Method for preparing umbilical cord mesenchymal stem cells by using anticoagulant storage and transportation liquid

Abstract

The invention discloses a method for preparing umbilical cord mesenchymal stem cells by using an anticoagulant storage and transportation liquid, and relates to the technical field of umbilical cord tissue preservation and treatment.The method comprises the following steps that S1, collected umbilical cord tissues are placed in a storage and transportation bottle containing the anticoagulant storage and transportation liquid; s2, the umbilical cord tissue is taken out, the outer surface is washed with the anticoagulant storage and transportation liquid, and the two ends are cut off; s3, cutting the umbilical cord into small sections of 1-3cm, and washing; s4, dissecting blood vessels, removing amniotic membranes, tearing off Wharton's jelly, and cutting the Wharton's jelly into small blocks; s5, spreading the tissue blocks in a culture bottle for primary culture; s6, the culture medium is replaced on the sixth day of culture, P0-generation cells are digested, subcultured and collected on the twelfth day for cell counting and motility rate detection, the P0-generation cells are cultured to the P1 generation, storage and transportation liquid is used in the storage and transportation process of the umbilical cord tissue and the separation and preparation process of the umbilical cord mesenchymal stem cells, so that the risks of blood agglutination and bacterial contamination in the umbilical cord tissue are reduced, and the survival rate of the umbilical cord tissue is increased. Agglutinated blood clots can be effectively removed and dissolved, and the number and motility rate of umbilical cord mesenchymal stem cells are increased.

Description

Technical Field

[0001] The present invention relates to the technical field of umbilical cord tissue preservation and processing, and more particularly to a method for preparing umbilical cord mesenchymal stem cells by utilizing an anticoagulant blood storage and transportation fluid. Background Art

[0002] Umbilical cord mesenchymal stem cells (UCMSCs) are multipotent stem cells found in the umbilical cord tissue of newborns. They possess self-renewal and multiple differentiation potentials, capable of developing into osteoblasts, chondrocytes, adipocytes, endothelial cells, and other cell types. Their strong proliferation capacity and low immunogenicity hold promise for broad clinical application.

[0003] However, umbilical cord mesenchymal stem cells are typically isolated from the umbilical cord's Wharton's jelly. During the umbilical cord collection process, residual blood easily remains within the tissue, and clots easily form after prolonged storage and transportation. These clots lead to a series of serious problems: clots impede the diffusion of oxygen and nutrients to tissue cells (including the target stem cells) and hinder the excretion of metabolic waste, leading to a deterioration of the local microenvironment, directly impacting the vitality and subsequent proliferation and differentiation potential of the stem cells. Clots provide a favorable attachment and propagation matrix for microorganisms such as bacteria and viruses, significantly increasing the risk of biocontamination during storage, transportation, and subsequent operations. Clots increase the viscosity of the umbilical cord tissue, making its structure cohesive and unclear, seriously interfering with the subsequent dissection of structures such as blood vessels and the amniotic membrane, as well as the pure extraction of the Wharton's jelly. This increases the difficulty and risk of injury, and may reduce the purity of the resulting stem cells. Existing techniques often require additional physical manipulation (such as peeling with tweezers) to remove formed clots, which can easily cause mechanical damage to the fragile umbilical cord tissue and the target cells within it.

[0004] Therefore, to address the above problems, a method for preparing umbilical cord mesenchymal stem cells using anticoagulant blood storage and transportation fluid is proposed. Summary of the Invention

[0005] In order to overcome the above-mentioned defects of the prior art, the present invention provides a method for preparing umbilical cord mesenchymal stem cells using an anticoagulant blood storage and transportation fluid to solve the problems raised in the above-mentioned background technology.

[0006] To achieve the above object, the present invention provides the following technical solution: a method for preparing umbilical cord mesenchymal stem cells using anticoagulant blood storage and transportation fluid, comprising the following steps: S1. Place the collected umbilical cord tissue in a storage bottle containing anticoagulant storage solution and store at 2-8°C; S2. Remove the umbilical cord tissue, rinse the outer surface with the anticoagulant blood storage and transportation solution, cut off the two ends, and then rinse until there is no blood clot; S3. Cut the umbilical cord into 1-3 cm segments and vertically flush the blood vessels three times with anticoagulant storage and transport solution. S4. Dissect the blood vessels and remove the amniotic membrane. Tear off the Wharton's jelly, wash it with anticoagulant storage and transport solution, and then cut it into 1-3 mm³ Wharton's jelly pieces. S5. Spread the tissue pieces in a culture flask containing culture medium and culture them in primary culture at 37°C and 5% CO2. S6. On the 6th day of culture, the culture medium was replaced. On the 12th day, the P0 cells were digested and subcultured to collect the cells for cell counting and viability detection. The culture medium was 1×10 6 The cells were re-plated at a density of 1 / 4 cell / bottle and cultured for 2 more days. The P0 cells were subcultured to the P1 cells.

