CN115501254A

CN115501254A - Composition comprising cord blood concentrated cells

Info

Publication number: CN115501254A
Application number: CN202211314454.7A
Authority: CN
Inventors: 肖海蓉; 刘冰; 徐勇
Original assignee: BOYALIFE Inc
Current assignee: BOYALIFE Inc
Priority date: 2022-10-26
Filing date: 2022-10-26
Publication date: 2022-12-23

Abstract

The present invention relates to a composition comprising umbilical cord blood concentrated cells, comprising concentrated cells, granulocyte macrophage stimulating factor, supernatant concentrate and optionally excipients; the proportion of concentrated cells is that the concentrated cells are CD45 ⁺ The cell number is 4x10^6 cells: 10-15 ng granulocyte-macrophage stimulating factor: 75-125 mu L of supernatant concentrated solution. The concentrated cells and the supernatant concentrated solution are obtained by a PXP cell automatic separation system and a tangential flow ultrafiltration system. Also relates to the use of said composition for the preparation of a medicament for the treatment of premature ovarian failure, and to a method for preparing said composition, comprising bringing into association a defined amountThe concentrated cells, the granulocyte-macrophage stimulating factor, the supernatant concentrated solution, glutamine, sodium selenite and optional excipients are mixed to prepare a sterile preparation. The compositions of the present invention exhibit excellent biological and/or chemical effects.

Description

Composition comprising cord blood concentrated cells

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. Specifically, the present invention relates to a method for treating premature ovarian failure and premature ovarian failure by using a cell therapeutic agent prepared by rapidly isolating umbilical cord blood concentrated cells (umbilical cord blood concentrate) from umbilical cord blood, and then formulating it with GM-CSF (Granulocyte Macrophage stimulating Factor, also known as Granulocyte Macrophage Colony stimulating Factor, granulocyte/macro-collagen-stimulating Factor, gmCSF) to prepare the cell therapeutic agent. The method can effectively improve the efficiency of separating the cord blood concentrated cells from the cord blood, and provides a method for safely, efficiently and simply obtaining the cord blood concentrated cell preparation for treating premature ovarian failure and ovarian insufficiency, such as premature ovarian insufficiency. The invention also provides a new effective way for treating premature ovarian failure by combining the prepared umbilical cord blood concentrated cells, supernatant concentrated solution and GM-CSF.

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 have an 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), diagnostic criteria for POF: FSH levels are elevated >40IU/L twice with 4 weeks or more between at least 4 months of scanty menstruation or amenorrhea. 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 effect 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 and the like in addition to symptoms such as scanty menstruation, amenorrhea, infertility and the like, and can accelerate female aging, cause postmenopausal diseases such as osteoporosis, cardiovascular diseases, dementia and the like, and influence the quality of life and the life span 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 treatment for POF is Hormone Replacement Therapy (HRT). Although the therapy has a certain relieving effect on the clinical symptoms of POF, HRT cannot fundamentally repair damaged ovaries and recover the functions of the ovaries. 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.

Umbilical cord blood (umbilical cord blood) is blood remaining in the placenta and umbilical cord after the fetus is delivered, the umbilical cord is ligated and severed, and is generally discarded. In recent decades, cord blood contains Hematopoietic Stem Cells (HSCs) that can reconstitute the human Hematopoietic and immune systems, and can be used for Hematopoietic stem cell transplantation to treat over 80 diseases. Thus, cord blood has become an important source of hematopoietic stem cells, particularly hematopoietic stem cells of unrelated blood relationship. Is also a very important human biological resource. Umbilical cord blood contains a large amount of Mesenchymal Stem Cells (MSCs), which are vital seeds, and can differentiate into various cells of the human body, resulting in various fruits, such as blood cells, nerve cells, bone cells, and the like. With the development of science and technology, medical experts developed methods for treating diseases using stem cells in umbilical cord blood. Stem cells are a population of cells with self-renewal, high proliferation and multiple differentiation potential. These cells can maintain the characteristics and number of the cells themselves by division, and can further differentiate into various tissue cells, thereby playing a positive role in tissue repair and the like. In recent thirty years of medical research, cord blood contains abundant hematopoietic stem cells, can rebuild human hematopoietic and immune systems, and can be used for hematopoietic stem cell transplantation and treating diseases of the blood system, the immune system, genetic metabolism and congenital diseases. Therefore, the cord blood becomes an important source of hematopoietic stem cells, has been widely used in clinic, and 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 subset of cells with multiple differentiation potentials that differentiate to form bone, cartilage, fat, nerve and myoblasts, and MSCs act as a support for HSCs by paracrine secretion of multiple growth factors, maintaining the stability of hematopoietic microenvironment.

Umbilical cord blood concentrated cells (UCBC) are a concentrate of nucleated cells obtained by centrifuging and separating umbilical cord blood. The umbilical cord blood concentrated cells (UCBC) contain enriched Hematopoietic Stem Cells (HSCs) and Mesenchymal Stem Cells (MSCs) and a large amount 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. UCBC contains 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).

UCBC has anti-inflammatory, immunoregulatory, angiogenesis promoting, and tissue regeneration and repair promoting effects. Preclinical and preliminary clinical studies prove that UCBC improves ovarian function by improving ovarian microenvironment, promoting angiogenesis, promoting follicular development, increasing the number of antral follicles and promoting ovulation, and is a potential treatment method for treating POF patients.

However, the cord blood is separated by density gradient centrifugation method in the traditional manual operation mode, and the problems of complicated operation, long time consumption, easy pollution, poor result repeatability and the like are very troublesome. One skilled in the art would expect a method of processing cord blood to obtain cord blood concentrate, i.e., concentrated cells, that is simple, time consuming, less susceptible to contamination, and reproducible results. This technical advance has been achieved in the present research team in chinese patent application No. 2021116067919.

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 granulocytic and monocytic cell lines, promote growth, induce differentiation and enhance phagocytic function and bactericidal action; inducing dendritic cell maturation and functional distribution. The granulocyte macrophage stimulating factor used in clinic is usually recombinant human granulocyte macrophage stimulating factor, which 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 when leukopenia is caused, and can accelerate recovery of neutropenia caused by infection.

