CN116270451A - Fresh mesenchymal stem cell injection and preparation method thereof - Google Patents
Fresh mesenchymal stem cell injection and preparation method thereof Download PDFInfo
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- CN116270451A CN116270451A CN202310348778.0A CN202310348778A CN116270451A CN 116270451 A CN116270451 A CN 116270451A CN 202310348778 A CN202310348778 A CN 202310348778A CN 116270451 A CN116270451 A CN 116270451A
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Abstract
The invention discloses a fresh mesenchymal stem cell injection and a preparation method thereof, wherein the cell density in the mesenchymal stem cell injection is 1 multiplied by 10 6 ‑5×10 7 individual/mL; human serum albumin body finally contained in stem cell injection1-5% of product ratio, 0.5-2% of low molecular weight heparin sodium injection, 1-20% of compound amino acid injection, 0.3-0.7% of glutamine, 0.3-0.7% of anhydrous glucose and the balance of compound electrolyte solution; the invention can inhibit potential coagulation reaction and embolism formation by adding low molecular weight heparin sodium injection, thereby improving cell therapy.
Description
Technical Field
The invention relates to the technical field of cell injection, in particular to fresh mesenchymal stem cell injection and a preparation method thereof.
Background
In recent years, the techniques of stem cell therapy, immune cell therapy, gene editing and the like are developed from basic theory to deep clinical treatment, and a new treatment idea and method are provided for serious and refractory diseases.
Stem cells are a class of cells with self-replicating and multipotent differentiation potential, and mesenchymal stem cells (mesenchymal stem cell, MSCs) are important members of the stem cell family, and have been widely focused on their ability to expand in vitro, multipotent differentiation potential, and immunomodulation. Adipose tissue in which adipose-derived mesenchymal stem cells (ADSCs) are located is abundant in human body, and has been a research hot spot for cell therapy in recent years due to the advantage of small trauma to donors.
Cell therapy is used to achieve therapeutic effects by injecting living cells into a patient. Systemic infusion of mesenchymal stem cells has become a promising strategy for disease treatment and tissue regeneration. Strategies to increase the therapeutic efficiency of ADSCs cells are critical to facilitate their clinical use. The survival rate of ADSCs is closely related to the clinical treatment effect, and researches show that the dosage range of the preparation of the stem cells is also related to the clinical treatment effect. High doses of ADSCs can lead to a series of symptoms of respiratory failure and heart failure, producing serious adverse effects such as thrombosis.
Heparin anticoagulation treatment can effectively prevent coagulation induced by mesenchymal stem cells and acute adverse reactions of large-dose mesenchymal stem cell infusion. In addition, heparin treatment reduces pulmonary embolism of bone marrow mesenchymal stem cells, and enhances migration and maintenance of mesenchymal stem cells to target organs in cell therapy.
Thus, the addition of heparin sodium in cell therapy can improve BMSC cell therapy by inhibiting BMSC-induced coagulation response. Heparin anticoagulation therapy is a practical strategy for the safety and efficacy of ADSCs treatment.
Disclosure of Invention
The aim of the application is to provide a fresh mesenchymal stem cell injection, which can inhibit potential coagulation reaction and embolism formation by adding low molecular weight heparin sodium injection, thereby improving cell therapy.
In order to solve the technical problems, the invention adopts a technical scheme that: the invention provides a fresh mesenchymal stem cell injection, wherein the cell density in the mesenchymal stem cell injection is 1 multiplied by 10 6 -5×10 7 individual/mL; the volume ratio of human serum albumin finally contained in the stem cell injection is 1-5%, the volume ratio of low molecular weight heparin sodium injection is 0.5-2%, the volume ratio of compound amino acid injection is 1-20%, the mass volume ratio of glutamine is 0.3-0.7%, the mass volume ratio of anhydrous glucose is 0.3-0.7%, and the balance is compound electrolyte solution.
Further, the mesenchymal stem cell injection has a cell density of 1×10 7 -5×10 7 The volume ratio of human serum albumin finally contained in the stem cell injection is 5%, the volume ratio of the low molecular weight heparin sodium injection is 0.5-2%, the volume ratio of the compound amino acid injection is 3%, the mass volume ratio of glutamine is 0.5% (g/L), the mass volume ratio of anhydrous glucose is 0.4% (g/L), and the balance is the compound electrolyte solution.
