CN115531521B - Cell therapeutic agent, preparation method and application thereof - Google Patents
Cell therapeutic agent, preparation method and application thereof Download PDFInfo
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Abstract
The present invention relates to cell therapeutic agents and methods of making and using the same. In one aspect, the cell composition of the invention comprises bone marrow concentrated cells, granulocyte macrophage-stimulating factor, supernatant concentrate, and optionally an excipient; wherein, the ratio of the concentrated cells to granulocyte macrophage stimulating factor and supernatant concentrate is that the concentrated cells count 4x10 x 6 cells by CD45 + cells: 10-15 ng granulocyte macrophage stimulating factor: 75-125 mu L of supernatant concentrate. Also relates to a preparation method of the cell composition and application of the cell composition in preparing medicines for treating premature ovarian failure. The present invention provides a combination therapeutic agent comprising bone marrow concentrated cells, granulocyte macrophage stimulating factor, and supernatant concentrate exhibiting excellent biological effects.
Description
Technical Field
The invention belongs to the field of biotechnology and biological medicine, and relates to a method for treating premature ovarian failure (Premature ovarian failure, POF) by using a cell therapeutic agent.
Background
Premature ovarian failure (Premature ovarian failure, POF) refers to the phenomenon of amenorrhea and infertility in women before age 40 due to ovarian failure. POF is a disease characterized by amenorrhea, infertility, estrogen deficiency, follicular reduction, and gonadotrophin elevation, accompanied by a range of low estrogen symptoms such as: hot flashes, excessive sweating, flushing of the face, hyposexuality, etc., seriously affect the physical and mental health of women. Furthermore, women with POF have an increased risk of osteoporosis, cardiovascular disease and senile dementia. POF is one of the important causes of female infertility. The incidence of POF in women of childbearing age is about 1-3%, and there is a trend toward rising and younger.
Diagnostic criteria for POF according to the guidelines of the European Society for Human Reproduction and Embryology (ESHRE): at least 4 months of menorrhagia or amenorrhea, two FSH levels, more than 4 weeks apart, are elevated by >40IU/L. Premature ovarian failure is of unknown etiology, and may be associated with genetic and autoimmune diseases, environmental factors, and iatrogenic and idiopathic conditions, there is no effective treatment. Hormone Replacement Therapy (HRT) is one of the most common treatments for POF, but is not ideal and has been shown to increase the risk of venous thrombosis, breast and ovarian cancer. POF can also show symptoms of hectic fever, hyperhidrosis, anxiety, depression, palpitation, insomnia and other climacteric symptoms besides the symptoms of amenorrhea, infertility and the like, and can accelerate female aging, so that postmenopausal diseases such as osteoporosis, cardiovascular diseases, dementia and the like can be caused, and the quality of life and the service life of females can be influenced.
The etiology of POF is complex and not yet fully elucidated, and may be related to autoimmune response, infection, genetic factors, therapeutic effects of chemotherapy, radiotherapy, surgery, etc., and endocrine dysfunctions, and there is no effective treatment. Currently, the most common treatment for POF is hormone replacement therapy (horone REPLACEMENT THERAPY, HRT). Although the therapy has a certain relieving effect on the clinical symptoms of POF, HRT can not fundamentally repair damaged ovaries and restore the ovarian function. Furthermore, studies have shown that long-term HRT treatment increases the risk of heart disease and stroke, possibly increasing the risk of breast and ovarian cancer. Thus, 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 erythrocytes, leukocytes and platelets in the blood circulation. Mesenchymal stem cells (MESENCHYMAL STEM CELL, MSCs) in bone marrow are a cell subset with multiple differentiation potential for differentiation into bone, cartilage, fat, nerve and myoblast cells, and MSCs support HSCs by paracrine secretion of multiple growth factors, maintaining the stability of the hematopoietic microenvironment of bone marrow.
Bone marrow concentrated cells (BMACs) are concentrates of nucleated cells obtained by centrifuging and separating bone marrow. Bone marrow concentrated cells (BMACs) contain enriched Hematopoietic Stem Cells (HSCs) and Mesenchymal Stem Cells (MSCs), as well as a large number of various cell growth factors. HSCs can differentiate into erythrocytes, leukocytes and platelets in the blood circulation. MSCs are a subpopulation of cells with multiple differentiation potential that differentiate to form bone, cartilage, fat, neural, and myoblast cells. BMAC 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 morphogenic proteins (BMP-2, BMP-7), and interleukins (IL-1, IL-6, IL-8).
The marrow concentrated cells have antiinflammatory, immunoregulatory, angiogenesis promoting, and tissue regeneration and repair promoting effects. Preclinical and preliminary clinical studies have demonstrated that BMAC improves ovarian function by improving ovarian microenvironment, promoting angiogenesis, promoting follicular development, increasing the number of sinus follicles, and promoting ovulation, thus being a potential therapeutic approach for treating POF patients.
However, conventionally, the bone marrow puncture fluid is separated by a density gradient centrifugation method by a manual operation, and the bone marrow puncture fluid has the problems of complex operation, long time consumption, easy pollution, poor result repeatability and the like. Those skilled in the art desire a method of treating a bone marrow aspirate to obtain a bone marrow concentrate, i.e., a bone marrow concentrated cell, that is simple to operate, takes a short time, is less susceptible to contamination, and has good reproducibility of results. This technical advance has been achieved in chinese patent application number 2021116067849 of the research team.
Granulocyte-macrophage colony stimulating factor (GM-CSF) is produced primarily by T cells and macrophages and is capable of inducing colony growth of granulocyte precursor and macrophage precursor cells, and is therefore abbreviated as granulocyte-macrophage colony stimulating factor. The main biological roles of GM-CSF in vivo are to maintain the survival of granulocyte and monocyte lineage cells, 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 recombinant human granulocyte-macrophage stimulating factor, is generally suitable for cancer chemotherapy and leucopenia caused by myelosuppression therapy, is also suitable for treating leucopenia of patients with bone marrow failure, can prevent potential infection complications when leucopenia is caused, 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 further desirable to provide a method of treating premature ovarian failure, such as using a bone marrow concentrated cell therapeutic agent, such as a combination therapeutic agent of bone marrow concentrated cells and GM-CSF, which may further comprise preparing a sideproduct supernatant concentrate from the concentrated cells.
Disclosure of Invention
An object of the present invention is to provide a method for preparing bone marrow concentrated cells, which is expected to have one or more of the advantages of simple operation, short time consumption, less susceptibility to contamination, good reproducibility of results, etc.; or it is an object of the present invention to provide a novel method for treating premature ovarian failure by formulating bone marrow concentrated cells and GM-CSF into a combined therapeutic agent. It has been unexpectedly found that the present invention can achieve one or more of the above objects by preparing marrow-concentrated cells using a closed PXP cell autosegregation system, and/or by formulating the obtained marrow-concentrated cells with GM-CSF to treat premature ovarian failure, which may further comprise preparing a sideproduct supernatant concentrate from the concentrated cells, and the present invention has been completed based on such findings.
To this end, a first aspect of the present invention provides a method for preparing bone marrow concentrated cells, comprising the steps of:
(1) Providing biological sample bone marrow puncture liquid, and placing the biological sample bone marrow puncture liquid in a sterile bag containing an anticoagulant for standby;
(2) Removing the protective cap on the input tube of the automatic cell separation system, connecting the syringe to the input Guan Luer locking joint, passing through the thrombus filter at a slow and stable speed, transferring the anticoagulated biological sample into a disposable sterile separation cup, and shaking the mixed sample along a horizontal axis; the cell autosegregation system is a closed PXP segregation system, which 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;
(3) The disposable separating cup is placed in the control module, the state of the control module before centrifugation is displayed as 0, the separating cup/control module assembly is weighed, after balancing, the disposable separating cup is placed in the programmable centrifugal machine, and parameters of the centrifugal machine are set according to the following procedures:
(4) Starting the centrifugal machine to centrifuge, wherein the process is as follows:
4a) Stage P1 cells in biological samples are separated into the upper and lower three components in disposable separation cups by centrifugation density stratification: a red blood cell layer, a cell concentration layer, and a plasma layer;
4b) The P2 phase enables most of red blood cells to enter a red blood cell recovery cabin;
4c) Stage P3 further delaminates cells in the process chamber and stage P4 reduces centrifugal force to further remove erythrocytes;
4d) The cell concentrate layer and the plasma are further layered in the P5 stage, the centrifugal force is reduced in the P6 stage, the cell concentrate layer is transferred to a recovery chamber through a conveying pipe, and the plasma is remained in a central chamber;
(5) After centrifugation is completed, confirming that a window of a control module displays P which is a qualified state, taking out a separation cup from the control module, connecting an injector to an output pipe which is communicated with a recovery cabin and the separation cup, and collecting the obtained marrow concentrated cells;
Optionally (I)
(6) Placing the separation cup and the control module on a separation base to transmit data and processing the data captured during centrifugation with a DataTrak software processing system;
and/or optionally, continuing the following steps to prepare a (plasma) supernatant concentrate:
(7) Separating supernatant (i.e. plasma layer) in the central compartment with a syringe, centrifuging for 20min at 2000g to remove cell debris, and filtering with 0.22 μm sterile filter membrane;
(8) Flushing the pipeline of a tangential flow ultrafiltration system (Shibi pure KR2i type tangential flow ultrafiltration system) with ultrapure water, installing a MidiKros filter of 100kD and 100cm2, and ultrafiltering and concentrating the filtrate obtained in the step (7) to 1/15 volume of the initial biological sample volume to obtain supernatant concentrate.
