Preparation method and application of human umbilical cord mesenchymal stem cells

Abstract

The application discloses a preparation method and application of human umbilical cord mesenchymal stem cells. The technical scheme improves the extraction method and subculture process of cells in many aspects. Specifically, the umbilical cord tissue block is attached to a cell culture bottle, Ham's F-12 culture medium is added, and the cell culture bottle is placed in a cell culture box for culture at 37 DEG C for 2.5-3.5 days; the culture is collected, filtered, and the liquid phase is taken, the culture medium is replaced after 36-48 hours of culture, the unattached cells are discarded, and the culture is continued for 2-3 days; the cells are collected, resuspended in L-DMEM culture medium, added dropwise into a centrifuge tube containing Percoll separation solution along the tube wall, and the middle interface white film layer is sucked after centrifugation; L-DMEM complete culture medium containing 10% fetal bovine serum, sodium penicillin and streptomycin is added, the medium is replaced after 48 hours of inoculation and culture, the unattached cells are removed, and then trypsin-EDTA digestion is carried out. The application fully improves the extraction and amplification efficiency of human umbilical cord mesenchymal stem cells, and has good technical effects and outstanding technical advantages.

Description

Technical Field

[0001] This invention belongs to the field of cell culture technology, specifically a method for preparing and applying human umbilical cord mesenchymal stem cells. Background Technology

[0002] Mesenchymal stem cells (MSCs) are pluripotent stem cells, possessing all the common characteristics of stem cells, namely self-renewal and multipotent differentiation capabilities. They are most widely used clinically, and when combined with hematopoietic stem cells, they can improve transplant success rates and accelerate hematopoietic reconstitution. When patients receive high-dose chemotherapy, infusing MSCs along with hematopoietic stem cells can significantly accelerate the recovery time of blood cells, with no adverse reactions. MSCs are not only found in bone marrow but also in skeletal muscle, umbilical cord blood, umbilical cord tissue, placental tissue, and adipose tissue. Due to their wide range of tissue differentiation potentials, they have high clinical application value.

[0003] Mesenchymal stem cells (MSCs) are attracting increasing attention due to their multipotent differentiation potential, ability to support hematopoiesis and promote hematopoietic stem cell engraftment, immune regulation, and ease of isolation and culture. With the increasing maturity of MSCs and related technologies, clinical research has been conducted in many countries. As seed cells, they are mainly used clinically to treat various refractory diseases caused by tissue and organ damage that the body cannot repair naturally; as immunomodulatory cells, they are used to treat immune rejection and autoimmune diseases. While significant progress has been made in MSC research, some challenges remain. These include significant individual differences in expansion capacity; the potential risk of tumor cell contamination; and the time required for culture, which may prevent timely adaptation to the needs of the patient's condition. These limitations restrict the use of autologous MSCs. In contrast, placental and umbilical cord-derived MSCs possess characteristics such as high differentiation potential, strong proliferation capacity, low immunogenicity, convenient sourcing, no ethical restrictions, and ease of industrial production, making them potentially the most promising pluripotent stem cells for clinical application. Summary of the Invention

[0004] The present invention aims to address the technical deficiencies of the prior art by providing a method for preparing and applying human umbilical cord mesenchymal stem cells, thereby solving the technical problem of low expansion efficiency of human umbilical cord mesenchymal stem cells by conventional preparation methods.

[0005] To achieve the above technical objectives, the present invention adopts the following technical solution: A method for preparing and applying human umbilical cord mesenchymal stem cells includes: taking umbilical cords from full-term newborns, washing them with PBS solution, cutting off the arteries and veins, washing them again with PBS solution, cutting them into tissue blocks, attaching them to cell culture flasks, adding Ham's F-12 medium, and culturing them in a cell culture incubator at 37°C for 2.5-3.5 days; collecting the culture, filtering it, taking the liquid phase, culturing for 36-48 hours, changing the medium, discarding non-adherent cells, continuing to culture for 2-3 days, collecting the cells, resuspending them in L-DMEM medium, adding Percoll separation solution dropwise to a centrifuge tube, centrifuging, aspirating the white membrane layer at the intermediate interface, adding L-DMEM complete medium containing 10% fetal bovine serum, penicillin sodium, and streptomycin, inoculating, and culturing in a 37°C, 5% CO2 saturated humidity incubator for 48 days. Change the medium after h to remove non-adherent cells. Change the medium every 2 days thereafter. When the cells reach 80%~90% confluence, digest them with trypsin-EDTA.

