steps of mixing a prescribed amount of skeleton-concentrated cells, granulocyte-macrophage stimulating factor, supernatant concentrate, glutamine, sodium selenite, and optionally an excipient to prepare a sterile preparation; wherein the content of the first and second substances,

the ratio of the concentrated cells to the granulocyte-macrophage stimulating factor is that the concentrated cells are CD45 ⁺ Counting the number of cells to 4x10^6 cells: 10 to 15ng granulocyte macrophage stimulating factor;

(8) And (3) rinsing a pipeline of the tangential flow ultrafiltration system by using ultrapure water, installing a 100kD 100cm2 MidiKros filter, and carrying out ultrafiltration concentration on the filtrate obtained in the step (7) to 1/15 volume of the initial biological sample amount to obtain a supernatant concentrated solution.

10. Use of the cell composition according to any one of claims 1 to 8 or the cell composition prepared by the method according to claim 9 in the preparation of a medicament for treating premature ovarian failure.