According to the method of the first aspect of the invention, the biological sample provided in the step (1) has a volume of 20-200 ml.
The method according to the first aspect of the invention, wherein in step (1) an additional 1ml sample is withdrawn for detection.
The method according to the first aspect of the present invention, wherein the anticoagulant used in step (1) is sodium citrate solution.
The method according to the first aspect of the invention, wherein the anticoagulant used in step (1) is a 3.2% sodium citrate solution.
The method according to the first aspect of the 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 invention, wherein the volume ratio of anticoagulant used in step (1) to biological sample is 1:12.
The method according to the first aspect of the invention, wherein the anticoagulant used in step (1) is formulated by: adding sodium citrate, histidine and phosphatidylcholine into proper water, heating to 60 ℃, stirring to dissolve, adding water to a full amount, filtering with a microporous filter membrane of 0.22 mu m, and performing hot-press sterilization at 121 ℃.
The method according to the first aspect of the invention further comprises the steps of: (6) The separation cup and control module were placed on a separation base to transmit data and the data captured during centrifugation was processed with a DataTrak software processing system.
Further, the second aspect of the present invention provides a bone marrow concentrated cell prepared by a method comprising the steps of:
(1) Providing biological sample bone marrow puncture liquid, and placing the biological sample bone marrow puncture liquid in a sterile bag containing an anticoagulant for standby;
(2) Removing the protective cap on the input tube of the automatic cell separation system, connecting the syringe to the input Guan Luer locking joint, passing through the thrombus filter at a slow and stable speed, transferring the anticoagulated biological sample into a disposable sterile separation cup, and shaking the mixed sample along a horizontal axis; the cell autosegregation system is a closed PXP segregation system, which 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;
(3) The disposable separating cup is placed in the control module, the state of the control module before centrifugation is displayed as 0, the separating cup/control module assembly is weighed, after balancing, the disposable separating cup is placed in the programmable centrifugal machine, and parameters of the centrifugal machine are set according to the following procedures:
(4) Starting the centrifugal machine to centrifuge, wherein the process is as follows:
4a) Stage P1 cells in biological samples are separated into the upper and lower three components in disposable separation cups by centrifugation density stratification: a red blood cell layer, a cell concentration layer, and a plasma layer;
4b) The P2 phase enables most of red blood cells to enter a red blood cell recovery cabin;
4c) Stage P3 further delaminates cells in the process chamber and stage P4 reduces centrifugal force to further remove erythrocytes;
4d) The cell concentrate layer and the plasma are further layered in the P5 stage, the centrifugal force is reduced in the P6 stage, the cell concentrate layer is transferred to a recovery chamber through a conveying pipe, and the plasma is remained in a central chamber;
(5) After centrifugation is completed, confirming that a window of a control module displays P which is a qualified state, taking out a separation cup from the control module, connecting an injector to an output pipe which is communicated with a recovery cabin and the separation cup, and collecting the obtained marrow concentrated cells;
Optionally (I)
(6) Placing the separation cup and the control module on a separation base to transmit data and processing the data captured during centrifugation with a DataTrak software processing system;
and/or optionally, continuing the following steps to prepare a (plasma) supernatant concentrate:
(7) Separating supernatant (i.e. plasma layer) in the central compartment with a syringe, centrifuging for 20min at 2000g to remove cell debris, and filtering with 0.22 μm sterile filter membrane;
(8) Flushing the pipeline of a tangential flow ultrafiltration system (Shibi pure KR2i type tangential flow ultrafiltration system) with ultrapure water, installing a MidiKros filter of 100kD and 100cm2, and ultrafiltering and concentrating the filtrate obtained in the step (7) to 1/15 volume of the initial biological sample volume to obtain supernatant concentrate.
According to the marrow concentrated cell of the second aspect of the present invention, the biological sample provided in the step (1) has a volume of 20-200 ml.
The bone marrow concentrated cells according to the second aspect of the invention, wherein 1ml of the sample is additionally withdrawn for detection in step (1).
According to the second aspect of the invention, the bone marrow concentrated cells wherein the anticoagulant used in the step (1) is sodium citrate solution.
According to the second aspect of the invention, the bone marrow concentrated cells wherein the anticoagulant used in step (1) is 3.2% sodium citrate solution.
The bone marrow concentrated cells according to the second aspect of the invention, wherein the anticoagulant used in the step (1) is 3.2% sodium citrate solution supplemented with 0.5mg/ml histidine and 0.1mg/ml phosphatidylcholine.
According to a second aspect of the invention, the bone marrow concentrated cells used in step (1) have a volume ratio of anticoagulant to biological sample of 1:12.
According to the second aspect of the invention, the preparation method of the anticoagulant used in the step (1) comprises the following steps: adding sodium citrate, histidine and phosphatidylcholine into proper water, heating to 60 ℃, stirring to dissolve, adding water to a full amount, filtering with a microporous filter membrane of 0.22 mu m, and performing hot-press sterilization at 121 ℃.
The bone marrow concentrated cell according to the second aspect of the present invention, further comprising the steps of: (6) The separation cup and control module were placed on a separation base to transmit data and the data captured during centrifugation was processed with a DataTrak software processing system.
Further, the third aspect of the present invention provides the use of bone marrow concentrated cells prepared by a method comprising the steps of:
(1) Providing biological sample bone marrow puncture liquid, and placing the biological sample bone marrow puncture liquid in a sterile bag containing an anticoagulant for standby;
(2) Removing the protective cap on the input tube of the automatic cell separation system, connecting the syringe to the input Guan Luer locking joint, passing through the thrombus filter at a slow and stable speed, transferring the anticoagulated biological sample into a disposable sterile separation cup, and shaking the mixed sample along a horizontal axis; the cell autosegregation system is a closed PXP segregation system, which 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;
(3) The disposable separating cup is placed in the control module, the state of the control module before centrifugation is displayed as 0, the separating cup/control module assembly is weighed, after balancing, the disposable separating cup is placed in the programmable centrifugal machine, and parameters of the centrifugal machine are set according to the following procedures:
(4) Starting the centrifugal machine to centrifuge, wherein the process is as follows:
4a) Stage P1 cells in biological samples are separated into the upper and lower three components in disposable separation cups by centrifugation density stratification: a red blood cell layer, a cell concentration layer, and a plasma layer;
4b) The P2 phase enables most of red blood cells to enter a red blood cell recovery cabin;
4c) Stage P3 further delaminates cells in the process chamber and stage P4 reduces centrifugal force to further remove erythrocytes;
4d) The cell concentrate layer and the plasma are further layered in the P5 stage, the centrifugal force is reduced in the P6 stage, the cell concentrate layer is transferred to a recovery chamber through a conveying pipe, and the plasma is remained in a central chamber;
(5) After centrifugation is completed, confirming that a window of a control module displays P which is a qualified state, taking out a separation cup from the control module, connecting an injector to an output pipe which is communicated with a recovery cabin and the separation cup, and collecting the obtained marrow concentrated cells;
Optionally (I)
(6) Placing the separation cup and the control module on a separation base to transmit data and processing the data captured during centrifugation with a DataTrak software processing system;
and/or optionally, continuing the following steps to prepare a (plasma) supernatant concentrate:
(7) Separating supernatant (i.e. plasma layer) in the central compartment with a syringe, centrifuging for 20min at 2000g to remove cell debris, and filtering with 0.22 μm sterile filter membrane;
(8) Flushing the pipeline of a tangential flow ultrafiltration system (Shibi pure KR2i type tangential flow ultrafiltration system) with ultrapure water, installing a MidiKros filter of 100kD and 100cm2, and ultrafiltering and concentrating the filtrate obtained in the step (7) to 1/15 volume of the initial biological sample volume to obtain supernatant concentrate.