The existing methods for treating premature ovarian failure still need to be improved. Therefore, it would be desirable to provide a method for treating premature ovarian failure, such as a method for treating premature ovarian failure using a therapeutic agent of concentrated cells from cord blood, wherein the therapeutic agent of the combination may further comprise a supernatant concentrate of the by-product of the preparation of the concentrated cells.

Disclosure of Invention

The invention aims to provide a method for preparing a concentrated cell preparation of umbilical cord blood, which is expected to have the advantages of simple operation, short time consumption, difficult pollution, good result repeatability and the like in one or more aspects; alternatively, it is an object of the present invention to provide a novel method for treating premature ovarian failure by formulating cord blood concentrated cells and GM-CSF as a combination therapeutic. It has been surprisingly found that one or more of the above objects can be achieved by the present invention, which is based on the discovery that cord blood concentrated cells are prepared using a closed PXP cell autosegregation system, and/or premature ovarian failure is treated by formulating the obtained cord blood concentrated cells with GM-CSF into a combination therapeutic agent, which may further comprise a byproduct supernatant concentrate from the preparation of the concentrated cells.

To this end, the present invention provides in a first aspect a process for preparing a concentrated cell preparation from cord blood, comprising the steps of:

(1) Providing a biological sample of umbilical cord blood, placing it in a sterile bag containing an anticoagulant for use;

(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;

(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 for centrifugation, and carrying out the following processes:

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 umbilical cord blood concentrated cell preparation;

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 of:

(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 (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.

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 350ml.

The method according to the first aspect of the present invention, wherein 1ml of the sample is additionally withdrawn in 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 3.6% sodium citrate solution.

The method according to the first aspect of the present invention, wherein the anticoagulant used in step (1) is a 3.6% sodium citrate solution supplemented with 0.5mg/ml histidine and 0.15mg/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 appropriate amount of water, heating to 60 deg.C, stirring to dissolve, adding water to full volume, filtering with 0.22 μm microporous membrane, and sterilizing at 121 deg.C 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 the data captured during centrifugation was processed with the DataTrak software processing system.

Further, the second aspect of the present invention provides a concentrated umbilical cord blood cell preparation, 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:

program number	Acceleration	Speed reduction	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

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

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

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

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

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

The concentrated cell preparation of cord blood according to the second aspect of the present invention, wherein 1ml of the sample is additionally withdrawn in the step (1) for examination.

The concentrated cell preparation of cord blood according to the second aspect of the present invention, wherein the anticoagulant used in the step (1) is a sodium citrate solution.

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

The concentrated cell preparation of umbilical cord blood according to the second aspect of the present invention, wherein the anticoagulant used in the step (1) is 3.6% sodium citrate solution supplemented with 0.5mg/ml histidine and 0.15mg/ml phosphatidylcholine.

The concentrated cell preparation of umbilical cord blood 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 concentrated cell preparation of umbilical cord blood according to the second aspect of the present invention, wherein the anticoagulant used in the step (2) is prepared by: adding sodium citrate, histidine and phosphatidyl choline into appropriate amount of water, heating to 60 deg.C, stirring to dissolve, adding water to full volume, filtering with 0.22 μm microporous membrane, and sterilizing at 121 deg.C under hot pressure.

The concentrated cell preparation of cord blood 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 process the data captured during centrifugation with a DataTrak software processing system.

Further, the third aspect of the present invention provides the use of a concentrated cell preparation of cord blood 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, and consists 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;

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 the injector to an output pipe of the separation cup communicated with the recovery cabin, and collecting the obtained umbilical cord blood concentrated cell preparation;

optionally (c) is

(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 use according to the third aspect of the present invention, wherein the volume of the biological sample provided in step (1) is 20 to 350ml.

The use according to the third aspect of the present invention, wherein 1ml of the sample is additionally withdrawn in 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 3.6% sodium citrate solution.

The use according to the third aspect of the invention, wherein the anticoagulant used in step (1) is a 3.6% sodium citrate solution supplemented with 0.5mg/ml histidine and 0.15mg/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 formulated by: adding sodium citrate, histidine and phosphatidylcholine into appropriate amount of water, heating to 60 deg.C, stirring to dissolve, adding water to full volume, filtering with 0.22 μm microporous membrane, and sterilizing at 121 deg.C under hot pressure.

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 the data captured during centrifugation was processed with the DataTrak software processing system.

Further, in a fourth aspect, the present invention provides a method for treating premature ovarian failure, the method comprising administering to a subject in need thereof a concentrated cell preparation comprising a therapeutically effective amount of cord blood prepared by a method comprising the steps of:

(1) Providing a biological sample umbilical cord blood, placing it in a sterile bag containing an anticoagulant for 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 separator cup, b) a control module, c) a separation base for transmitting data, d) a DataTrak software processing system;

optionally (c) is

and/or optionally, continuing the steps of:

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 350ml.

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

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 3.6% sodium citrate solution.

The method according to the fourth aspect of the present invention, wherein the anticoagulant used in step (1) is a 3.6% sodium citrate solution supplemented with 0.5mg/ml histidine and 0.15mg/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 phosphatidyl choline into appropriate amount of water, heating to 60 deg.C, stirring to dissolve, adding water to full volume, filtering with 0.22 μm microporous membrane, and sterilizing at 121 deg.C under hot pressure.

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 process the data captured during centrifugation with a DataTrak software processing system.

Further, the fifth aspect of the present invention provides a cell composition made of concentrated cells of cord blood, which comprises the concentrated 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 count is 5x10^6 cells: 10-15 ng granulocyte-macrophage stimulating factor; for example, in a ratio of concentrating cells to CD45 ⁺ The cell count 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 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: 0.1-0.5 mg:5 to 20 mu g.

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: 0.2-0.3 mg: 10-15 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:0.3mg:10 μ g.

A cell composition according to the fifth aspect of the invention, comprising: CD45 ⁺ Concentrated cells with the cell number of 4-6 x10^6, 10-15 ng of gmCSF, 0.1-0.5 mg of glutamine, 5-20 mu g of 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 cell number of 4-6x10 ^6 and gm of 10-15 ngCSF, 0.2-0.3 mg glutamine, 10-15 mug sodium selenite and 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 5x10^6, 12.5ng of gmCSF, 0.2-0.3 mg of glutamine, 10-15 mu g of sodium selenite and a proper amount of normal 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, 0.3mg of glutamine, 10 mu g of sodium selenite and a proper amount of normal saline to 1mL.

