Stem cell preparation for knee joint treatment and preparation method thereof

Abstract

The invention discloses a stem cell preparation for knee joint treatment and a preparation method thereof, and relates to the technical field of stem cell preparations. According to the stem cell preparation for knee joint treatment and the preparation method thereof, through double-antibiotic cleaning and alcohol disinfection, the disinfection effect is guaranteed, and the toxicity of chemical reagents to cells can be reduced; umbilical artery and vein are removed, only Wharton's jelly is reserved, vascular endothelial cells or blood cells are prevented from being mixed, it is ensured that the separated cells are mainly mesenchymal stem cells, and the cell purity is improved; the Wharton's jelly is cut into small blocks and inoculated and cultured, stem cells naturally climb out by means of the wall adhering characteristic of tissue blocks, damage of enzyme digestion to cell surface antigens is avoided, and cell dryness and functional integrity are maintained.

Description

Technical Field

[0001] The present invention relates to the technical field of stem cell preparations, and in particular to a stem cell preparation for knee joint treatment and a preparation method thereof. Background Art

[0002] Stem cell preparations for knee joint treatment are typically biological agents with mesenchymal stem cells (MSCs) as their core ingredient. Stem cells can suppress knee synovial inflammation and promote chondrocyte regeneration by secreting anti-inflammatory factors (such as IL-10 and TGF-β). Their immunomodulatory function can reduce the activation of immune cells (such as T cells and macrophages) within the joint cavity, alleviating the progression of diseases like rheumatoid arthritis. Furthermore, UC-MSCs can home to the site of injury and directly participate in cartilage repair. Through their multidirectional differentiation potential, immunomodulatory ability, and tissue repair properties, they exert therapeutic effects on knee joint diseases such as osteoarthritis, cartilage damage, and synovitis.

[0003] The continuous improvement of stem cell theory and the rapid development of technology are bound to produce epoch-making results in disease treatment and biomedicine. Humanity holds high hopes for the isolation and in vitro cultivation of stem cells to treat clinical diseases; however, the preparation technology for stem cell preparations is particularly lacking. To this end, the present invention provides a stem cell preparation for knee joint treatment and its preparation method to address the aforementioned issues. Summary of the Invention

[0004] In view of the deficiencies of the prior art, the present invention provides a stem cell preparation for knee joint treatment and a preparation method thereof, which solves the problems mentioned in the above background technology.

[0005] To achieve the above objectives, the present invention is implemented through the following technical solutions: A method for preparing a stem cell preparation for knee joint treatment, comprising the following preparation steps:

[0006] Step 1: Take the umbilical cord of a full-term healthy fetus and test the umbilical cord for Anti-HIV, HBSAg, CMV (IgM)-Ab, HBcAg, TP-Ab, and HCV-Ab. If the results are all negative, keep it for future use.

[0007] Step 2: Wash the umbilical cord three times in a D-PBS solution containing 1% penicillin and 1% streptomycin sulfate. Then, completely immerse the umbilical cord in a 75% alcohol solution for disinfection. Remove the cord immediately and transfer it to a D-PBS solution containing 0.1% penicillin and 0.1% streptomycin sulfate for three washes to remove any residual alcohol.

[0008] Step 3: Cut the umbilical cord along the umbilical vein, remove one umbilical vein and two umbilical arteries, and the remaining white jelly is Wharton's jelly, which should be rinsed twice with saline.

[0009] Step 4: Cut the Wharton's gel into tissue blocks and place them in a 50 mL centrifuge tube. Add 20 mL of complete culture medium to the centrifuge tube, resuspend the tissue blocks evenly in the complete culture medium, and then inoculate them into a T-75 cm 2 Gently shake the culture flask to evenly distribute the tissue blocks, then place it in a 37°C 5% CO2 saturated humidity incubator for culture, and add culture medium to 15 mL per bottle;

[0010] Step 5: Change the medium after 5 days, observe the cell growth status after 14 days, and subculture after reaching 80% confluence. When the cells crawl out of the tissue block and grow to 80%-90% confluence, inoculate the washed tissue block into a new culture flask for continued culture, and then digest and subculture the primary umbilical cord mesenchymal stem cells. When the cells that crawl out of the second tissue block adhere to the wall grow to 80%-90% confluence, repeat the above method for a third tissue adherence culture;

[0011] Step 6: After reaching 80%-90% confluence, discard the culture medium, rinse twice with PBS, add digestion solution, and digest at 37°C for 1-3 minutes. When the cells become round and the gaps between them increase under the microscope, add complete culture medium to terminate the digestion.