[0007] Preferably, the anticoagulant blood storage and transportation fluid includes PBS buffer, glucose, 20% human serum albumin, sodium heparin, nattokinase, penicillin and streptomycin, the amount of the PBS buffer is 90-110 ml, the amount of glucose is 1 g-15 g, the amount of 20% human serum albumin is 1 ml-20 ml, the amount of sodium heparin is 5-100 mg, the amount of nattokinase is 2000 FU-6000 FU, the amount of penicillin is 0.5 mg-2.5 mg, and the amount of streptomycin is 0.5 mg-2.5 mg.

[0008] Preferably, the preparation method of the PBS buffer is: A1. Dissolve 8.0 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4, and 0.24 g KH2PO4 in 800 ml of distilled water. A2. Use hydrochloric acid to adjust the solution to pH 7.4; A3. Finally, dilute the volume to 1 L with distilled water.

[0009] Preferably, the preparation method of the anticoagulant blood storage and transportation solution is as follows: B1. Add glucose, human serum albumin, heparin sodium, nattokinase, penicillin, and streptomycin in 1000 ml PBS buffer in appropriate proportions and fully dissolve. B2. Filter the prepared storage and transport solution through a 0.22 μm filter and dispense it into umbilical cord storage and transport bottles, 100 ml per bottle, and store at 4°C until use.

[0010] Preferably, in step S4, the washing operation includes: First, using the anticoagulant storage and transportation solution to stir and wash the Wharton's gel once; Second, wash twice with saline.

[0011] Preferably, in step S4, after flushing three times, the umbilical cord tissue is transferred to a new culture dish, half of the anticoagulant storage and transport solution is added to the culture dish, and one vein and two arteries are dissected, the amniotic membrane is removed with tissue forceps, and Wharton's jelly is torn off.

[0012] Preferably, in step S5, the tissue blocks are pipetted using a 10 ml pipette with a head removed and evenly spread on the bottom of a culture flask containing 25 ml of culture medium, wherein the culture flask is a T175 culture flask.

[0013] Preferably, in step S6, the digestion is performed using a trypsin substitute, and the digestion time is 1 minute, and the digestion is terminated with an equal volume of fresh culture medium.

[0014] Preferably, in step S6, the synergistic ratio of heparin sodium and nattokinase is: 800-1200FU of nattokinase per 10mg of heparin sodium.

[0015] Technical effects and advantages of the present invention: Compared to existing technologies, this method for preparing umbilical cord mesenchymal stem cells using anticoagulant storage and transport fluid innovatively combines sodium heparin (an anticoagulant) with nattokinase (a thrombolytic enzyme) in the umbilical cord tissue storage and transport fluid. Sodium heparin effectively inhibits coagulation, preventing the formation of new clots; while nattokinase directly acts on the fibrin network, dissolving existing clots. This dual mechanism of "prevention + clearance" works synergistically to fundamentally address the core issue of clot formation.

[0016] Compared with the existing technology, this method of preparing umbilical cord mesenchymal stem cells using anticoagulant storage and transportation fluid contains PBS buffer to maintain osmotic pressure and pH stability, glucose to provide basic energy, and human serum albumin to provide necessary nutrients. Together, they create a relatively stable and nutritionally supportive environment for ex vivo umbilical cord tissue and its cells (especially target stem cells) during storage and transportation, helping to maintain their basic activity. At the same time, penicillin and streptomycin are added to provide broad-spectrum antibacterial protection during storage, transportation and initial processing, significantly reducing the risk of microbial contamination that may be caused by blood clots.