According to the use of the third aspect of the present invention, the biological sample provided in the step (1) has a volume of 20-200 ml.
The use according to the third aspect of the invention wherein in step (1) an additional 1ml sample is withdrawn for detection.
The use according to the third aspect of the invention wherein the anticoagulant used in step (1) is sodium citrate solution.
The use according to the third aspect of the invention wherein the anticoagulant used in step (1) is a 3.2% sodium citrate solution.
The use according to the third aspect of the 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 use according to the third aspect of the invention wherein the volume ratio of anticoagulant used in step (1) to biological sample is 1:12.
The use according to the third aspect of the present invention, wherein the anticoagulant used in step (1) is formulated by: adding sodium citrate, histidine and phosphatidylcholine into proper water, heating to 60 ℃, stirring to dissolve, adding water to a full amount, filtering with a microporous filter membrane of 0.22 mu m, and performing hot-press sterilization at 121 ℃.
The use according to the third aspect of the invention further comprises the steps of: (6) The separation cup and control module were placed on a separation base to transmit data and the data captured during centrifugation was processed with a DataTrak software processing system.
Further, a fourth aspect of the present invention provides a method of treating premature ovarian failure, the method comprising administering to a subject in need thereof a cell therapeutic agent comprising a therapeutically effective amount of bone marrow concentrated cells prepared by a method comprising the steps of:
(1) Providing biological sample bone marrow puncture liquid, and placing the biological sample bone marrow puncture liquid in a sterile bag containing an anticoagulant for standby;
(2) Removing the protective cap on the input tube of the automatic cell separation system, connecting the syringe to the input Guan Luer locking joint, passing through the thrombus filter at a slow and stable speed, transferring the anticoagulated biological sample into a disposable sterile separation cup, and shaking the mixed sample along a horizontal axis; the cell autosegregation system is a closed PXP segregation system, which 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;
(3) The disposable separating cup is placed in the control module, the state of the control module before centrifugation is displayed as 0, the separating cup/control module assembly is weighed, after balancing, the disposable separating cup is placed in the programmable centrifugal machine, and parameters of the centrifugal machine are set according to the following procedures:
(4) Starting the centrifugal machine to centrifuge, wherein the process is as follows:
4a) Stage P1 cells in biological samples are separated into the upper and lower three components in disposable separation cups by centrifugation density stratification: a red blood cell layer, a cell concentration layer, and a plasma layer;
4b) The P2 phase enables most of red blood cells to enter a red blood cell recovery cabin;
4c) Stage P3 further delaminates cells in the process chamber and stage P4 reduces centrifugal force to further remove erythrocytes;
4d) The cell concentrate layer and the plasma are further layered in the P5 stage, the centrifugal force is reduced in the P6 stage, the cell concentrate layer is transferred to a recovery chamber through a conveying pipe, and the plasma is remained in a central chamber;
(5) After centrifugation is completed, confirming that a window of a control module displays P which is a qualified state, taking out a separation cup from the control module, connecting an injector to an output pipe which is communicated with a recovery cabin and the separation cup, and collecting the obtained marrow concentrated cells;
Optionally (I)
(6) Placing the separation cup and the control module on a separation base to transmit data and processing the data captured during centrifugation with a DataTrak software processing system;
and/or optionally, continuing the following steps to prepare a (plasma) supernatant concentrate:
(7) Separating supernatant (i.e. plasma layer) in the central compartment with a syringe, centrifuging for 20min at 2000g to remove cell debris, and filtering with 0.22 μm sterile filter membrane;
(8) Flushing the pipeline of a tangential flow ultrafiltration system (Shibi pure KR2i type tangential flow ultrafiltration system) with ultrapure water, installing a MidiKros filter of 100kD and 100cm2, and ultrafiltering and concentrating the filtrate obtained in the step (7) to 1/15 volume of the initial biological sample volume to obtain supernatant concentrate.
According to the method of the fourth aspect of the present invention, the biological sample provided in the step (1) has a volume of 20 to 200ml.
The method according to the fourth aspect of the present invention, wherein in step (1) 1ml of the sample is additionally withdrawn for detection.
The method according to the fourth aspect of the present invention, wherein the anticoagulant used in step (1) is sodium citrate solution.
The method according to the fourth aspect of the invention, wherein the anticoagulant used in step (1) is 3.2% sodium citrate solution.
The method according to the fourth aspect of the present invention, wherein the anticoagulant used in step (1) is 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 invention, wherein the volume ratio of anticoagulant used in step (1) to biological sample is 1:12.
The method according to the fourth aspect of the invention, wherein the anticoagulant used in step (1) is formulated by: adding sodium citrate, histidine and phosphatidylcholine into proper water, heating to 60 ℃, stirring to dissolve, adding water to a full amount, filtering with a microporous filter membrane of 0.22 mu m, and performing hot-press sterilization at 121 ℃.
The method according to the fourth aspect of the present invention further comprises the steps of: (6) The separation cup and control module were placed on a separation base to transmit data and the data captured during centrifugation was processed with a DataTrak software processing system.
Further, a fifth aspect of the present invention provides a cell composition made from bone marrow concentrated cells, comprising 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 concentrated cells to granulocyte macrophage stimulating factor is that the concentrated cells are 5x10≡6 cells by CD45 + cell number: 10-15 ng granulocyte macrophage stimulating factor; for example, the ratio is that the concentrated cells count 5×10≡6 cells in terms of CD45 + cells: 12.5ng granulocyte macrophage stimulating factor.
The cell composition according to the fifth aspect of the present invention, wherein the excipient is physiological saline or 5% dextrose solution.
According to the cell composition of 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 10-15 ng/ml, for example, 12.5ng/ml.
The cell composition according to the fifth aspect of the invention, wherein the granulocyte macrophage-stimulating factor is a human granulocyte macrophage-stimulating factor.
The cell composition according to the fifth aspect of the 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 invention, wherein the concentrated cells are as described in any one of the embodiments of the second aspect of the 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 granulocyte macrophage stimulating factor to glutamine and sodium selenite in the composition is 12.5ng: 0.1-0.5 mg: 5-20 mug.
The cell composition according to the fifth aspect of the present invention, further comprising glutamine and sodium selenite, wherein the weight ratio of granulocyte macrophage stimulating factor to glutamine and 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 granulocyte macrophage stimulating factor to glutamine and sodium selenite in the composition is 12.5ng:0.2mg:15 mug.
The cell composition according to the fifth aspect of the invention, comprising: concentrated cells with the cell number of 4-6 x 10-6 of CD45 +, gmCSF with the cell number of 10-15 ng, glutamine with the cell number of 0.1-0.5 mg, sodium selenite with the cell number of 5-20 mug, and physiological saline with the cell number of 5-20 mug with the cell number of 2-6 x 10-6.
The cell composition according to the fifth aspect of the invention, comprising: concentrated cells with the cell number of 4-6 x 10-6 of CD45 +, gmCSF with the cell number of 10-15 ng, glutamine with the cell number of 0.2-0.3 mg, sodium selenite with the cell number of 10-15 mug and a proper amount of physiological saline to 1mL.
The cell composition according to the fifth aspect of the invention, comprising: concentrated cells with the cell number of 5x10 times of CD45 +, gmCSF with the cell number of 12.5ng, glutamine with the cell number of 0.2 to 0.3mg, sodium selenite with the cell number of 10 to 15 mug and physiological saline with the cell number of 10 times of 5g to 1mL.
The cell composition according to the fifth aspect of the invention, comprising: concentrated cells with the cell number of CD45 + x10 x 6, gmCSF with the cell number of 12.5ng, 0.2mg of glutamine, 15 mug 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 from bone marrow concentrated cells, comprising 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 concentrated cells to granulocyte macrophage stimulating factor is 4x10≡6 cells by CD45 + cell number: 10-15 ng granulocyte macrophage stimulating factor; for example, the ratio is that the concentrated cells count 4x10≡6 cells by CD45 + cells: 12.5ng granulocyte macrophage stimulating factor.