[0006] Preferably, the process also includes the following steps: aspirating waste liquid, rinsing with PBS solution free of calcium and magnesium ions, adding trypsin containing EDTA to the culture flask, shaking to cover the bottom with trypsin, and placing it in a 37°C incubator to digest the cells; observing under a microscope until the cell layer is completely detached, adding MEF complete culture medium to stop digestion; pipetting the cells to prepare a single-cell suspension; passage at a ratio of 1:2 to 1:3; and culturing in a 37°C incubator.

[0007] Preferably, the particle size of the tissue block does not exceed 5 mm.

[0008] Preferably, during the 2.5 to 3.5 days of cultivation, the pressure inside the cultivation container is maintained at 1.1 to 1.2 atmospheres.

[0009] Preferably, the filtration is achieved using a 200-mesh filter.

[0010] Preferably, the final concentration of the Percoll separation solution added is 1.073 g / mL.

[0011] Preferably, the centrifugation speed is 1500~1800 rpm and the centrifugation time is 4~6 min.

[0012] Preferably, the culture flask is placed on a shaker at a speed of 60-80 rpm during the cell digestion process.

[0013] Preferably, the concentration of the single-cell suspension is 10. 4 ~10 5 per mL.

[0014] Based on the above technical solutions, the present invention further provides the application of preparing immunomodulatory drugs using the human umbilical cord mesenchymal stem cells prepared above.

[0015] This invention discloses a method for preparing and applying human umbilical cord mesenchymal stem cells. The technical solution improves the cell extraction method and passage culture process in several aspects. Specifically, the umbilical cord tissue block is attached to a cell culture flask, Ham's F-12 medium is added, and the flask is cultured at 37°C for 2.5–3.5 days. The culture is collected, filtered, and the liquid phase is taken. After culturing for 36–48 hours, the medium is changed, and non-adherent cells are discarded. The culture is continued for 2–3 days, and the cells are collected and resuspended in L-DMEM medium. Percoll separation solution is added dropwise to a centrifuge tube, and after centrifugation, the white membrane layer at the intermediate interface is aspirated. L-DMEM complete medium containing 10% fetal bovine serum, penicillin sodium, and streptomycin is added. After inoculation, the medium is changed after 48 hours of culture, and non-adherent cells are removed. The cells are then digested with trypsin-EDTA. This invention significantly improves the extraction and expansion efficiency of human umbilical cord mesenchymal stem cells, and the process is easy to implement, exhibiting good technical effects and outstanding technical advantages. Detailed Implementation

[0016] The specific embodiments of the present invention will be described in detail below. To avoid excessive and unnecessary detail, well-known structures or functions will not be described in detail in the following embodiments. The approximate language used in the following embodiments is for quantitative purposes, indicating that a certain degree of variation in quantity is permissible without changing the basic function. Unless otherwise defined, the technical and scientific terms used in the following embodiments have the same meaning as commonly understood by those skilled in the art to which this invention pertains.