Further, the sixth aspect of the present invention provides a cell composition prepared from the concentrated cells of umbilical cord blood, which comprises the concentrated 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 such that the enriched cells are CD45 ⁺ Counting the number of cells to 4x10^6 cells: 10-15 ng granulocyte macrophage stimulating factor; for example, the ratio of the concentration of 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 expressed as CD45 ⁺ Counting the number of cells to 4x10^6 cells: 75-125 mul of supernatant concentrated solution; for example, in a ratio of concentrating cells to CD45 ⁺ The cell number is 4x10^6 cells: mu.L of the 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 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: 0.1-0.5 mg: 5-20 mu g.

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: 0.2-0.3 mg: 10-15 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:0.3mg:10 μ g.

A cell composition according to a sixth aspect of the invention, comprising: CD45 ⁺ Concentrated cells with the cell number of 3-5x10 ^6, 10-15 ng of gmCSF, 75-125 mu L of supernatant concentrated solution, 0.1-0.5 mg of glutamine, 5-20 mu g of sodium selenite and proper amount of normal saline to 1mL.

A cell composition according to a sixth aspect of the invention, comprising: CD45 ⁺ Concentrated cells with the cell number of 3-5x10 ^6, 10-15 ng of gmCSF, 80-120 mu L of supernatant concentrated solution, 0.2-0.3 mg of glutamine, 10-15 mu g 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 concentrated cells, 12.5ng of gmCSF, 100 mu L of supernatant concentrated solution, 0.2-0.3 mg of glutamine, 10-15 mu g of selenious acidSodium and normal saline are properly added to 1mL.

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

The unexpected finding that the supernatant concentrate obtained by combining the supernatant concentrate with concentrated cells and gmCSF has an excellent effect of treating premature ovarian failure, for example, the desired effect can be achieved by using a lower dose of concentrated cells, is unexpected in the prior art.

Further, in a seventh aspect, the present invention provides the use of the cell composition of any one of the fifth or sixth aspects 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 of 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. Of the various process steps described above, although specific steps are described in some detail or in language specific to the process steps described in the examples of the following detailed description, those skilled in the art will be able to fully appreciate the above-described process steps from the detailed disclosure of the invention as a whole.

Any embodiment of any aspect of the invention may be combined with other embodiments, as long as they do not contradict. Furthermore, in any embodiment of any aspect of the invention, any feature may be applicable to that feature in other embodiments, so long as they do not contradict. The invention is further described below.

All documents cited herein are incorporated herein by reference in their entirety and to the extent they do not conform to the teachings of the present invention, the statements made therein shall control. Further, the various terms and phrases used herein have the ordinary meaning as is known to those skilled in the art, and it is intended that such terms and phrases be interpreted as having a more complete description and interpretation herein, unless otherwise expressly stated otherwise, unless expressly stated otherwise.

The invention uses a PXP automatic cell rapid processing system, applies a closed system with automatic separation and concentration, and safely, efficiently and simply obtains the cord blood concentrated cells, thereby laying a foundation for the clinical application of the cord blood concentrated cells for treating POF patients. The invention provides a method for quickly separating and obtaining umbilical cord blood concentrated cells. The umbilical cord blood concentrated cells obtained by the invention can be used as an active ingredient for treating ovarian injury, and can promote angiogenesis and follicular development, so that the ovarian function is improved.

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 has a pressing need for 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

The automatic separation system is 80065-01, the supplier is Shenzhen Boya perception medical technology Limited, and the producer is ThermoGenesis 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 operate with little or no red blood cell contaminationIn general, less than 5% of the starting sample, rapidly achieves very high recovery rates of stem and progenitor cells.

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, umbilical cord blood) within 30 minutes at 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 GMP process control and reporting information for customers.

The PXP system is registered by medical equipment products of the Ministry of health of multinational countries at present, the clinical application scene of the equipment system is wide, the PXP system is mainly used for the cell therapy of the orthopedic diseases, the efficient and high-quality autologous bone marrow stem cell preparation in an operating room environment is realized through the PXP system, the cell therapy of the orthopedic diseases in a treatment center is brought with a new step, and the long-standing competitiveness is strived for the treatment center. The present invention uses the PXP system for processing bone marrow extracts, can rapidly and automatically process umbilical cord blood cells in real time, ensures the recovery rate of mononuclear cells (MNCs), can process multiple umbilical cord blood units simultaneously, and does not require a cell separation medium or a precipitant. Similar to the above-mentioned bone marrow stem cells, umbilical cord blood cells can also be used for the treatment of premature ovarian failure.

Advantages of the PXP system used in particular experiments of the present invention include, but are not limited to: the method has the advantages of high recovery rate of stable and excellent MNC (mononuclear cells) and CD34+ and CD45+ cells, rapid processing of cord blood samples within 30 minutes, high erythrocyte removal rate of more than 95 percent, an automatic closed sterile system, rapid and accurate data tracking and document recording, uploading of sample processing data to a computer through DataTrak software, and providing production record and report information meeting GMP requirements.

Recent studies have shown that cord tissue and cord blood also contain mesenchymal stem cells and can be successfully isolated, with cord blood-derived stem cells commonly referred to as hematopoietic stem cells. The mesenchymal stem cells from the source not only keep the biological characteristics of the mesenchymal stem cells, but also have more primitive isolated stem cells and stronger proliferation and differentiation capacities. The functional activity of immune cells is low, and the risk of triggering immune response and causing graft-versus-host disease is greatly reduced. The probability of infection and transmission of latent viruses and microorganisms is low. The collecting process is simple, and has no harm or injury to parturient and newborn. The above reasons are enough to make umbilical cord mesenchymal stem cells an ideal substitute for bone marrow mesenchymal stem cells.