The cell composition according to the sixth aspect of the invention, wherein the ratio of concentrated cells to supernatant concentrate is 4x10≡6 cells by CD45 + cell number: 75-125 mu L of supernatant concentrate; for example, the ratio is that the concentrated cells count 4x10≡6 cells by CD45 + cells: 100 [ mu ] L supernatant concentrate.
The cell composition according to the sixth aspect of the present invention, wherein the excipient is physiological saline or 5% dextrose 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 10-15 ng/ml, for example 12.5ng/ml.
The cell composition according to the sixth aspect of the invention, wherein the granulocyte macrophage-stimulating factor is a human granulocyte macrophage-stimulating factor.
The cell composition according to the sixth aspect of the 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 invention, wherein the concentrated cells and supernatant concentrate are prepared according to the method of any one of the embodiments of the first aspect of the 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 granulocyte macrophage stimulating factor to glutamine and sodium selenite in the composition is 12.5ng: 0.1-0.5 mg: 5-20 mug.
The cell composition according to the sixth aspect of the present invention, further comprising glutamine and sodium selenite, wherein the weight ratio of granulocyte macrophage stimulating factor to glutamine and 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 granulocyte macrophage stimulating factor to glutamine and sodium selenite in the composition is 12.5ng:0.2mg:15 mug.
The cell composition according to the sixth aspect of the invention, comprising: CD45 + cells 3-5 x10 times 6 concentrated cells, 10-15 ng gmCSF, 75-125 mu L supernatant concentrate, 0.1-0.5 mg glutamine, 5-20 mu g sodium selenite and a proper amount of physiological saline to 1mL.
The cell composition according to the sixth aspect of the invention, comprising: CD45 + cells 3-5 x10 times 6 concentrated cells, 10-15 ng gmCSF, 80-120 mu L supernatant concentrate, 0.2-0.3 mg glutamine, 10-15 mu g sodium selenite and a proper amount of physiological saline to 1mL.
The cell composition according to the sixth aspect of the invention, comprising: concentrated cells with the cell number of 4x10 x 6 of CD45 +, gmCSF with the cell number of 12.5ng, 100 mu L supernatant concentrate, 0.2-0.3 mg glutamine, 10-15 mu g sodium selenite and a proper amount of physiological saline to 1mL.
The cell composition according to the sixth aspect of the invention, comprising: CD45 + cells 4x10 x 6 concentrated cells, 12.5ng gmCSF, 100 mu L supernatant concentrate, 0.2mg glutamine, 15 mu g sodium selenite, and physiological saline to 1mL.
The unexpected finding that a cell composition prepared by combining a supernatant concentrate obtained by the present invention with concentrated cells, gmCSF, after preparing concentrated cells using PXP system, that is, a supernatant concentrate obtained by treating a plasma fraction with tangential flow ultrafiltration system, has excellent effects of treating premature ovarian failure, such as a desired effect achieved by using a lower dose of concentrated cells, was unexpected in the prior art.
Further, the seventh aspect of the invention provides the use of a cell composition according to any of the fifth or sixth aspects of the invention in the manufacture of a medicament for the treatment of 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, comprising the step of mixing prescribed amounts of concentrated cells, granulocyte macrophage stimulating factor, supernatant concentrate, glutamine, sodium selenite, and optional excipients to formulate a sterile formulation.
As used herein, the phrase "concentrated cells with a CD45 + cell number of 5x 10A 6" refers to 5 times 10 to the 6 th power, and the remaining similar expressions have the same meaning.
The invention uses PXP automatic cell rapid processing system, applies automatic separation and concentration closed system, and safely, efficiently and simply obtains BMAC, thus laying foundation for clinical application of BMAC for treating POF patients. The invention provides a method for obtaining marrow concentrated cells by rapid separation, which uses a closed automatic cell separation system to separate marrow puncture liquid from human to obtain marrow concentrated cells. The marrow concentrated cells obtained by the invention can promote angiogenesis and follicular development as an active ingredient for treating ovarian injury, thereby improving the ovarian function.
Prior studies have demonstrated that contaminating erythrocytes are associated with a decrease in stem/progenitor cell function, and that erythrocyte contaminating cell concentrates are believed to decrease the effectiveness of cell therapy. To better exploit the potential of cell therapies, the industry is pressing to need new treatment systems that can increase target cell purity and contaminating erythrocyte removal rates.
The cell separation system used in the experiments of the present invention was PXP% autosegregation system, and its manufacturer was ThermoGenesis company in the united states. The innovative PXP system addresses many of the shortcomings of the current systems on the market. The PXP system enables clinicians to quickly obtain very high stem and progenitor cell recovery with little red blood cell contamination, typically less than 5% of the initial sample.
The PXP system addresses the need for rapid, efficient, sterile cell handling in an operating room environment by clinical institutions developing and using cell therapy techniques, and is a fairly efficient Point-of-Care product. As a leading-edge automatic cell rapid processing system, the PXP system does not need a cell separation medium or a precipitant, can process a plurality of samples simultaneously, has high recovery rate of MNC and CD34+ and CD45+ cells, can enable a clinician to extract stem cells from biological samples (such as bone marrow) within 30 minutes in a hospital surgical center or a clinic, and has the removal rate of red blood cells of more than 90 percent. The PXP system is also equipped with proprietary DataTrak software to track the captured data to facilitate GMP process control and reporting information to the customer.
The invention uses PXP system for processing bone marrow extract, can automatically process bone marrow cells in real time, ensures recovery rate of mononuclear cells (MNC), can process multiple bone marrow units at the same time, and does not need cell separation medium or precipitant.
The PXP system of the present invention is useful in specific assays, and advantages include, but are not limited to: stable and excellent MNC (mononuclear cells) and CD34+, CD45+ cell recovery rate, rapid processing of bone marrow samples within 30 minutes, red blood cell removal rate of more than 95%, automatic closed sterile system, rapid and accurate data tracking and document recording, and uploading of sample processing data to a computer through DataTrak software, and providing production record and report information meeting GMP requirements.
Bone marrow concentrates are widely used in the clinic of related subjects such as orthopedics, severe lower limb ischemia, etc., because they are rich in a large amount of stem cells and growth factors. The bone marrow concentrate is a nucleated cell concentrate obtained by centrifuging and separating bone marrow, and is rich in mesenchymal stem cells. As is well known, the world wide marrow concentrate market application area can be divided into bone surgery, wound healing, chronic pain, peripheral vascular disease, dermatological lesions, and others. Due to the exacerbation of global aging and the rising incidence of osteoarthritis, future use of bone disorders will dominate the overall bone marrow concentrate market. With the explosive development of the industry, advances in technology, and increase in available revenue, the future dermatological field is expected to grow at the highest complex annual growth rate (6.0%). The regenerative capacity of fibroblasts and keratinocytes in human skin has been used to develop cell therapies, while scientists are studying the plasticity of bone marrow derived extradermal cells in skin regeneration, all of which would accelerate the application of cell therapies in the dermatological field. Bone marrow concentrates are considered "platinum standards" for bone substitute materials. It has been found that cells, precursor cells, growth factors, platelets, etc. in bone marrow concentrates play an important role in bone regeneration. Endothelial cells in, for example, bone marrow concentrates play an important role in angiogenesis; the bone marrow concentrate contains a large amount of growth factors which can act on osteoblasts and osteoclasts, regulate the bone reconstruction process and promote new bone formation; platelets in the bone marrow concentrate can provide a faster and more efficient bone regeneration environment for mesenchymal stem cells.
According to the investigation results of the united nations and world health organization, more than 4 hundred million people worldwide suffer from arthritis, and the development of the orthopedics field is driven by such a huge patient population background. 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 to 5 to 6 weeks required for surgical treatment. The reduction in healing time is one of the factors driving the development of the bone marrow concentrate market. In recent years, advances in bone marrow concentrate processing and preparation technology have provided hospitals and clinics with effective options for improving the clinical outcome 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 to 40 years of research and development. The advent of bone marrow concentrates, platelet rich plasma and stem cell derivatives is becoming a new trend in future medical treatments. With the development of personalized medicine, the clinical requirement of cell therapy is more and more remarkable worldwide, and the release trend of the clinical application of the cell therapy is also more and more obvious. With the increasing coverage of clinical applications of cell therapy, the demands of medical institutions for new generation of cell processing and preparation solutions based on automated technology will increase increasingly, and these technology platforms will also have an important impact on the success of cell therapies.
The present invention achieves satisfactory results by using PXP systems for cell separation.