[0017] Example 1 A method for preparing and applying human umbilical cord mesenchymal stem cells, characterized by comprising: taking the umbilical cord of a full-term newborn, washing it with PBS solution, cutting off the arteries and veins, washing it again with PBS solution, cutting it into tissue blocks, attaching it to a cell culture flask, adding Ham's F-12 medium, and culturing it in a cell culture incubator at 37°C for 2.5-3.5 days; collecting the culture, filtering it, taking the liquid phase, culturing it for 36-48 hours, changing the medium, discarding non-adherent cells, continuing to culture for 2-3 days, collecting the cells, resuspending them in L-DMEM medium, adding Percoll separation solution dropwise to a centrifuge tube, centrifuging, aspirating the white membrane layer at the intermediate interface, adding L-DMEM complete medium containing 10% fetal bovine serum, penicillin sodium, and streptomycin, inoculating, and culturing in a 37°C, 5% CO2 saturated humidity incubator for 48 days. After h, the medium was changed to remove non-adherent cells. The medium was then changed every 2 days until the cells reached 80%–90% confluence, at which point they were digested with trypsin-EDTA. Testing showed that this method improved the expansion efficiency of human umbilical cord mesenchymal stem cells by 36.4%.

[0018] Example 2 A method for preparing and applying human umbilical cord mesenchymal stem cells, characterized by comprising: taking the umbilical cord of a full-term newborn, washing it with PBS solution, cutting off the arteries and veins, washing it again with PBS solution, cutting it into tissue blocks, attaching it to a cell culture flask, adding Ham's F-12 medium, and culturing it in a cell culture incubator at 37°C for 2.5-3.5 days; collecting the culture, filtering it, taking the liquid phase, culturing it for 36-48 hours, changing the medium, discarding non-adherent cells, continuing to culture for 2-3 days, collecting the cells, resuspending them in L-DMEM medium, adding Percoll separation solution dropwise to a centrifuge tube, centrifuging, aspirating the white membrane layer at the intermediate interface, adding L-DMEM complete medium containing 10% fetal bovine serum, penicillin sodium, and streptomycin, inoculating, and culturing in a 37°C, 5% CO2 saturated humidity incubator for 48 days. After h, change the medium to remove non-adherent cells. Change the medium every 2 days thereafter. When the cells reach 80%–90% confluence, digest them with trypsin-EDTA. The process also includes the following steps: aspirate waste liquid, rinse with calcium- and magnesium-free PBS solution, add trypsin containing EDTA to the culture flask, shake to cover the bottom with trypsin, and place in a 37°C incubator to digest the cells; observe under a microscope until the cell layer is completely detached, then add MEF complete culture medium to stop digestion; pipette the cells to prepare a single-cell suspension; passage at a ratio of 1:2–1:3; and culture in a 37°C incubator. During the 2.5–3.5 days of culture, maintain the pressure inside the culture vessel at 1.1–1.2 atmospheres. The final concentration of Percoll separation solution added is 1.073 g / mL. During the cell digestion process, the culture flask is placed on a shaker at 60–80 rpm. Application of the human umbilical cord mesenchymal stem cells prepared according to claim 1 in the preparation of immunomodulatory drugs. Tests showed that this method improved the expansion efficiency of human umbilical cord mesenchymal stem cells by 45.1%.

[0019] Example 3 A method for preparing and applying human umbilical cord mesenchymal stem cells, characterized by comprising: taking the umbilical cord of a full-term newborn, washing it with PBS solution, cutting off the arteries and veins, washing it again with PBS solution, cutting it into tissue blocks, attaching it to a cell culture flask, adding Ham's F-12 medium, and culturing it in a cell culture incubator at 37°C for 2.5-3.5 days; collecting the culture, filtering it, taking the liquid phase, culturing it for 36-48 hours, changing the medium, discarding non-adherent cells, continuing to culture for 2-3 days, collecting the cells, resuspending them in L-DMEM medium, adding Percoll separation solution dropwise to a centrifuge tube, centrifuging, aspirating the white membrane layer at the intermediate interface, adding L-DMEM complete medium containing 10% fetal bovine serum, penicillin sodium, and streptomycin, inoculating, and culturing in a 37°C, 5% CO2 saturated humidity incubator for 48 days. After h, change the medium to remove non-adherent cells. Change the medium every 2 days thereafter. When the cells reach 80%–90% confluence, digest them with trypsin-EDTA. The process also includes the following steps: aspirate waste liquid, rinse with calcium- and magnesium-free PBS solution, add trypsin containing EDTA to the culture flask, agitate to cover the bottom with trypsin, and incubate at 37°C to digest the cells; observe under a microscope until the cell layer is completely detached, then add MEF complete culture medium to stop digestion; pipette the cells to prepare a single-cell suspension; passage at a ratio of 1:2–1:3; and culture at 37°C. The particle size of the tissue blocks should not exceed 5 mm. During the 2.5–3.5 days of culture, maintain the pressure inside the culture container at 1.1–1.2 atmospheres. Filtration is achieved using a 200-mesh filter. The final concentration of Percoll separation solution added is 1.073 g / mL. Centrifugation is performed at 1500–1800 rpm for 4–6 minutes. During the cell digestion process, the culture flask is placed on a shaker at a speed of 60-80 rpm. The concentration of the single-cell suspension is 10. 4 ~10 5 The number of cells / mL was measured, and the method showed that it improved the expansion efficiency of human umbilical cord mesenchymal stem cells by 46.1%.