Further, the mesenchymal stem cells are derived from human fat mesenchymal stem cells, the cell generation number is between the generation P3 and the generation P10, and the activity of the mesenchymal stem cells is maintained above 90%.
Further, the mesenchymal stem cells are selected from human adipose mesenchymal stem cells, umbilical cord mesenchymal stem cells, placental mesenchymal stem cells, umbilical cord blood mesenchymal stem cells, bone marrow mesenchymal stem cells or dental pulp mesenchymal stem cells.
Further, the compound amino acid injection is amino acid injection 14AA, amino acid injection 18AA or amino acid injection 20AA.
Preferably compound amino acid injection 20AA.
The invention also provides a preparation method of the fresh mesenchymal stem cell injection, which is characterized by comprising the following steps of: the preparation method comprises the following steps:
step 1: dissolving anhydrous glucose and glutamine in a compound electrolyte injection according to a formula, adding the compound amino acid injection, human serum albumin and low molecular weight heparin sodium injection, uniformly mixing, continuously adding the compound electrolyte injection, and fixing the volume (for example, fixing the volume to 100 ml) to obtain a preservation solution, and adjusting the pH value to 6.0-8.0;
step 2: filtering with preservation solution filter membrane, collecting filtrate, and mixing the filtrate with the obtained mesenchymal stem cells to obtain the stem cell injection.
Further, the pore size of the filter membrane in the step 2 is 0.22. Mu.m.
Further, the preparation method of the mesenchymal stem cells in the step 2 comprises the following steps: the mesenchymal stem cells are adipose-derived mesenchymal stem cells;
step 21: collection, transport, handling and virus detection of adipose tissue of human origin, wherein the virus detection comprises: human Immunodeficiency Virus (HIV) detection, hepatitis B Virus (HBV) detection, hepatitis C Virus (HCV) detection, human T-lymphocytic leukemia virus (HTLV) detection, cytomegalovirus (CMV) detection, epstein Barr Virus (EBV) detection, treponema pallidum detection;
step 22: primary extraction and culture of human adipose-derived mesenchymal stem cells: dividing fresh adipose tissue into small blocks by a physical shearing method, and washing for a plurality of times by using a washing liquid to wash off blood color; adding collagenase to digest adipose tissues, and adding a stop solution to stop digestion; centrifuging to obtain a lower substrate part, and transferring into a culture flask for adherence culture;
step 23: subculturing human adipose-derived mesenchymal stem cells: when the cells reach more than 60 percent of fusion, adding cell digestive juice after washing twice, filtering to remove tissue small blocks, and obtaining primary mesenchymal stem cells after centrifuging and discarding the supernatant; the primary mesenchymal stem cells are resuspended by a serum-free culture medium and then inoculated in a culture bottle or a culture dish, and when the cell fusion degree reaches 80% -90%, the primary mesenchymal stem cells are collected and used as seed cells or are continuously subcultured;
step 24: identifying the three-lineage differentiation capacity of the extracted mesenchymal stem cells by flow cytometry;
step 25: when the fusion degree of each generation of cells reaches 80% -90%, centrifugally collecting mesenchymal stem cells, and continuing subculturing;
step 26: the mesenchymal stem cells amplified in step 25 were collected after centrifugation.
Further, the collagenase in step 23 is at least one selected from collagenase i, collagenase ii, collagenase iii, collagenase iv, and trypsin.
Further, the serum-free medium in step 23 is a DMEM-F12 basal medium with 10% serum replacement added thereto.
Further, CO is used in the adherence culture in the step 22 2 An incubator; the CO 2 The conditions of the incubator were: low oxygen conditions of 37 ℃, 5% oxygen, 5% carbon dioxide, 90% nitrogen.
Compared with the prior art, the invention has the following beneficial effects:
1. the human serum albumin, the compound amino acid injection, the compound electrolyte injection and the low molecular weight heparin sodium adopted by the invention are all clinical injection components, can provide nutrition for cells, are beneficial to metabolism of the cells and simulate living environment of the cells in vivo. The addition of the low molecular weight heparin sodium ensures that cells maintain a good cell dispersion state in the preservation process, reduces the phenomena of cell adhesion agglomeration and cell adhesion container wall, reduces the risk of intravascular cell agglomeration embolism possibly occurring during clinical cell infusion, simultaneously reduces cell loss caused by cell aggregation and filtration by an infusion filter, and does not cause adverse reactions such as clinical bleeding and the like when the trace heparin sodium is added, thereby improving the safety and the effectiveness of intravenous injection of mesenchymal stem cells.