GM-CSF can be human granulocyte macrophage stimulating factor or recombinant granulocyte macrophage stimulating factor, which has been loaded into several versions of the chinese pharmacopoeia and has been approved for clinical use with a number of brands of products. In the present invention, GM-CSF used in the test is a commercially available recombinant human granulocyte macrophage stimulating factor (S19991012, specification 750000 IU/75. Mu.g, 10 IU/ng) product, and the composition may be diluted with 0.9% sodium chloride injection to an appropriate concentration if necessary. GM-CSF (Granulocyte-macrophage Colony Stimulating Factor, granulocyte-macrophage colony stimulating factor, or granulocyte-macrophage stimulating factor), which acts on hematopoietic progenitor cells to promote their proliferation and differentiation, has the important effects of stimulating the maturation of granulocytes and mononuclear macrophages, promoting the release of mature cells to peripheral blood, and can promote multiple functions of macrophages and eosinophils. GM-CSF is a drug clinically used for leucocyte or granulocytopenia caused by various causes, and it excites hematopoietic function of bone marrow, stimulates proliferation of granulocytes, monocytes, T cells, and promotes maturation of monocytes and granulocytes. In addition, GM-CSF also overcomes the myelotoxicity caused by radiotherapy and chemotherapy, shortens the time of neutropenia in tumor chemotherapy, and makes patients easily endure chemotherapy. GM-CSF can enhance the anti-tumor and anti-infective immunity of the body by enhancing the functions of monocytes, granulocytes, eosinophils and macrophages.
The invention obtains positive effects by combining marrow concentrated cells obtained by PXP system separation with GM-CSF and verifying by using an premature ovarian failure model.
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. Those skilled in the art will appreciate that various changes and modifications can be made to the invention without departing from the spirit and scope thereof. The present invention generally and/or specifically describes the materials used in the test as well as the test methods. Although many materials and methods of operation are known in the art for accomplishing the objectives of the present invention, the present invention will be described in as much detail herein.
In the present invention, the cell separation system used in the specific experiments is PXP% autosegregation system, which may be also simply referred to as PXP system, PXP cell autosegregation system, PXP autosegregation system, PXP autosegregation system, etc. in the present invention, unless otherwise specified. The model number of the PXP system used in the experiment is 80065-01, the supplier is Shenzhen Boya perception medical science and technology Co., ltd, and the manufacturer is American ThermoGenesis company. In the present invention, the term "bone marrow concentrated cells" may also be referred to as "bone marrow concentrated cell preparation", if not in particular context, both have the same meaning.
Example 1: rapid isolation preparation of bone marrow concentrated cells (BMAC)
(1) Providing a biological sample bone marrow puncture fluid (capable of processing samples with the volume of 20-200 ml), placing the biological sample bone marrow puncture fluid in a sterile 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 added in a supplementary way, 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 proper water, heating to 60 ℃, stirring to dissolve, adding water to a full amount, filtering with a microporous filter membrane of 0.22 mu m, and performing hot-press sterilization at 121 ℃ to obtain the sodium citrate;
(2) Removing the protective cap on the input tube of the automatic cell separation system, connecting the syringe to the input Guan Luer locking joint, passing through the thrombus filter at a slow and stable speed, transferring the anticoagulated biological sample into a disposable sterile separation cup, and shaking the mixed sample along a horizontal axis; the cell autosegregation system is a closed PXP segregation system, which 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;
(3) The disposable separating cup is placed in the control module, the state of the control module before centrifugation is displayed as 0, the separating cup/control module assembly is weighed, after balancing, the disposable separating cup is placed in the programmable centrifugal machine, and parameters of the centrifugal machine are set according to the following procedures:
(4) Starting the centrifugal machine to centrifuge, wherein the process is as follows:
4a) Stage P1 cells in biological samples are separated into the upper and lower three components in disposable separation cups by centrifugation density stratification: a red blood cell layer, a cell concentration layer, and a plasma layer;
4b) The P2 phase enables most of red blood cells to enter a red blood cell recovery cabin;
4c) Stage P3 further delaminates cells in the process chamber and stage P4 reduces centrifugal force to further remove erythrocytes;
4d) The cell concentrate layer and the plasma are further layered in the P5 stage, the centrifugal force is reduced in the P6 stage, the cell concentrate layer is transferred to a recovery chamber through a conveying pipe, and the plasma is remained in a central chamber;
(5) After centrifugation is completed, confirming that a window of a control module displays P which is a qualified state, taking out a separation cup from the control module, connecting an injector to an output pipe which is communicated with a recovery cabin and the separation cup, and collecting the obtained marrow concentrated cells;
(6) The separation cup and control module were placed on a separation base to transmit data and the data captured during centrifugation was processed with a DataTrak software processing system.
The following steps were continued to prepare a (plasma) supernatant concentrate:
(7) Separating supernatant (i.e. plasma layer) in the central compartment with a syringe, centrifuging for 20min at 2000g to remove cell debris, and filtering with 0.22 μm sterile filter membrane;
(8) Flushing the pipeline of a tangential flow ultrafiltration system (Shibi pure KR2i type tangential flow ultrafiltration system) with ultrapure water, installing a MidiKros filter of 100kD and 100cm2, and ultrafiltering and concentrating the filtrate obtained in the step (7) to 1/15 volume of the initial biological sample volume to obtain supernatant concentrate.
In the present invention, unless otherwise specified, the tangential flow ultrafiltration system used is a Shi must pure KR2i type tangential flow ultrafiltration system; of course other brands of tangential flow ultrafiltration systems may be used.
This example 1 was prepared by cell separation and supernatant concentration of 10 parts of collected human bone marrow aspirate (obtained by methods well known in the art, for example, the biological samples of the present invention were obtained according to the method described in Song Jianhua (Song Jianhua, et al, autologous bone marrow concentrated stem cells for treatment of femoral neck bone fracture, journal of orthopaedics, 2008,14 (8): 467)), and 10 parts of bone marrow concentrated cells (BMAC) and 10 parts of supernatant concentrate were each labeled as No.1 to No.10.
Test example 1: analysis of MNC recovery in Bone Marrow Aspirate (BMA) and bone marrow concentrated cells (BMAC)
Cell separation was performed on 10 parts of the collected human bone marrow aspirate (volume before separation was in the range of 79 to 97 ml) using the method of example 1; next, referring to the method described in chinese patent application No. 2021116067849, cell detection was performed on each of the isolated fractions, results for 10 samples: the mean value of the input 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%, the mean value of the recovery rate of mononuclear cells (MNC) is 97.2%, and the average concentration of MNC is improved by 6.93 times. For example, the result of a certain portion of the puncture fluid (No. 2): volume before separation = 91.34ml, final volume = 12.97ml, erythrocyte removal = 98.3%, MNC recovery = 97.6%, MNC fold concentration = 7.04.
The results show that the use of PXP dynamic separation system can enrich MNC in bone marrow in a manner that is simple to operate, short in time consumption, not easy to pollute, and good in result repeatability, and simultaneously remove most of red blood cells.
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 for 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 of 10 bone marrow aspirate samples (BMA) prior to PXP treatments = 88.17 ±3.14%, cell viability of 10 bone marrow aspirate samples (BMAC) after PXP treatments = 97.42±1.24%, for example, results for a certain aspirate sample (No. 2): BMA cell viability = 89.64%, BMAC cell viability = 98.27%. The cell viability in the BMAC samples was shown to be significantly higher than the BMA cell viability.
Test example 3: CD45+, CD34+ cell count in BMA and BMAC samples
The 10 samples of test example 1 were examined. The numbers of cd45+ and cd34+ cells in all pre-and post-treatment bone marrow samples were analyzed on an FC500 flow cytometer using 7-AAD staining, as a result:
In terms of CD45+ viable cell number, a marrow aspirate sample (BMA) = (15.08+ -2.84) x10≡6/mL, a marrow concentrated cell sample (BMAC) = (106.41 + -6.94) x10≡6/mL, an increase of 7.1-fold;
Cd34+ viable cell count, bone marrow aspirate sample (BMA) = (127.42 ±10.36) x 10++3/mL, bone marrow concentrated cell sample (BMAC) = (882.63 ±16.41) x 10++3/mL, 6.9 fold increase.
Test example 4: marrow sample sterility test
10 Cell concentrate samples and 10 supernatant concentrate samples according to test example 1 were examined. Sterile testing was performed using gram stain, smears of BMA, BMAC samples were prepared, fixed with methanol, post-stain testing, results: no microorganisms were seen in the gram stain smears of the 10 BMA samples, no microorganisms were seen in the gram stain smears of the 10 BMAC samples, and no microorganisms were seen in the gram stain smears of the 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: cell level efficacy study
BMAC prepared by using 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., to promote neovascularization and endothelial cell migration.