Basic research has found that the stem cell types capable of differentiating into vascular endothelial cells include Endothelial Progenitor Cells (EPCs), bone marrow mononuclear cells (BMMNC), and peripheral blood mononuclear cells (PBMNC). However, these stem cells are limited in tissue origin and have limited numbers of extracts and amplifications. Such therapies are currently in preclinical research phase.

It has been proved by experiments that human CD34 ⁺ Cells (CD 34 is a marker of mature blood vessels) can alleviate the symptoms of CLI, improve the function of the affected limb, and prevent amputation. However, since the content of Wharton's jelly in umbilical cord Wharton's jelly is only about 5% -10%, how to produce the cells efficiently becomes an important prerequisite for its wide application.

Premature Ovarian Failure (POF) is a common endocrine disorder in women and affects female health. The incidence of women before age 40 is 1% -2%, and the incidence of women before age 30 is 0.1%. The diagnosis standards given by the Chinese medical society gynaecology and obstetrics society are: the amenorrhea time is more than or equal to 4-6 months, the follicle stimulating hormone of more than 4 weeks is more than 40U/L between two times, and the symptoms of estrogen reduction and menopause are accompanied. In POF patients, due to hypoovarianism and ovarian failure, hormone secretion is sharply reduced, which can cause menopause or menstrual rarefaction, and symptoms such as hectic fever, night sweat, dysphoria, anxiety, osteoporosis, temporary or permanent loss of fertility and the like are often accompanied, thus threatening physical and psychological health of women. The specific causes of most clinical premature ovarian failure are not clear, and at present, the specific causes include hereditary factors, autoimmune factors, iatrogenic factors, environmental factors and the like, and the incidence rate of iatrogenic secondary premature ovarian failure is on a gradual rise trend along with the wide application of chemotherapy to gynecological tumors. Aiming at the above causes, the current main measures for treating premature ovarian failure comprise hormone replacement therapy, immunoregulation, cryopreservation and transplantation of ovarian tissues, cryopreservation and transplantation of embryos and the like, and the clinical treatment methods can improve the premature ovarian failure symptoms and promote the fertility of patients, but cannot radically recover the ovarian function, and have ethical disputes and adverse reactions. Therefore, it is urgent to be in the academic world to find a treatment means capable of efficiently and safely restoring ovarian function.

A study conducted in the United states in 2019 showed that cord blood stem cells have shown encouraging regenerative capacity in POF. In the treatment experiment of this study, 18 female mice were divided into 3 groups on average, the first group was a control group (phosphate buffer injection alone), the second group was a chemotherapy group (combined chemotherapy with busulfan and cyclophosphamide, phosphate buffer injection), and the third group was an experimental group (bilateral ovarian umbilical cord blood stem cell injection based on the second group of chemotherapy drugs), after which the body weight and hormone levels of the mice were evaluated. Body weight data from mice after 8 weeks of treatment showed a significant increase in body weight after stem cell infusion in the cord blood stem cell group compared to a significant decrease in body weight after chemotherapy in the second group.

Starting at week 4, follicle Stimulating Hormone (FSH) levels in the group of cord blood stem cells infused were significantly elevated. FSH is a hormone secreted by anterior pituitary basophils and mainly acts to promote follicular maturation, thereby promoting proliferation and differentiation of granulosa cells of the follicle and allowing the entire ovary to grow. Breeding experiments were performed into 18 female mice, the procedure of the treatment experiment was repeated, starting 1 week after surgery, and 1 male mouse was placed in the same cage. The resulting pups were carefully checked for abnormalities and each animal was counted in each group. In the three-month test period, the mating rate of the mice in the group of cord blood stem cells infused to the control group is 100%, and the mating rate of the mice in the chemotherapy group is only 25%. The total number of pups in the cord blood stem cell infusion group was 26, and the chemotherapy group had only 2 pups. Although the total number of pups in the cord blood stem cell infusion group is significantly lower than that of the control group, the conception rate is equivalent to that of the control group. In this study, it was found that the ovaries damaged by chemotherapy in mice were positively treated after infusion of cord blood stem cells. The mice gain weight, hormone secretion function is good, and fertility is restored.

Cord blood is blood that remains in the placenta and umbilical cord after the fetus is delivered, ligated and severed, and is typically discarded. Research in recent ten years shows that cord blood contains hematopoietic stem cells which can restore human hematopoietic and immune systems, and the cord blood can be used for hematopoietic stem cell transplantation to treat more than 80 diseases. Thus, cord blood has become an important source of hematopoietic stem cells, particularly hematopoietic stem cells of unrelated blood relationship. Is also a very important human biological resource. Umbilical cord blood contains a large number of stem cells, which are life seeds, and which differentiate into various cells of the human body, resulting in various fruits, blood cells, nerve cells, bone cells, and the like. With the development of technology, medical professionals have developed methods for treating diseases by using stem cells in umbilical cord blood. Stem cells are a population of cells with self-renewal, high proliferation and multiple differentiation potential. These cells can maintain the characteristics and number of the cells themselves by division, and can further differentiate into various tissue cells, thereby playing a positive role in tissue repair and the like. In recent thirty years, medical research shows that cord blood contains very abundant Hematopoietic Stem Cells (HSCs), can rebuild the human hematopoietic and immune systems, and can be used for hematopoietic stem cell transplantation and treating the blood system, the immune system, genetic metabolic diseases and congenital diseases. Therefore, umbilical cord blood has become an important source of hematopoietic stem cells, has been widely used in clinic, and is a valuable human biological resource.

The 21 st century is a biological century, and cell therapy is used as a biological treatment means, and a brand new thought is opened for the treatment of human diseases. By the end of 5 months 2014, a total 928 clinical studies related to cord blood have been registered globally at the National Institutes of Health (NIH). Related diseases treated by the cord blood research institute comprise: autoimmune diseases: rheumatoid arthritis, lupus erythematosus, multiple sclerosis; cardiovascular and cerebrovascular diseases: congenital heart disease, heart repair and recovery, ischemic stroke; neurological disorders: cerebral palsy, hypoxic ischemic encephalopathy, autism, traumatic brain injury, spinal cord injury, hearing loss, amyotrophic lateral sclerosis, alzheimer's disease (senile dementia); malignant diseases: breast cancer, kidney cancer; congenital diseases: duchenne muscular dystrophy, becker-type muscular dystrophy, cystic fibrosis; other diseases: type I diabetes, AIDS, cartilage repair, and critical limb ischemia.