[0020] The embodiments of the present invention have been described in detail above, but the content described is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the scope of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing and applying human umbilical cord mesenchymal stem cells, characterized in that, include: Take the umbilical cord from a full-term newborn, wash it with PBS solution, cut off the artery and vein, wash it again with PBS solution, cut it into tissue blocks, attach them to cell culture flasks, add Ham's F-12 medium, and incubate in a cell culture incubator at 37°C for 2.5-3.5 days. Collect the culture, filter it, and take the liquid phase. Incubate for 36-48 hours, change the medium, discard non-adherent cells, and continue culturing for 2-3 days. Collect the cells, resuspend them in L-DMEM medium, add Percoll separation solution dropwise to the centrifuge tube, centrifuge, and aspirate the white membrane layer at the intermediate interface. Add L-DMEM complete medium containing 10% fetal bovine serum, penicillin sodium, and streptomycin. After inoculation, incubate in a 37°C, 5% CO2 saturated humidity incubator for 48 hours, change the medium, remove non-adherent cells, and change the medium every 2 days thereafter. When the cells reach 80%-90% confluence, digest them with trypsin-EDTA.

2. The method for preparing and applying human umbilical cord mesenchymal stem cells according to claim 1, characterized in that, It also includes the following steps: Remove the waste liquid, rinse with PBS solution free of calcium and magnesium ions, add trypsin containing EDTA to the culture flask, shake to cover the bottom with trypsin, and place in a 37°C incubator to digest the cells; observe under a microscope until the cell layer is completely detached, add MEF complete culture medium to stop digestion; pipette the cells to make a single-cell suspension; passage at a ratio of 1:2 to 1:3; and culture in a 37°C incubator.

3. The method for preparing and applying human umbilical cord mesenchymal stem cells according to claim 1, characterized in that, The particle size of the tissue block does not exceed 5 mm.

4. The method for preparing and applying human umbilical cord mesenchymal stem cells according to claim 1, characterized in that, During the 2.5 to 3.5 days of cultivation, the pressure inside the cultivation container was maintained at 1.1 to 1.2 atmospheres.

5. The method for preparing and applying human umbilical cord mesenchymal stem cells according to claim 1, characterized in that, The filtration is achieved using a 200-mesh filter.

6. The method for preparing and applying human umbilical cord mesenchymal stem cells according to claim 1, characterized in that, The final concentration of the Percoll separation solution added was 1.073 g / mL.

7. The method for preparing and applying human umbilical cord mesenchymal stem cells according to claim 1, characterized in that, The centrifugation speed is 1500~1800 rpm, and the centrifugation time is 4~6 min.

8. The method for preparing and applying human umbilical cord mesenchymal stem cells according to claim 2, characterized in that, During the cell digestion process, the culture flask is placed on a shaker rotating at 60-80 rpm.

9. The method for preparing and applying human umbilical cord mesenchymal stem cells according to claim 2, characterized in that, The concentration of the single-cell suspension is 10. 4 ~10 5 per mL.

10. The application of the human umbilical cord mesenchymal stem cells prepared according to claim 1 to prepare immunomodulatory drugs.