[0012] Step 7. When the cell density reaches 80% confluence, digest and collect the cells, resuspend them in freezing buffer, and adjust the cell concentration.

[0013] Preferably, in step 4, the complete culture medium is: 89% MSCBM + 10% UltraGROTM + 1% penicillin-streptomycin.

[0014] Preferably, in step 4, the Wharton's jelly is cut into 2-3 mm pieces. 3 organizational blocks.

[0015] Preferably, in step five, 0.25% trypsin-EDTA is used to digest and passage the primary umbilical cord mesenchymal stem cells.

[0016] Preferably, in step six, 0.25% trypsin-EDTA digestion solution is used.

[0017] Preferably, in step seven, the cell concentration is adjusted to 1×10 6 -5×10 6 cells / mL.

[0018] Preferably, in step seven, the freezing solution is: 90% FBS + 10% DMSO.

[0019] Preferably, in step seven, the freezing procedure is: 4°C, waiting; 1°C / min, sample temperature -5°C; 21°C / min, box temperature -54°C; 17°C / min, box temperature -17°C; 2°C / min, sample temperature -40°C; 10°C / min, sample temperature -80°C; transfer to a liquid nitrogen tank for long-term storage.

[0020] A stem cell preparation prepared using a method for preparing a stem cell preparation for knee joint treatment.

[0021] Beneficial effects

[0022] The present invention provides a stem cell preparation for knee joint treatment and its preparation method. Compared with the existing technology, it has the following advantages:

[0023] The stem cell preparation for knee joint treatment and the preparation method thereof are characterized by double antibiotic cleaning and alcohol disinfection, which not only ensures the disinfection effect but also reduces the toxicity of chemical reagents to cells; by removing the umbilical artery and vein and retaining only Wharton's jelly, the mixture of vascular endothelial cells or blood cells is avoided, ensuring that the separated cells are mainly mesenchymal stem cells, thereby improving cell purity; by cutting the Wharton's jelly into small pieces and inoculating and culturing them, the adherent properties of the tissue pieces themselves are utilized to allow stem cells to crawl out naturally, avoiding the damage of cell surface antigens by enzymatic digestion, and maintaining the stemness and functional integrity of the cells; after primary culture, the eluted tissue pieces are repeatedly inoculated and cultured, which can maximize the utilization of umbilical cord resources, significantly improve the stem cell yield, and reduce raw material waste.

[0024] In summary, this technical solution achieves the safe acquisition and expansion of umbilical cord mesenchymal stem cells through strict safety screening, efficient tissue block culture, and standardized passage and cryopreservation, providing high-purity and high-activity cell preparations for knee joint treatment. Its core value lies in balancing ease of operation and clinical applicability. At the same time, it improves the stem cell yield through multiple use of tissue blocks, making it suitable as a basic process for further optimization to meet clinical translation needs. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The cell morphology of the umbilical cord mesenchymal stem cells provided by the present invention after culturing for 1 day, 5 days and 14 days;

[0026] Figure 2 The present invention provides the surface conditions of rat femoral cartilage with and without injection of umbilical cord mesenchymal stem cell suspension. DETAILED DESCRIPTION

[0027] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0028] A method for preparing a stem cell preparation for knee joint treatment comprises the following steps:

[0029] Step 1: Take the umbilical cord of a full-term healthy fetus and test the umbilical cord for Anti-HIV, HBSAg, CMV (IgM)-Ab, HBcAg, TP-Ab, and HCV-Ab. If the results are all negative, keep it for future use.