To date, CD34 in cord blood was treated ⁺ The cells are usually isolated by Ficoll separation, hydroxyethyl starch separation and natural sedimentation of gelatin, and the CD34 obtained is subsequently further purified by immunomagnetic bead adsorption (MACS) ⁺ Cells to obtain satisfactory CD34 ⁺ A cell. The method can directly separate and purify primary CD34 from human umbilical cord blood ⁺ Cells, CD34 obtainable by the above method in view of limited supply of human cord blood ⁺ The number of cells is also extremely limited.

The cord blood cell extract 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 appearance of cord blood concentrated cells, platelet rich plasma and stem cell derivatives is becoming a new trend in future medical treatment. 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 a human granulocyte macrophage stimulating factor or a 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 of products. 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 drug used clinically for leukopenia or granulocytopenia due to various causes, and it stimulates hematopoietic function of bone marrow, stimulates proliferation of granulocytes, monocytes, T cells, and promotes 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 umbilical cord blood concentrated cells obtained by PXP system separation with GM-CSF, and uses an ovarian premature senility 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 purposes of this invention, the invention is nevertheless described herein in as detail as possible.

In the present invention, unless otherwise specified, the cell separation system used in the specific experiment is

The automatic separation system, which may also be referred to as a PXP system, a PXP cell automatic separation system, a PXP separation system, or the like in the present 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 the United states. In the present invention, the term "cord blood marrow concentrated cells" may also be referred to as "cord blood concentrated cell preparation", and both have the same meaning, unless otherwise specified.

Example 1: rapid separation preparation of umbilical cord blood concentrated cells (UCBC)

(1) Providing a biological sample umbilical cord blood (a sample with a volume of 20-350 ml can be processed), placing the umbilical cord blood into a sterile bag containing an anticoagulant for later use, and extracting 1ml of sample for detection; the anticoagulant is 3.6% sodium citrate solution, wherein 0.5mg/ml histidine and 0.15mg/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 phosphatidyl choline into appropriate amount of water, heating to 60 deg.C, stirring to dissolve, adding water to full volume, filtering with 0.22 μm microporous membrane, and sterilizing at 121 deg.C under hot pressure;

(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 the injector to an output pipe of the separation cup communicated with the recovery cabin, and collecting the obtained umbilical cord blood concentrated cells;

(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.

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 cord blood were subjected to cell separation and supernatant concentrate preparation to obtain 10 cord blood concentrated cells and 10 supernatant concentrates, each labeled as No.1 to No.10.

Test example 1: analysis of MNC recovery in cord blood concentrated cells

Using the method of example 1, 10 pooled cord blood (pre-isolation volume in the range of 172-193 ml) was subjected to cell isolation; then, referring to the method described in chinese patent application No. 2021116067919, cell detection was performed on each fraction, and results of 10 samples: the average value of input umbilical cord blood is 182.4ml, the average value of output umbilical cord blood concentrated cells is 23.6ml, the average value of Red Blood Cell (RBC) removal rate is 98.5%, the average value of single nuclear cell (MNC) recovery rate is 96.2%, and the MNC concentration is averagely improved by 7.73 times. For example, the results of a certain cord blood (No. 1): pre-separation volume =187.6ml, final volume =23.3ml, erythrocyte removal rate =98.1%, MNC recovery rate =96.8%, MNC concentration fold =8.05.

The results show that the PXP kinetic separation system can be used for enriching the MNC in the umbilical cord blood and removing most of red blood cells in a manner of simple operation, short time consumption, low possibility of pollution and good result repeatability.

Test example 2: cell viability in cord blood and cord blood concentrated cell samples

The 10 samples involved in test example 1 were examined. Cell viability is the most intuitive indicator of whether a cell has a biological function. Within 24-36 hours of sample collection (T <36 hours), the cell viability in cord blood, cord blood-concentrated cells was analyzed using FC500 flow cytometer using 7-AAD staining. Cell viability =87.32 ± 3.62% for 10 cord blood samples before PXP treatment and 98.19 ± 1.76% for 10 cord blood samples after PXP treatment, for example, results of a certain cord blood sample (No. 1): the cell viability of the cord blood =88.13%, and the cell viability of the cord blood concentrated cells =99.16%. The cell survival rate in the cord blood concentrated cell sample is shown to be significantly higher than that of the cord blood.

Test example 3: count of CD45+, CD34+ cells in cord blood and cord blood concentrated cell samples

The 10 samples involved in test example 1 were examined. The number of CD45+ and CD34+ cells and the cell viability rate of all the cord blood before and after treatment and the concentrated cell samples thereof were analyzed on FC500 flow cytometer by using 7-AAD staining method, and the results are:

in terms of CD45+ viable cell number, the cord blood sample = (12.24 +/-2.16) x10^6/mL, the cord blood concentrated cell sample = (91.13 +/-4.74) x10^6/mL is increased by 7.4 times;

in terms of the number of CD34+ living cells, the cord blood sample = (117.6 +/-12.4) x10^3/mL, and the cord blood concentrated cell sample = (802.4 +/-22.7) x10^3/mL are increased by 6.8 times.

Test example 4: cord blood and cord blood concentrated cell sample sterility testing

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 cord blood, cord blood concentrated cell samples were prepared, fixed with methanol, tested after staining, results: no microorganism is seen in any of the gram-stained sample smears of 10 cord blood samples, no microorganism is seen in any of the gram-stained sample smears of 10 cord blood concentrated cell samples, and no microorganism is seen in any of the gram-stained sample smears of 10 supernatant concentrated solution samples.

The process for preparing the umbilical cord blood concentrated cells by using the PXP system has the characteristics of rapidness, sealing and no bacteria in the whole process.

Test example 5: cellular level validation study

The present invention relates to a concentrated umbilical cord blood cell prepared by PXP system, which is an injection of cell preparation, wherein it contains several stem cell components, including Hemopoietic Stem Cells (HSCs), mesenchymal Stem Cells (MSCs), endothelial Progenitor Cells (EPCs), and several cytokines, such as Vascular Endothelial Growth Factor (VEGF), stromal cell derived factor (SDF-1), endostatin (Entostatin), etc., to promote neovascularization and endothelial cell migration.