2. The invention adopts the low molecular weight heparin sodium injection to reduce the risk of vascular embolism existing in the intravenous injection of the mesenchymal stem cell preparation; and compound amino acid injection and anhydrous glucose are introduced, so that the mesenchymal stem cell preparation can be used by intravenous infusion.
3. More importantly, the invention maintains the preservation environment of the cell preparation by adding substances such as human serum albumin, compound amino acid injection, anhydrous dextrose, glutamine and the like, simulates the in-vivo cell growth environment as much as possible, maintains the activity of the cell preparation, and improves the curative effect of the cell preparation.
3. The solution medium of the mesenchymal stem cell preparation is a compound electrolyte solution and a glucose solution, so that the osmotic pressure of cells can be kept, and the survival of the cells is facilitated.
4. The mesenchymal stem cell preparation is very favorable for survival of mesenchymal stem cells, is safe in clinical infusion, can keep high activity for a long time in the preservation solution, is convenient for clinical transportation without severe limitation of time, and solves the problem that the cells need to be transported for a long time when being used by patients in different places.
5. The mesenchymal stem cells adopted by the invention can be derived from human adipose tissue, the extraction wound of the adipose tissue is small, the yield is large, and the preparation system is easy to control the quality and easy to industrialize. The mesenchymal stem cells extracted by the invention have good activity and strong expansion capability.
The foregoing description is only an overview of the present invention, and is intended to provide a better understanding of the present invention, as it is embodied in the following description, with reference to the preferred embodiments of the present invention and the accompanying drawings.
Drawings
FIG. 1 is a photograph of mesenchymal stem cells of 7 th generation before harvest.
Detailed Description
The following specific embodiments of the invention are described in order to provide those skilled in the art with an understanding of the present disclosure. The invention may be embodied in other different forms, i.e., modified and changed without departing from the scope of the invention.
The sources of reagents in the following examples are as follows: human serum albumin was purchased from Hualan bioengineering, inc.; the compound electrolyte injection is purchased from Shanghai Baite medical supplies Co., ltd; the compound amino acid injection 20AA was purchased from Guangdong Korea pharmaceutical Co., ltd; glutamine was purchased from Jiangsu Shenhua pharmaceutical Co., ltd; anhydrous dextrose was purchased from the pharmaceutical company limited of the Weifang Shengtai; low molecular weight heparin sodium injection was purchased from kululu pharmaceutical limited.
The volume ratio of the invention refers to the ratio of the volume of each component to the final volume of the solution medium in the mesenchymal stem cell preparation of the invention; the mass-to-volume ratio refers to the ratio of the mass of each component to the final volume of the solution medium in the mesenchymal stem cell preparation of the present invention, for example: the volume ratio of the human serum albumin solution is 5 percent, namely, each 100 milliliters of the solution medium contains 5 grams of human serum albumin.
It will be appreciated that in order to facilitate the metering of the components of the mesenchymal stem cell preparation of the present invention and the formulation of the preparation, the metering of the mesenchymal stem cells of the present invention is performed by using the number of the mesenchymal stem cells per unit volume of the solution, i.e. the concentration of the mesenchymal stem cells, which does not exclude that the mesenchymal stem cells may be metered in other equivalent metering manners; some components in the invention are metered by mass-to-volume ratio, namely the ratio of the mass of each component to the volume of the solution medium, which cannot exclude that other equivalent metering modes can be adopted for metering, such as density, weight percentage, weight parts and the like, and all the components which are converted by unit fall into the numerical range of the invention belong to the protection scope of the invention. The metering mode of the invention can be converted into other equivalent metering modes if necessary so as to enable the protection scope of the invention to be understood more easily.
The concentration of the low molecular weight heparin sodium injection is determined according to the weight of a user, and specifically according to the following steps: the concentration of low molecular weight heparin sodium was determined at a ratio of 100U/kg.