This test example was examined on 10 samples of test example 1, and the biological efficacy of stem cells of BMAC was evaluated by CFU colony forming ability, and cytokine enriched in BMAC was quantitatively detected by ELISA.
5.1 Stem cell biological efficacy-CFU colony Forming experiment
Bone marrow stem cell biological efficacy (Potency Assays), cell stem properties in BMAC-mixed cells were characterized by identifying and analyzing the colony forming ability of progenitor/stem cells through an in vitro CFU colony forming experiment. CFU-F (stromal progenitor cells) were used to analyze the efficacy of various stem cells in BMA and BMAC samples using CFU-H (hematopoietic progenitor/stem cells), resulting in:
CFU-H (hematopoietic progenitor/stem cells), bone marrow aspirate sample (BMA) = (34.3+ -4.1) x 10++3/mL, bone marrow concentrated cell sample (BMAC) = (213.6+ -19.3) x 10++3/mL, 6.2 fold increase;
CFU-F (stromal progenitor cells), bone Marrow Aspirate (BMA) = (41.7+ -5.3) x 10++3/mL, bone marrow concentrate (BMAC) = (302.4+ -28.1) x 10++3/mL, 7.3 fold increase.
The results indicate that the PXP system is able to efficiently enrich for bone marrow stem cells while maintaining the biological efficacy of bone marrow stem cells.
5.2 Quantitative cytokine analysis
The PXP system produced BMAC injections containing various cytokines. Enzyme-linked immunosorbent assay (ELISA) quantitative assay of transforming growth factor- β (TGF- β), vascular Endothelial Growth Factor (VEGF) and Hepatocyte Growth Factor (HGF) levels in BMA and BMAC samples, results:
TGF-beta of BMA and BMAC are 34.1.+ -. 3.1pg/ml and 218.3.+ -. 17.2pg/ml, respectively,
VEGF for BMA and BMAC was 17.4+ -2.6 pg/ml and 151.6+ -13.4 pg/ml, respectively,
HGF of BMA and BMAC were 194.3.+ -. 24.2pg/ml and 1312.4.+ -. 44.7pg/ml, respectively.
The results showed that TGF- β, VEGF and HGF in BMAC were significantly higher than TGF- β, VEGF and HGF levels in BMA (p < 0.01), indicating that PXP system was able to efficiently concentrate enriched cell growth factors.
Test example 6: effectiveness of BMAC in treating Premature Ovarian Failure (POF)
In chinese patent application No. 2021116067849, a study of effectiveness in treating Premature Ovarian Failure (POF) was performed using BMAC concentrate. This test example uses the BMAC concentrate prepared above in combination with GM-CSF (also referred to herein as gmCSF) for the efficacy study of POF.
(1) Establishment of POF mouse model
Female C57BL/6 mice of 8 weeks old were given an intraperitoneal injection of 50mg/kg/day Cyclophosphamide (CTX), followed by an intraperitoneal injection for 15 days at the same time daily, and an Premature Ovarian Failure (POF) mouse model was established. The control group did not perform any treatment. BMAC cell transplantation treatment was performed after the completion of POF molding, and model animals were randomly grouped.
(2) The reserve function of the ovaries is assessed by means of hormone levels, follicular numbers and fertility tests.
A. Hormone levels
Grouping animals:
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 given 200. Mu.l of BMAC concentrated cell composition (200. Mu.l of BMAC concentrated cell composition was sample No.2 of example 1) by intravenous injection into each animal tail on day1 after POF modeling, and diluted with sterile physiological saline to prepare a solution having a concentration of 4X10≡6 cells/200. Mu.l in terms of CD45 + cells;
BMAC + gmCSF treated mice, 200 μl of BMAC concentrate gmCSF composition was given by tail vein injection to each animal on day 1 after POF modeling, respectively;
BMAC+supernatant concentrate+ gmCSF treatment group mice were given 200 μl of BMAC+supernatant concentrate+ gmCSF composition by tail intravenous injection on day 1 after POF modeling, respectively;
The POF model group is injected with an equal volume of physiological saline; the control group was not treated by injection;
Each group was equally given diet and water after cell transplantation.
Note that: the BMAC concentrate gmCSF composition (which may be abbreviated as BMAC-gmCSF composition) administered in the BMAC + gmCSF treatment group above comprises per 200 μl: the No.2 concentrated solution sample obtained in the example 1 is properly prepared into a composition by fixing the volume of gmCSF with the cell number of CD45 + of 1x 10-6 and 2.5ng and sterile physiological saline;
The bmac+supernatant concentrate + gmCSF composition administered to the bmac+supernatant concentrate + gmCSF treatment group above, comprising per 200 μl: a proper amount of No.2 concentrated solution sample obtained in the embodiment 1 is calculated as CD45 + cells number to be 0.8x10ζ6 and 2.5ng gmCSF, no.2 supernatant concentrated solution obtained in the embodiment 1 is 20 [ mu ] L, and sterile normal saline is used for constant volume to obtain a composition;
the composition is stored at the temperature of 2-4 ℃ after being prepared and is injected and administrated within 4 hours, and gmCSF is a commercially available freeze-dried powder injection.
10 Mice were collected from the orbit of each group 14 days and 28 days after BMAC transplantation, and serum was isolated and stored at-20 ℃. The results of the enzyme-linked immunosorbent assay (ELISA) for the analysis of the levels of estradiol (E2) and Follicle Stimulating Hormone (FSH) (specific methods are described in the Japanese paper (Xiang Li, et al, human placental mesenchymal stem cell transplantation by decreasing the expression of superoxide dismutase 1 and uncoupling protein-2 to increase ovarian function, journal of Chinese reproduction and contraception, 2018, 02)) are shown in the following table.
The results show that: compared with the POF model group, the serum level of E2 in the mice of BMAC group was increased at 28d, and the FSH level was decreased, all with significant differences (P < 0.05); in addition, it has been found that by using a 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. follicular count of mouse ovarian tissue
Each group of 10 mice was sacrificed 28 days after BMAC transplantation, the left ovarian tissue of the mice was fixed in 4% paraformaldehyde, the fixed tissue was subjected to serial alcohol dehydration, xylene transparency, paraffin embedding, serial sections, section thickness 5um, HE staining, and microscopic observation.
The results show that: compared with a control group, the number of primary follicles, secondary follicles and mature follicles of mice in the POF model group is obviously reduced, and the number of closed follicles is obviously increased; the number of follicles at each stage is recovered to different degrees 28 days after BMAC treatment, so that the growth of granulosa cells is increased, the apoptosis is reduced, the morphology of ovarian epithelial cells is stable, the number of primary follicles, secondary follicles and mature follicles is obviously increased, and the number of closed follicles is obviously reduced. The follicular counts at each stage 28 days post BMAC transplantation were significantly different from the POF group, and the specific results are shown in the table below.
C. observation of fertility of mice
On day 28 post BMAC transplantation, male and female mice were treated at 2:1 proportion cage-matched feeding, counting the fertility rate of mice, comparing the litter size of the mice, observing the repair effect of BMAC transplantation on the ovary function of the mice, and showing that the BMAC group is significantly different from the POF group. The results of the mice litter size comparison are shown in the following table.
According to the results, the BMAC transplantation treatment can obviously improve the reserve function of damaged ovaries of POF mice, increase the number of follicles, increase the oestrogen, recover the fertility of the mice, and provide experimental basis for applying BMAC to the clinical treatment of POF.