In this test example, 10 samples of test example 1 were examined to evaluate the biological efficacy of stem cells in cord blood concentrated cells by CFU colony forming ability, and cytokines abundant in cord blood concentrated cells were quantitatively detected by ELISA.

5.1 Stem cell biological potency-CFU colony formation assay

Cord blood stem cell biological Potency (Poteny Assays), characterization of the cell desiccation in cord blood enriched cell pool cells by in vitro CFU colony formation assay, analysis of the colony forming ability of progenitor/stem cells. The efficiency of CFU-H (hematopoietic progenitor/stem cells), CFU-F (stromal progenitor cells) was analyzed for various stem cells in cord blood and cord blood concentrated cell samples, and the results were:

CFU-H (hematopoietic progenitors/stem cells), cord blood sample = (24.7 + -3.3) x10^3/mL, cord blood concentrated cell sample = (183.2 + -17.5) x10^3/mL, increase 7.4-fold;

CFU-F (matrix progenitor cells), cord blood sample = (44.8 + -7.1) x10^3/mL, cord blood concentrated cell sample = (316.3 + -32.6) x10^3/mL, increase 7.1-fold.

The result shows that the PXP system can effectively enrich the cord blood stem cells, and maintain the biological efficacy of the stem cells.

5.2 quantitative analysis of cytokines

The umbilical cord blood concentrated cell preparation injection prepared by the PXP system contains a plurality of cytokines. Enzyme-linked immunosorbent assay (ELISA) quantitative assay the transforming growth factor-beta (TGF-beta), vascular Endothelial Growth Factor (VEGF) and Hepatocyte Growth Factor (HGF) levels in cord blood and cord blood concentrated cell samples were analyzed, and results:

the TGF-beta of the cord blood and the cord blood concentrated cells is respectively 25.6 +/-3.3 pg/ml and 203.5 +/-15.7 pg/ml,

the VEGF of the cord blood and the cord blood concentrated cells are respectively 19.8 +/-4.7 pg/ml and 172.4 +/-17.2 pg/ml,

HGF of cord blood and cord blood concentrated cells is 171.6 + -21.3 pg/ml and 1124.8 + -38.3 pg/ml respectively.

The results show that the TGF-beta, VEGF and HGF in the umbilical cord blood concentrated cells are obviously higher than the TGF-beta, VEGF and HGF levels in the umbilical cord blood (p < 0.01), and the PXP system can effectively concentrate and enrich the cell growth factors.

Test example 6: effectiveness of umbilical cord blood concentrated cells in treating Premature Ovarian Failure (POF)

In Chinese patent application No. 2021116067919, the effectiveness of umbilical cord blood concentrated cells for treating Premature Ovarian Failure (POF) is studied. In this test example, the cord blood concentrated cells prepared above were used in combination with GM-CSF (also referred to herein as gmCSF) to investigate the effectiveness of POF.

(1) Establishment of 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 molding is finished, umbilical cord blood concentrated cell transplantation treatment is carried out, and model animals are randomly grouped.

(2) Ovarian reserve function is assessed by hormone levels, follicle number and fertility tests.

A. Hormone levels

Animal grouping:

control group (n = 20),

POF model group (n = 20),

Cord blood concentrated cell treatment group (n = 20),

Cord blood concentrated cell + gmCSF treatment group (n = 20),

Cord blood concentrated cells + supernatant concentrate + gmCSF treatment group (n = 20).

Cord blood concentrated cell treatment group mice were treated with 200. Mu.l of cord blood concentrated cell composition by tail vein injection on day 1 after POF modeling (the 200. Mu.l of cord blood concentrated cell composition was the No.1 cord blood concentrated cell sample referred to in example 1, and was prepared to be CD45 by dilution with sterile physiological saline) ⁺ The cell concentration is 4x10^6 cells/200 mu l solution);

cord blood concentrated cell + gmCSF treatment group mice were administered with 200. Mu.l of cord blood concentrated cell gmCSF composition by tail vein injection for each animal on day 1 after POF modeling, respectively;

cord blood concentrated cells, supernatant concentrated solution and gmCSF treatment group mice are respectively injected with 200 mu l of cord blood concentrated cells, supernatant concentrated solution and gmCSF composition by tail vein on 1 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: the cord blood concentrated cell gmCSF composition (which may be simply referred to as cord blood cell gmCSF composition) administered in the above cord blood concentrated cell + gmCSF treatment group, which comprises per 200 μ L: a proper amount of the No.1 umbilical cord blood concentrated cell sample obtained in example 1 was CD45 ⁺ The cell count is 1x10^6, 2.5ng of gmCSF and the volume of sterile physiological saline is fixed to obtain the composition;

the cord blood concentrated cell + supernatant concentrate + gmCSF composition administered in the above cord blood concentrated cell + supernatant concentrate + gmCSF treatment group, which comprises per 200 μ L: a suitable amount of the No.1 concentrated cell sample obtained in example 1 was CD45 ⁺ 0.8x10^6 in cell count, 2.5ng of gmCSF, 20 mu L of No.1 supernatant concentrate obtained in example 1, and constant volume with sterile normal saline to obtain a composition;

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

10 mice were collected from each group 14 days and 28 days after transplantation with the cord blood concentrated cell preparation, blood was collected from the orbit, and serum was separated and stored at-20 ℃. Enzyme-linked immunosorbent assay (ELISA) was used to analyze the levels of estradiol (E2) and Follicle Stimulating Hormone (FSH) (the specific method is described in the paper (allelochemicals, et al, the methods for transplanting human placental mesenchymal stem cells to improve ovarian function by reducing the expression of superoxide dismutase 1 and uncoupling protein-2, china journal of 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 umbilical cord blood concentrated cell group is increased at 28d, the FSH level is reduced, and the levels are all significantly different (P < 0.05); in addition, it has been found that the amount of cord blood concentrated cells can be significantly reduced and substantially the same effect can be obtained by using in combination with GM-CSF and/or supernatant concentrate.