[0030] Step 2: Wash the umbilical cord three times in a D-PBS solution containing 1% penicillin and 1% streptomycin sulfate. Then, completely immerse the umbilical cord in a 75% alcohol solution for disinfection. Remove the cord immediately and transfer it to a D-PBS solution containing 0.1% penicillin and 0.1% streptomycin sulfate for three washes to remove any residual alcohol.

[0031] Step 3: Cut the umbilical cord along the umbilical vein, remove one umbilical vein and two umbilical arteries, and the remaining white jelly is Wharton's jelly, which should be rinsed twice with saline.

[0032] Step 4: Cut Wharton's glue into 2-3mm pieces 3 The tissue blocks were then placed in a 50 mL centrifuge tube. 20 mL of 89% MSCBM + 10% UltraGROTM + 1% penicillin-streptomycin complete medium was added to the centrifuge tube. The tissue blocks were evenly resuspended in the complete medium and then inoculated into T-75 cm 2 Gently shake the culture flask to evenly distribute the tissue blocks, then place it in a 37°C 5% CO2 saturated humidity incubator for culture, and add culture medium to 15 mL per bottle;

[0033] Step 5: Change the medium after 5 days, observe the cell growth status after 14 days, and subculture after reaching 80% confluence. After the cells crawl out of the tissue block and grow to 80%-90% confluence, inoculate the washed tissue block into a new culture flask and continue culturing. Then, use 0.25% trypsin-EDTA to digest and subculture the primary umbilical cord mesenchymal stem cells. When the cells that crawl out of the second tissue block adhere to the wall and grow to 80%-90% confluence, repeat the above method for a third tissue adherence culture;

[0034] Step 6: After reaching 80%-90% confluence, discard the culture medium, rinse twice with PBS, add 0.25% trypsin-EDTA digestion solution, and digest at 37°C for 3 minutes. When the cells become round and the gaps between them increase under the microscope, add complete culture medium to terminate the digestion.

[0035] Step 7: When the cell density reaches 80% confluence, digest and collect the cells, resuspend them in 90% FBS + 10% DMSO freezing solution, and adjust the cell concentration to 5×10 6 cells / mL, where the freezing program is as follows: 4°C, waiting; 1°C / min, sample temperature -5°C; 21°C / min, chamber temperature -54°C; 17°C / min, chamber temperature -17°C; 2°C / min, sample temperature -40°C; 10°C / min, sample temperature -80°C; transfer to liquid nitrogen tank for long-term storage.

[0036] like Figure 1 As shown, on the first day of primary tissue culture, the cells were seeded in a sterile culture dish, and the primary tissue adhered closely to the culture dish. On the fifth day of primary culture, the umbilical cord mesenchymal stem cells were small spindle-shaped adherent cells that crawled out from around the primary tissue. On the 14th day, the spindle-shaped, elongated cells were closely arranged, with a fusion degree of about 85%-90%, and grew densely in parallel or spiral shapes.

[0037] Experimental steps:

[0038] (1) Aspirate 5% sodium pentobarbital and anesthetize the rat using a 1 ml syringe at a rate of 3-4 μl / g;

[0039] (2) After 2-3 minutes, the rats were anesthetized, the skin was prepared, and after disinfection, a 1-2 cm deep incision was made on the medial surface of the right knee joint using a scalpel. Tissue forceps were used to free the subcutaneous fat and expose the patellar ligament.

[0040] (3) Use an insulin needle to draw an appropriate amount of human umbilical cord mesenchymal stem cell suspension, insert the needle along the lower edge of the patellar ligament on the medial side of the knee joint, and slowly push the syringe to inject the human umbilical cord mesenchymal stem cells into the rat joint cavity;

[0041] (6) After surgery, a mixture of gentamicin (2 mg / kg) and ceftriaxone (50 mg / kg) was administered once every 24 hours for five days, and the rats were observed.

[0042] like Figure 2 As shown, the experimental group (injected with human umbilical cord mesenchymal stem cell suspension) had obvious cartilage repair, while the control group (not injected with human umbilical cord mesenchymal stem cell suspension) had a smooth and complete femoral surface, the soft group was transparent and shiny, and the solvent group had obvious wear at the contact between the inner side of the femur and the tibial plateau.