Test example 7: BMAC-gmCSF compositions
The BMAC-gmCSF composition used in test example 6 above was administered by injection as soon as possible after formulation, and the inventors found that the composition in a liquid state showed a tendency of decreasing the biological activity of GM-CSF after 12 hours and 24 hours of standing at 4 ℃ and that this tendency of decreasing the 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: using 5 BMACs of Nos. 1 to 5 obtained in example 1, 5 compositions in a liquid state were prepared according to the following formulations, and were designated as compositions aNo.1 to aNo.5, respectively: BMAC comprising CD45 + cells 5x10≡6, gmCSF of 12.5ng (i.e. 125 IU), sterile saline in an amount of 1mL;
Formula a1: using the 5 BMACs of No.1 to No.5 obtained in example 1 and the 5 supernatant concentrates, 5 compositions in liquid state were prepared as follows, and were designated as compositions a1No.1 to a1No.5, respectively: BMAC comprising CD45 + cells 4x10≡6, gmCSF of 12.5ng (i.e. 125 IU), 100 [ mu ] L of supernatant concentrate, and a proper amount of sterile physiological saline to 1mL;
formula b: preparing a composition in a liquid state according to the formula a without adding BMAC, and marking the composition as a composition b;
formula b1: the supernatant concentrate of No.1 was used in accordance with the above formulation a1, but BMAC was not added, to prepare a composition in a liquid state, designated as composition b1;
Formula c: using 5 BMACs of nos. 1 to 5 obtained in example 1, 5 compositions in liquid state were prepared according to the above formulation a, but with glutamine (final concentration 0.2 mg/ml) and sodium selenite (final concentration 15 μg/ml) added, and were designated as composition cn 1 to composition cn 5, respectively;
formula c1: using 5 BMACs of No.1 to No.5 obtained in example 1 and 5 supernatant concentrates, 5 compositions in a liquid state were prepared as compositions c1No.1 to composition c1No.5, respectively, according to the above-mentioned formulation a1, but with the addition of glutamine (to a final concentration of 0.2 mg/ml) and sodium selenite (to a final concentration of 15. Mu.g/ml);
formula d: using 5 BMACs No.1 to No.5 obtained in example 1, 5 compositions in liquid state were prepared according to the above-mentioned formulation a with glutamine (to a final concentration of 0.2 mg/ml) and were designated as compositions dNO.1 to dNO.5, respectively;
Formula d1: using 5 BMACs of No.1 to No.5 obtained in example 1 and 5 supernatant concentrates, 5 compositions in a liquid state were prepared according to the above-mentioned formulation a1, except that glutamine (to a final concentration of 0.2 mg/ml) was also added, and they were designated as compositions d1No.1 to d1No.5, respectively; formula e: using 5 BMACs of nos. 1 to 5 obtained in example 1, 5 compositions in liquid state were prepared according to the above formulation a but with sodium selenite (to a final concentration of 15 μg/ml), respectively designated composition No.1 to composition No.5;
Formula e1: using 5 BMACs of No.1 to No.5 obtained in example 1 and 5 supernatant concentrates, 5 compositions in a liquid state were prepared according to the above-mentioned formulation a1, except that sodium selenite (to a final concentration of 15. Mu.g/ml) was also added, and they were designated as compositions E1No.1 to E1No.5, respectively.
The preparation method of the above-mentioned various compositions is a conventional method well known to those skilled in the art, for example, under aseptic operation conditions, gmCSF in the form of a prescribed amount of lyophilized powder and optionally glutamine and optionally sodium selenite are quantitatively dissolved to a prescribed volume with sterile physiological saline, BMAC is diluted with sterile physiological saline to a proper concentration in terms of CD45 + cells, the two solutions are diluted with sterile physiological saline to a prescribed concentration in a formulation ratio, and the mixture is bottled in glass.
Placing each composition of the 10 formulas at 4 ℃, sampling at 0h, 12h and 24h respectively, and measuring the biological activity (IU/ml) of each composition at a specified time according to the biological activity measurement method of the recombinant human granulocyte macrophage stimulating factor of 3526 of the four appendices of the 2015 edition of Chinese pharmacopoeia; for a composition, the percent obtained by dividing the biological activity for 12h or 24h by the biological activity for 0h and multiplying by 100% is the residual percent of the biological activity of the composition at that time point gmCSF.
Results:
The biological activity of all the compositions from formula a to formula e and formula a1 to formula e1 is in the range of 121.3-129.6 IU/ml at 0h, for example, the biological activity of composition aNo.1 at 0h is 126.4IU/ml;
The percentage residues of the compositions of formula b and formula b1 for 12 hours were 97.4% and 96.8%, respectively,
The percentage of 12h residues of all the compositions of formulation c and formulation c1 are in the range of 97 to 102%, for example the percentage of 12h residues of composition cNo.1 is 98.6%,
The residual percentage of the composition 12h of all the formula a and the formula a1, the formula d and the formula d1, the formula e and the formula e1 is in the range of 81-88%, for example, the residual percentage of the composition aNo.1 at 12h is 85.3%;
the 24h residual percentages of the compositions of formula b and formula b1 are 94.7% and 95.4%, respectively,
The 24h residual percentage of all the compositions of formulation c and formulation c1 is in the range of 91 to 95%, for example the 24h residual percentage of composition cNo.1 is 93.6%,
The residual percentages of all the compositions of formula a and formula a1, formula d and formula d1, formula e and formula e1 for 24h are in the range of 64 to 73%, for example 70.4% for composition aNo.1 for 24 h.
These results indicate that gmCSF in the cell-containing combinations have a rapid decrease in biological activity, which can be significantly overcome when trace amounts of glutamine and sodium selenite are added to the compositions.
In addition, the number of CD45+ living cells was determined as in test example 3 above, resulting in:
All the compositions of formula a, formula c, formula d and formula e have CD45+ viable cell numbers within the range of 472-524 x 10-4/ml at 0h, e.g., composition aNo.1 has CD45+ viable cell numbers of 511.7x10-4/ml at 0h,
The total composition of the formula a1, the formula c1, the formula d1 and the formula e1 has the CD45+ living cell number of 387-416 x10 x 4/ml in 0h, for example, the CD45+ living cell number of the composition a1No.1 in 0h is 394.5x10 x 4/ml,
All the compositions of formula a, formula c, formula d and formula e have a CD45+ viable cell number in the range of 144 to 212 x 10-4/ml at 24 hours, for example, the CD45+ viable cell number of composition aNo.1 at 24 hours is 196.7x10-4/ml;
All the compositions of the formula a1, the formula c1, the formula d1 and the formula e1 have the CD45+ living cell number of 121-158 x 10-4/ml in 24h, for example, the composition a1No.1 has the CD45+ living cell number of 142.2x10-4/ml in 24 h.
These results indicate that there is no significant difference in the number of cd45+ living cells at different time points for each composition, and that glutamine and sodium selenite are not expected to affect the biological activity of the cells. Thus, while the BMAC + gmCSF composition of formula a and the BMAC + supernatant concentrate + gmCSF composition of formula a1 can exhibit excellent biological effects in the treatment of premature ovarian failure, the stability of the gmCSF biological activity in the composition can be significantly improved when supplemented with small amounts of glutamine and sodium selenite, and the living cells in the composition are not significantly different, and such an improvement in gmCSF biological activity stability would be of great significance for therapeutic applications.