B. Follicle count in mouse ovarian tissue

28 days after the transplantation of the umbilical cord blood concentrated cell group, 10 mice are respectively taken from each group and killed, the left ovary tissue of each mouse is taken and fixed in 4 percent paraformaldehyde, the fixed tissue is dehydrated by series alcohol, xylene is transparent, paraffin is embedded, the continuous section is carried out, the section thickness is 5 mu m, HE staining is carried out, and the 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; 28 days after the umbilical cord blood concentrated cell group 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 count of each follicle in 28 days after the transplantation of the umbilical cord blood concentrated cell group is obviously different from that of the POF group, and specific results are shown in the following table.

P <0.01 compared to POF model group.

C. Observation of mouse fertility

On day 28 after the transplantation of the cord blood concentrated cell group, male and female mice were transplanted as 2:1 proportion is bred in a cage, the fertility rate of the mice is counted, the litter size of the mice is compared, the restoration effect of the transplantation of the cord blood concentrated cell group on the ovary function of the mice is observed, and the result shows that the cord blood concentrated cell group has obvious difference compared with the POF group. Results of comparisons of litter sizes of mice are shown in the following table.

According to the results, the storage function of the damaged ovary of the POF mouse can be obviously improved by the transplantation treatment of the cord blood concentrated cell group, the number of follicles is increased, the female progestogen is increased, the fertility of the mouse is recovered, and an experimental basis is provided for the application of the cord blood concentrated cell group to the clinical treatment of the POF.

Test example 7: umbilical cord blood concentrated cell + gmCSF composition

The cord blood concentrated cell + gmCSF composition used in test example 6 above was administered by injection as soon as possible after preparation, 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 to stand 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 as follows.

Formula a: 5 kinds of compositions in a liquid state were prepared by using 5 kinds of umbilical cord blood-concentrated cells obtained in example 1, and the compositions were designated as composition aNo.1 to composition aNo.5, respectively, according to the following formulation: comprising CD45 ⁺ The cell number is 5x10^6, the umbilical cord blood concentrated cells, 12.5ng (namely 125 IU) of gmCSF and a proper amount of sterile physiological saline are 1mL;

formulation a1: 5 kinds of compositions in a liquid state were prepared according to the following formulation using 5 kinds of umbilical cord blood-concentrated cells and 5 kinds of supernatant concentrated solutions obtained in examples 1 to 5, and the compositions were designated as compositions a1No.1 to a1No.5: comprising CD45 ⁺ 4x10^6 umbilical cord blood concentrated cells, 12.5ng (namely 125 IU) gmCSF, 100 mu L supernatant concentrated solution and proper amount of sterile physiological saline to 1mL;

and (b) a formula: preparing a composition in a liquid state according to the formula a without adding the cord blood concentrated cells, and marking as a composition b;

formulation b1: a composition in a liquid state was prepared as composition b1 using the supernatant concentrate of No.1 without adding concentrated cells of cord blood according to the above formulation a 1;

and (c) a formula: using 5 kinds of umbilical cord blood-concentrated cells of Nos. 1 to 5 obtained in example 1, 5 kinds of compositions in a liquid state were prepared in accordance with the above formulation a except that glutamine (to a final concentration of 0.3 mg/ml) and sodium selenite (to a final concentration of 10. Mu.g/ml), and they were designated as composition cNo.1 to composition cNo.5, respectively;

the formula c1: 5 kinds of compositions in a liquid state were prepared by using 5 kinds of umbilical cord blood-concentrated cells and 5 kinds of supernatant concentrates obtained in example 1, and adding glutamine (to a final concentration of 0.3 mg/ml) and sodium selenite (to a final concentration of 10. Mu.g/ml) in the same manner as in the above formulation a1, and they were respectively designated as composition c1No.1 to composition c1No.5;

and (3) formula d: 5 kinds of compositions in a liquid state, designated as composition dNO.1 to composition dNO.5, were prepared using 5 kinds of umbilical cord blood-concentrated cells of Nos. 1 to 5 obtained in example 1 in accordance with the above formulation a except that glutamine was added (to a final concentration of 0.3 mg/ml);

formulation d1: 5 kinds of compositions in a liquid state were prepared by using 5 kinds of umbilical cord blood-concentrated cells and 5 kinds of supernatant concentrates obtained in example 1 in the same manner as in the above formulation a1 except that glutamine was added (to a final concentration of 0.3 mg/ml), and they were designated as composition d1No.1 to composition d1No.5;

and a formula e: 5 kinds of umbilical cord blood-concentrated cells No.1 to No.5 obtained in example 1 were used, and according to the above formulation a, sodium selenite (to a final concentration of 10. Mu.g/ml) was further added to prepare 5 kinds of liquid compositions, which were respectively designated as composition eNo.1 to composition eNo.5;

formulation e1: 5 kinds of compositions in a liquid state were prepared by using 5 kinds of umbilical cord blood-concentrated cells and 5 kinds of supernatant concentrates obtained in example 1 in the same manner as in the above formulation a1 except that sodium selenite was added (to a final concentration of 10. Mu.g/ml), and they were designated as composition e1No.1 to composition e1No.5, respectively.

The above-mentioned various compositions are prepared by conventional methods well known to those skilled in the art, for example, by quantitatively dissolving a prescribed amount of gmCSF in a lyophilized powder state and optionally glutamine and optionally sodium selenite in a sterile physiological saline to a prescribed volume under aseptic conditions, and further diluting the cord blood concentrated cells with a sterile physiological saline to CD45 ⁺ And (3) diluting the two solutions to a specified concentration by using sterile normal saline according to a formula ratio, and subpackaging by using glass bottles to obtain the cell-based injection.