The preparation method of the mesenchymal stem cell injection comprises the following steps:
step 1: dissolving anhydrous glucose and glutamine in a compound electrolyte injection according to a formula, adding the compound amino acid injection, human serum albumin and low molecular weight heparin sodium injection, uniformly mixing, continuously adding the compound electrolyte injection, and fixing the volume (for example, fixing the volume to 100 ml) to obtain a preservation solution, and adjusting the pH value to 6.0-8.0;
step 2: filtering the preservation solution with a 0.22 mu m filter membrane, collecting filtrate, and mixing the filtrate with the harvested mesenchymal stem cells to obtain the stem cell injection.
The preservation solution comprises the following raw materials in percentage by volume: the volume ratio of the human serum albumin is 1-5% (v/v), the volume ratio of the low molecular weight heparin sodium injection is 0.5-2% (v/v), the volume ratio of the compound amino acid injection is 1-20% (v/v), the mass-volume ratio of the glutamine is 0.1-10% (g/L), and the mass-volume ratio of the anhydrous glucose is 0.1-10% (g/L).
The preparation method of the mesenchymal stem cells in the step 2 comprises the following steps: the mesenchymal stem cells are adipose-derived mesenchymal stem cells;
step 21: collection, transport, handling and virus detection of adipose tissue of human origin, wherein the virus detection comprises: human Immunodeficiency Virus (HIV) detection, hepatitis B Virus (HBV) detection, hepatitis C Virus (HCV) detection, human T-lymphocytic leukemia virus (HTLV) detection, cytomegalovirus (CMV) detection, epstein Barr Virus (EBV) detection, treponema pallidum detection;
step 22: primary extraction and culture of human adipose-derived mesenchymal stem cells: dividing fresh adipose tissue into small blocks by a physical shearing method, and washing for a plurality of times by using a washing liquid to wash off blood color; adding collagenase to digest adipose tissues, and adding a stop solution to stop digestion; centrifuging to obtain a lower substrate part, and transferring into a culture flask for adherence culture;
step 23: subculturing human adipose-derived mesenchymal stem cells: when the cells reach more than 60 percent of fusion, adding cell digestive juice after washing twice, filtering to remove tissue small blocks, and obtaining primary mesenchymal stem cells after centrifuging and discarding the supernatant; the primary mesenchymal stem cells are resuspended by a serum-free culture medium and then inoculated in a culture bottle or a culture dish, and when the cell fusion degree reaches 80% -90%, the primary mesenchymal stem cells are collected and used as seed cells or are continuously subcultured;
step 24: identifying the three-lineage differentiation capacity of the extracted mesenchymal stem cells by flow cytometry;
step 25: when the fusion degree of each generation of cells reaches 80% -90%, centrifugally collecting mesenchymal stem cells, and continuing subculturing;
step 26: the mesenchymal stem cells amplified in step 25 were collected after centrifugation.
The obtained mesenchymal stem cells had a concentration of 5×10 6 -5×10 7 When the mesenchymal stem cells are derived from human adipose-derived mesenchymal stem cells, the cell generation number is between the generation P3 and the generation P10, and the activity of the mesenchymal stem cells is maintained above 90%, for example, the microscopic photograph of the cell generation number at the generation P7 is shown in FIG. 1.
The contents of the components and the index of the stem cell injection in comparative example 1, example 1 to example 3 are shown in the following table:
as can be clearly seen from the above comparative examples and examples, the cell activities of the examples and comparative examples are relatively high, and the requirements are met; in comparative example 1, the injection of low molecular weight heparin sodium is adopted to reduce the mesenchymal stem cell preparation without coagulation, thus eliminating the risk of vascular embolism existing in intravenous injection.
The foregoing description is only illustrative of the present invention and is not intended to limit the scope of the invention, and all equivalent structures made by the description of the invention and the accompanying drawings, or direct or indirect application in other related technical fields, are included in the scope of the invention.
Claims (10)
1. A fresh mesenchymal stem cell injection, which is characterized in that: the roomCell density in the injection of mesenchymal stem cells is 1×10 6 -5×10 7 individual/mL; the volume ratio of human serum albumin finally contained in the stem cell injection is 1-5%, the volume ratio of low molecular weight heparin sodium injection is 0.5-2%, the volume ratio of compound amino acid injection is 1-20%, the mass volume ratio of glutamine is 0.3-0.7%, the mass volume ratio of anhydrous glucose is 0.3-0.7%, and the balance is compound electrolyte solution.