[0043] At the same time, the contents not described in detail in this specification belong to the existing technology well known to those skilled in the art.

[0044] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a stem cell preparation for knee joint treatment, characterized in that: The method comprises the following preparation steps: Step 1: Take the umbilical cord of a full-term healthy fetus and test the umbilical cord for Anti-HIV, HBSAg, CMV (IgM)-Ab, HBcAg, TP-Ab, and HCV-Ab. If the results are all negative, keep it for future use. Step 2: Wash the umbilical cord three times in a D-PBS solution containing 1% penicillin and 1% streptomycin sulfate. Then, completely immerse the umbilical cord in a 75% alcohol solution for disinfection. Remove the cord immediately and transfer it to a D-PBS solution containing 0.1% penicillin and 0.1% streptomycin sulfate for three washes to remove any residual alcohol. Step 3: Cut the umbilical cord along the umbilical vein, remove one umbilical vein and two umbilical arteries, and the remaining white jelly is Wharton's jelly, which should be rinsed twice with saline. Step 4: Cut the Wharton's gel into tissue blocks and place them in a 50 mL centrifuge tube. Add 20 mL of complete culture medium to the centrifuge tube, resuspend the tissue blocks evenly in the complete culture medium, and then inoculate them into a T-75 cm 2 Gently shake the culture flask to evenly distribute the tissue blocks, then place it in a 37°C 5% CO2 saturated humidity incubator for culture, and add culture medium to 15 mL per bottle; Step 5: Change the medium after 5 days, observe the cell growth status after 14 days, and subculture after reaching 80% confluence. When the cells crawl out of the tissue block and grow to 80%-90% confluence, inoculate the washed tissue block into a new culture flask for continued culture, and then digest and subculture the primary umbilical cord mesenchymal stem cells. When the cells that crawl out of the second tissue block adhere to the wall grow to 80%-90% confluence, repeat the above method for a third tissue adherence culture; Step 6: After reaching 80%-90% confluence, discard the culture medium, rinse twice with PBS, add digestion solution, and digest at 37°C for 1-3 minutes. When the cells become round and the gaps between them increase under the microscope, add complete culture medium to terminate the digestion. Step 7. When the cell density reaches 80% confluence, digest and collect the cells, resuspend them in freezing buffer, and adjust the cell concentration.

2. The method for preparing a stem cell preparation for knee joint treatment according to claim 1, characterized in that: In step 4, the complete culture medium is: 89% MSCBM + 10% UltraGRO™ + 1% penicillin-streptomycin.

3. The method for preparing a stem cell preparation for knee joint treatment according to claim 1, characterized in that: In step 4, cut the Wharton's jelly into 2-3 mm pieces. 3 organizational blocks.

4. The method for preparing a stem cell preparation for knee joint treatment according to claim 1, characterized in that: In the step 5, 0.25% trypsin-EDTA is used to digest and passage the primary umbilical cord mesenchymal stem cells.

5. The method for preparing a stem cell preparation for knee joint treatment according to claim 1, characterized in that: In step six, 0.25% trypsin-EDTA digestion solution is used.

6. The method for preparing a stem cell preparation for knee joint treatment according to claim 1, characterized in that: In step 7, the cell concentration was adjusted to 1×10 6 -5×10 6 cells / mL.

7. The method for preparing a stem cell preparation for knee joint treatment according to claim 1, characterized in that: In step 7, the freezing solution is: 90% FBS + 10% DMSO.

8. The method for preparing a stem cell preparation for knee joint treatment according to claim 1, characterized in that: In step seven, the freezing procedure is: 4°C, waiting; 1°C / min, sample temperature -5°C; 21°C / min, box temperature -54°C; 17°C / min, box temperature -17°C; 2°C / min, sample temperature -40°C; 10°C / min, sample temperature -80°C; transfer to a liquid nitrogen tank for long-term storage.

9. A stem cell preparation prepared by the method for preparing a stem cell preparation for knee joint treatment according to any one of claims 1 to 8.

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