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 appendix of the four parts of the 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 and multiplying 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 of the formula a to the formula e and the formula a1 to the formula e1 in 0h ranges from 121.2 IU/ml to 128.6IU/ml, for example, the biological activity of the composition aNo.1 in 0h is 124.3IU/ml;

the 12h residual percentages of the compositions of formulation b and formulation b1 were 98.6% and 97.4% respectively,

the 12h residual percentage of the total composition of formulation c and of formulation c1 is in the range 94 to 99% for example 95.3% for the 12h residual percentage of composition cNo.1,

the 12h residual percentage for all compositions of formulation a and formulation a1, formulation d and formulation d1, formulation e and formulation e1 ranged from 80 to 86%, e.g., composition aNo.1 had a residual percentage of 83.4% at 12 h;

the 24h residual percentages of the compositions of formulation b and formulation b1 were 95.3% and 94.2% respectively,

the 24h residual percentage of the total composition of formulation c and formulation c1 is in the range of 88 to 92% for example 90.1% for 24h residual percentage of composition cNo.1,

the residual percentages for 24h for all compositions of formulation a and formulation a1, formulation d and formulation d1, formulation e and formulation e1 were in the range of 61 to 69%, for example composition a No.1 at 24h was 67.2%.

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 number of CD45+ living cells of all the compositions of formula a, formula c, formula d and formula e at 0h is in the range of 476-513x10 ^4/ml, for example, the number of CD45+ living cells of composition aNo.1 at 0h is 486.3x10^4/ml,

the CD45+ viable cell count of all the compositions of the formula a1, the formula c1, the formula d1 and the formula e1 at 0h is in the range of 392-431x10 ^4/ml, for example, the CD45+ viable cell count of the composition a1No.1 at 0h is 416.8x10^4/ml,

the number of CD45+ living cells of all the compositions of the formula a, the formula c, the formula d and the formula e in 24h is in the range of 121-187x10 ^4/ml, for example, the number of CD45+ living cells of the composition aNo.1 in 24h is 156.2x10^4/ml;

the CD45+ number of living cells at 24h of all the compositions of formula a1 and formula c1, formula d1, formula e1 are in the range of 137-168x 10^4/ml, for example the CD45+ number of living cells at 24h of composition a1No.1 is 154.6x10^4/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 concentrated cord blood cells + gmCSF composition of formula a and the concentrated cord blood cells + supernatant concentrate + gmCSF composition of formula a1 can exhibit excellent biological effects for treating premature ovarian failure, the stability of the biological activity of gmCSF 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 the living cells in the composition, and the improvement of the stability of the biological activity of gmCSF will be very significant for therapeutic applications.

In addition, since gmCSF is inexpensive and readily available, the amount of cells can be significantly reduced by combining with cord blood concentrated cells having poor availability while still obtaining excellent biological effects 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 composition comprising umbilical cord blood concentrated cells comprising concentrated cells, granulocyte macrophage stimulating factor, supernatant concentrate, and optionally an excipient; 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: 10-15 ng granulocyte-macrophage stimulating factor; the concentrated cell and the supernatant concentrateIn a ratio of concentrating the cells to CD45 ⁺ The cell number is 4x10^6 cells: 75-125 mul of supernatant concentrated solution.

2. The composition of claim 1, wherein said concentrated cell and supernatant concentrate are prepared by the following steps:

(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;

(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.

3. A composition according to claim 1, wherein:

the volume of the biological sample provided in the step (1) is 20-350 ml;

the anticoagulant used in the step (1) is 3.6% sodium citrate solution;

the anticoagulant used in step (1) is 3.6% sodium citrate solution, which is supplemented with 0.5mg/ml histidine and 0.15mg/ml phosphatidylcholine.

4. According to claim1, wherein the ratio of said condensed cells to granulocyte macrophage stimulating factor is such that the condensed cells are CD45 ⁺ The cell number is 4x10^6 cells: 12.5ng granulocyte macrophage stimulating factor.

5. The composition of claim 1, wherein the ratio of concentrated cells to supernatant concentrate is such that the concentrated cells are CD45 ⁺ The cell number is 4x10^6 cells: 100 μ L of the supernatant concentrate.

6. A composition according to claim 1, wherein:

the excipient is normal saline or 5% glucose solution;

the excipient is normal saline, and the concentration of the granulocyte macrophage stimulating factor in the composition is 10-15 ng/ml, such as 12.5ng/ml;

the granulocyte macrophage stimulating factor is human granulocyte macrophage stimulating factor;

the granulocyte macrophage stimulating factor is recombinant human granulocyte macrophage stimulating factor.

7. A composition according to claim 1, wherein:

it also contains glutamine and sodium selenite;

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

wherein the composition further comprises glutamine and sodium selenite, and the weight ratio of the granulocyte macrophage stimulating factor to the glutamine and the sodium selenite in the composition is 12.5ng: 0.2-0.3 mg: 10-15 mug;

the weight ratio of the granulocyte macrophage stimulating factor to the glutamine and the sodium selenite in the composition is 12.5ng:0.3mg:10 μ g.

8. A composition according to claim 1, comprising: CD45 ⁺ Concentrated cells having a cell number of 3 to 5x10^6, 10 to 15ng gmCSF, 75 to 125 mu L of supernatant concentrated solution, 0.1 to 0.5mg of glutamine, 5 to 20 mu g of sodium selenite and proper amount of normal saline to 1mL; alternatively, it comprises: CD45 ⁺ The cell number is 3-5x10 ^6, the concentrated cells are 10-15 ng, the gmCSF is 10-15 ng, the supernatant concentrated solution is 80-120 mu L, the glutamine is 0.2-0.3 mg, the sodium selenite is 10-15 mu g, and the proper amount of the normal saline is 1mL; alternatively, it comprises: CD45 ⁺ 4x10^6 cells, 12.5ng of gmCSF, 100 mu L of supernatant concentrate, 0.2-0.3 mg of glutamine, 10-15 mu g of sodium selenite and proper amount of normal saline to 1mL; alternatively, it comprises: CD45 ⁺ 4x10^6 concentrated cells, 12.5ng of gmCSF, 100 mu L of supernatant concentrated solution, 0.3mg of glutamine, 10 mu g of sodium selenite and proper amount of normal saline to 1mL.

9. Use of a composition according to any one of claims 1 to 8 in the manufacture of a medicament for the treatment of premature ovarian failure.

10. A process for preparing a composition according to any one of claims 1 to 8, comprising the step of bringing into admixture defined amounts of concentrated cells, granulocyte macrophage stimulating factor, supernatant concentrate, glutamine, sodium selenite, and optionally excipients, to formulate a sterile preparation.