2. The fresh mesenchymal stem cell injection according to claim 1, wherein: the cell density in the mesenchymal stem cell injection is 1 multiplied by 10 7 -5×10 7 The volume ratio of human serum albumin finally contained in the stem cell injection is 5%, the volume ratio of the low molecular weight heparin sodium injection is 0.5-2%, the volume ratio of the compound amino acid injection is 3%, the mass volume ratio of glutamine is 0.5%, the mass volume ratio of anhydrous glucose is 0.4%, and the balance is the compound electrolyte solution.
3. The fresh mesenchymal stem cell injection according to claim 1, wherein: the mesenchymal stem cells are derived from human fat mesenchymal stem cells, the cell generation number is between the generation number P3 and the generation number P10, and the activity of the mesenchymal stem cells is maintained to be more than 90%.
4. The fresh mesenchymal stem cell injection according to claim 1, wherein: the mesenchymal stem cells are selected from human adipose mesenchymal stem cells, umbilical cord mesenchymal stem cells, placenta mesenchymal stem cells, umbilical cord blood mesenchymal stem cells, bone marrow mesenchymal stem cells or dental pulp mesenchymal stem cells.
5. The fresh mesenchymal stem cell injection according to claim 1, wherein: the compound amino acid injection is amino acid injection 14AA, amino acid injection 18AA or amino acid injection 20AA.
6. A method for preparing the fresh mesenchymal stem cell injection according to claim 1, which is characterized in that: the preparation method comprises the following steps:
step 1: dissolving anhydrous glucose and glutamine in a compound electrolyte injection according to a formula, adding the compound amino acid injection, human serum albumin and low molecular weight heparin sodium injection, uniformly mixing, continuously adding the compound electrolyte injection, fixing the volume to obtain a preservation solution, and adjusting the pH value to be 6.0-8.0;
step 2: filtering with preservation solution filter membrane, collecting filtrate, and mixing the filtrate with the obtained mesenchymal stem cells to obtain the stem cell injection.
7. The method for preparing the fresh mesenchymal stem cell injection according to claim 6, wherein the method comprises the following steps: the pore size of the filter membrane in step 2 was 0.22. Mu.m.
8. The method for preparing the fresh mesenchymal stem cell injection according to claim 6, wherein the method comprises the following steps: the preparation method of the mesenchymal stem cells in the step 2 comprises the following steps: the mesenchymal stem cells are adipose-derived mesenchymal stem cells;
step 21: collection, transport, handling and virus detection of adipose tissue of human origin, wherein the virus detection comprises: human Immunodeficiency Virus (HIV) detection, hepatitis B Virus (HBV) detection, hepatitis C Virus (HCV) detection, human T-lymphocytic leukemia virus (HTLV) detection, cytomegalovirus (CMV) detection, epstein Barr Virus (EBV) detection, treponema pallidum detection;
step 22: primary extraction and culture of human adipose-derived mesenchymal stem cells: dividing fresh adipose tissue into small blocks by a physical shearing method, and washing for a plurality of times by using a washing liquid to wash off blood color; adding collagenase to digest adipose tissues, and adding a stop solution to stop digestion; centrifuging to obtain a lower substrate part, and transferring into a culture flask for adherence culture;
step 23: subculturing human adipose-derived mesenchymal stem cells: when the cells reach more than 60 percent of fusion, adding cell digestive juice after washing twice, filtering to remove tissue small blocks, and obtaining primary mesenchymal stem cells after centrifuging and discarding the supernatant; the primary mesenchymal stem cells are resuspended by a serum-free culture medium and then inoculated in a culture bottle or a culture dish, and when the cell fusion degree reaches 80% -90%, the primary mesenchymal stem cells are collected and used as seed cells or are continuously subcultured;
step 24: identifying the three-lineage differentiation capacity of the extracted mesenchymal stem cells by flow cytometry;
step 25: when the fusion degree of each generation of cells reaches 80% -90%, centrifugally collecting mesenchymal stem cells, and continuing subculturing;
step 26: the mesenchymal stem cells amplified in step 25 were collected after centrifugation.
9. The method for preparing the fresh mesenchymal stem cell injection according to claim 8, wherein the method comprises the following steps: the collagenase in step 23 is at least one selected from collagenase I, collagenase II, collagenase III, collagenase IV and trypsin.
10. The method for preparing the fresh mesenchymal stem cell injection according to claim 8, wherein the method comprises the following steps: the serum-free medium in step 23 was a 10% serum replacement added to DMEM-F12 basal medium.
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