In addition, since gmCSF is inexpensive and readily available, the amount of cells can be significantly reduced by combining with BMAC having poor availability while still achieving excellent biological effects for the treatment of 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 characteristics 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 (10)
1. A cell composition consisting of bone marrow concentrated cells, granulocyte macrophage stimulating factor, supernatant concentrate, glutamine, sodium selenite and physiological saline as an excipient; wherein,
The ratio of bone marrow concentrated cells to granulocyte macrophage-stimulating factor is 4x10 6 cells by CD45 + cells: 10-15 ng granulocyte macrophage stimulating factor;
The ratio of the bone marrow concentrated cells to the supernatant concentrated solution is that the bone marrow concentrated cells are 4x10 6 cells in terms of CD45 + cells number: 75-125 mu L of supernatant concentrate;
The weight ratio of granulocyte macrophage-stimulating factor to glutamine and sodium selenite in the composition is 12.5ng: 0.1-0.5 mg: 5-20 mug;
The marrow concentrated cells and supernatant concentrate are prepared by a method comprising the following steps:
(1) Providing biological sample bone marrow puncture liquid, and placing the biological sample bone marrow puncture liquid in a sterile bag containing an anticoagulant for standby; the anticoagulant is 3.2% sodium citrate solution, wherein 0.5mg/ml histidine and 0.1mg/ml phosphatidylcholine are added in a supplementary way;
(2) Removing a protective cap on an input pipe of the automatic cell separation system, connecting a syringe to an input Guan Luer locking joint, enabling the syringe to pass through a thrombus filter at a slow and stable speed, transferring anticoagulated biological sample marrow puncturing liquid to a disposable sterile separation cup, and shaking and mixing the biological sample marrow puncturing liquid along a horizontal axis; the cell autosegregation system is a closed PXP segregation system, which 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;
(3) The disposable separating cup is placed in the control module, the state of the control module before centrifugation is displayed as 0, the separating cup/control module assembly is weighed, after balancing, the disposable separating cup is placed in the programmable centrifugal machine, and parameters of the centrifugal machine are set according to the following procedures:
procedure P1: acceleration 9, deceleration 9, relative centrifugal force 2000, time 8.5min,
Program P2: acceleration 9, deceleration 9, relative centrifugal force 50, time 2min,
Procedure P3: acceleration 9, deceleration 9, relative centrifugal force 500, time 2min,
Procedure P4: acceleration 9, deceleration 9, relative centrifugal force 50, time 1min,
Program P5: acceleration 9, deceleration 9, relative centrifugal force 250, time 0.5min,
Program P6: acceleration 9, deceleration 9, relative centrifugal force 50, time 1min;
(4) Starting the centrifugal machine to centrifuge, wherein the process is as follows:
4a) Cells in the bone marrow puncture liquid of the biological sample are separated into the following upper and lower three components in a disposable separation cup through centrifugal density delamination in the P1 phase: a red blood cell layer, a cell concentration layer, and a plasma layer;
4b) The P2 phase enables most of red blood cells to enter a red blood cell recovery cabin;
4c) Stage P3 further delaminates cells in the process chamber and stage P4 reduces centrifugal force to further remove erythrocytes;
4d) The cell concentrate layer and the plasma are further layered in the P5 stage, the centrifugal force is reduced in the P6 stage, the cell concentrate layer is transferred to a recovery chamber through a conveying pipe, and the plasma is remained in a central chamber;
(5) After centrifugation is completed, confirming that a window of a control module displays P which is a qualified state, taking out a separation cup from the control module, connecting an injector to an output pipe which is communicated with a recovery cabin and the separation cup, and collecting the obtained marrow concentrated cells;
(6) Placing the separation cup and the control module on a separation base to transmit data and processing the data captured during centrifugation with a DataTrak software processing system;
(7) Separating the supernatant in the central compartment (i.e. plasma layer) with a syringe, centrifuging for 20min at 2000g to remove cell debris, and filtering with 0.22 μm sterile filter membrane;
(8) Flushing a pipeline of a tangential flow ultrafiltration system with ultrapure water, installing a MidiKros filter with a 100kD of 100cm 2, and ultrafiltering and concentrating the filtrate obtained in the step (7) to 1/15 volume of the initial biological sample volume to obtain supernatant concentrate.
2. The cell composition of claim 1, wherein the ratio of bone marrow-concentrated cells to granulocyte macrophage-stimulating factor is 4x10 6 cells by CD45 + cells: 12.5ng granulocyte macrophage stimulating factor.
3. The cell composition of claim 1, wherein the ratio of bone marrow-concentrated cells to supernatant concentrate is 4x10 6 cells by CD45 + cells count: 100 [ mu ] L supernatant concentrate.
4. The cellular composition of claim 1, wherein the weight ratio of granulocyte macrophage stimulating factor to glutamine and sodium selenite in the composition is 12.5ng: 0.2-0.3 mg: 10-15 mug.
5. The cell composition according to claim 1, wherein the concentration of granulocyte macrophage stimulating factor in the composition is 10-15 ng/ml.
6. The cellular composition of claim 1, wherein the granulocyte macrophage-stimulating factor is a human granulocyte macrophage-stimulating factor.
7. The cell composition of claim 1, which consists of: CD45 + cells 4x10 6 bone marrow concentrated cells, 12.5ng granulocyte macrophage stimulating factor, 100 mu L supernatant concentrated solution, 0.2-0.3 mg glutamine, 10-15 mu g sodium selenite and a proper amount of physiological saline to 1mL.
8. The cell composition of claim 1, which consists of: bone marrow concentrated cells with the cell number of 4x10 6 of CD45 +, granulocyte macrophage stimulating factor of 12.5ng, supernatant concentrated solution of 100 mu L, 0.2mg glutamine, 15 mu g sodium selenite and physiological saline with a proper amount to 1mL.
9. A method for producing the cell composition according to any one of claims 1 to 8, comprising a step of mixing predetermined amounts of bone marrow-concentrated cells, granulocyte macrophage stimulating factor, supernatant concentrate, glutamine, sodium selenite, and physiological saline as an excipient to prepare a sterile preparation; wherein,
The ratio of bone marrow concentrated cells to granulocyte macrophage-stimulating factor is 4x10 6 cells by CD45 + cells: 10-15 ng granulocyte macrophage stimulating factor;
The ratio of the bone marrow concentrated cells to the supernatant concentrated solution is that the bone marrow concentrated cells are 4x10 6 cells in terms of CD45 + cells number: 75-125 mu L of supernatant concentrate;
The weight ratio of granulocyte macrophage-stimulating factor to glutamine and sodium selenite in the composition is 12.5ng: 0.1-0.5 mg: 5-20 mug;
The marrow concentrated cells and supernatant concentrate are prepared by a method comprising the following steps:
(1) Providing biological sample bone marrow puncture liquid, and placing the biological sample bone marrow puncture liquid in a sterile bag containing an anticoagulant for standby; the anticoagulant is 3.2% sodium citrate solution, wherein 0.5mg/ml histidine and 0.1mg/ml phosphatidylcholine are added in a supplementary way;
(2) Removing a protective cap on an input pipe of the automatic cell separation system, connecting a syringe to an input Guan Luer locking joint, enabling the syringe to pass through a thrombus filter at a slow and stable speed, transferring anticoagulated biological sample marrow puncturing liquid to a disposable sterile separation cup, and shaking and mixing the biological sample marrow puncturing liquid along a horizontal axis; the cell autosegregation system is a closed PXP segregation system, which 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;
(3) The disposable separating cup is placed in the control module, the state of the control module before centrifugation is displayed as 0, the separating cup/control module assembly is weighed, after balancing, the disposable separating cup is placed in the programmable centrifugal machine, and parameters of the centrifugal machine are set according to the following procedures:
procedure P1: acceleration 9, deceleration 9, relative centrifugal force 2000, time 8.5min,
Program P2: acceleration 9, deceleration 9, relative centrifugal force 50, time 2min,
Procedure P3: acceleration 9, deceleration 9, relative centrifugal force 500, time 2min,
Procedure P4: acceleration 9, deceleration 9, relative centrifugal force 50, time 1min,
Program P5: acceleration 9, deceleration 9, relative centrifugal force 250, time 0.5min,
Program P6: acceleration 9, deceleration 9, relative centrifugal force 50, time 1min;
(4) Starting the centrifugal machine to centrifuge, wherein the process is as follows:
4a) Cells in the bone marrow puncture liquid of the biological sample are separated into the following upper and lower three components in a disposable separation cup through centrifugal density delamination in the P1 phase: a red blood cell layer, a cell concentration layer, and a plasma layer;
4b) The P2 phase enables most of red blood cells to enter a red blood cell recovery cabin;
4c) Stage P3 further delaminates cells in the process chamber and stage P4 reduces centrifugal force to further remove erythrocytes;
4d) The cell concentrate layer and the plasma are further layered in the P5 stage, the centrifugal force is reduced in the P6 stage, the cell concentrate layer is transferred to a recovery chamber through a conveying pipe, and the plasma is remained in a central chamber;
(5) After centrifugation is completed, confirming that a window of a control module displays P which is a qualified state, taking out a separation cup from the control module, connecting an injector to an output pipe which is communicated with a recovery cabin and the separation cup, and collecting the obtained marrow concentrated cells;
(6) Placing the separation cup and the control module on a separation base to transmit data and processing the data captured during centrifugation with a DataTrak software processing system;
(7) Separating the supernatant in the central compartment (i.e. plasma layer) with a syringe, centrifuging for 20min at 2000g to remove cell debris, and filtering with 0.22 μm sterile filter membrane;
(8) Flushing a pipeline of a tangential flow ultrafiltration system with ultrapure water, installing a MidiKros filter with a 100kD of 100cm 2, and ultrafiltering and concentrating the filtrate obtained in the step (7) to 1/15 volume of the initial biological sample volume to obtain supernatant concentrate.
10. Use of a cell composition according to any one of claims 1 to 8 for the preparation of a medicament for the treatment of premature ovarian failure.
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| CN112538456A (en) * | 2019-09-20 | 2021-03-23 | 北京干细胞与再生医学研究院 | Pluripotent stem cells, pharmaceutical composition, preparation method and application thereof |
| CN111643526A (en) * | 2020-07-14 | 2020-09-11 | 贵州金元泰生物科技有限公司 | Mesenchymal stem cell preparation for delaying ovarian aging and preventing and treating senile dementia and depression |
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