[0017] Compared to existing technologies, this method for preparing umbilical cord mesenchymal stem cells using anticoagulant storage and transport fluid involves immersing the umbilical cord in the anticoagulant storage and transport fluid from the start of storage and transport, and continuing to use this fluid during subsequent key steps such as flushing, internal blood vessel cleaning, and Wharton's Jelly washing. This comprehensive application ensures that the anticoagulant, thrombolytic, nutritional support, and antibacterial effects are maintained throughout the entire pretreatment process. The thrombolytic effect of the storage and transport fluid eliminates or greatly reduces the need for forceful physical removal of clots, making the process of cleaning the interior of blood vessels and removing residual blood clots more gentle, effectively reducing the risk of mechanical damage to the umbilical cord tissue and stem cells caused by physical manipulation. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a data table of the proportion examples of the anticoagulant storage and transportation solution of the present invention.

[0019] Figure 2 This is a schematic diagram of tissue culture in physiological saline on day 0 of the present invention.

[0020] Figure 3 This is a schematic diagram of a Wharton's gum block without blood clot residue after being prepared using the storage and transportation liquid in the present invention.

[0021] Figure 4 This is a schematic diagram of cells crawling out of physiological saline on the 6th day of culture in the present invention.

[0022] Figure 5 This is a comparison diagram of the cells crawling out of the first ratio on the 6th day of culture in the present invention.

[0023] Figure 6 This is a comparison diagram of the cells crawling out of the second mixture on the 6th day of culture in the present invention.

[0024] Figure 7 This is a comparison diagram of the cells crawling out of the third ratio on the 6th day of culture in the present invention. DETAILED DESCRIPTION

[0025] 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. Example 1

[0026] As attached Figures 1 to 7 A method for preparing umbilical cord mesenchymal stem cells using an anticoagulant blood storage and transportation solution includes the following steps: S1. Place the collected umbilical cord tissue in a storage bottle containing anticoagulant storage solution and store at 2-8°C; S2. Remove the umbilical cord tissue, rinse the outer surface with anticoagulant storage and transport solution, cut off both ends, and rinse again until there are no blood clots; S3. Cut the umbilical cord into 1-3 cm segments and vertically flush the blood vessels three times with anticoagulant storage and transport solution. S4. Dissect the blood vessels and remove the amniotic membrane. Tear off the Wharton's jelly, wash it with anticoagulant storage and transport solution, and then cut it into 1-3 mm³ Wharton's jelly pieces. S5. Spread the tissue pieces in a culture flask containing culture medium and culture them in primary culture at 37°C and 5% CO2. S6. On the 6th day of culture, the culture medium was replaced. On the 12th day, the P0 cells were digested and subcultured to collect the cells for cell counting and viability detection. The culture medium was 1×10 6 The cells were re-plated at a density of 1 / 4 cell / bottle and cultured for 2 more days. The P0 cells were subcultured to the P1 cells.

[0027] Specifically, S1. Place the collected umbilical cord in an umbilical cord storage bottle, cover the bottle, and store at 4 degrees Celsius. The umbilical cord storage bottle contains umbilical cord tissue storage solution, which has an anticoagulant effect and can prevent blood coagulation inside and outside the residual umbilical cord tissue; S2. In a clean bench, remove the umbilical cord tissue from the storage bottle and place it in a 150 cm2 cell culture dish. Rinse the outer surface of the umbilical cord thoroughly with clean umbilical cord storage and transportation solution. Use pointed straight scissors to cut off approximately 1 cm of length from each end of the umbilical cord and discard. Rinse the umbilical cord tissue with storage and transportation solution again until the outer surface of the tissue is clean and free of blood clots. S3. Use pointed straight scissors to cut the umbilical cord into small segments of about 2 cm in length. Use tissue forceps to hold one end of the umbilical cord segment so that the umbilical cord segment is kept perpendicular to the culture dish. Use a Pasteur pipette to absorb the storage and transportation solution to vertically rinse the inside of the umbilical cord segment, and rinse 3 times. The storage and transportation solution has a certain effect of dissolving blood clots, which can separate the blood congestion in the umbilical cord tissue and blood vessels from the tissue or dissolve it, and avoid damaging the umbilical cord tissue when using tissue forceps to force blood clots from the umbilical cord blood vessels. Transfer the umbilical cord tissue to a new culture dish, add 1 / 2 storage and transportation solution to the culture dish, and maintain the activity of tissues and cells during tissue separation. Dissect one vein and two arteries, remove the amniotic membrane with tissue forceps, and tear off the Wharton's jelly; S4-S5. Place the Wharton's gel in a 50ml centrifuge tube filled with storage and transport solution. Stir with forceps and thoroughly wash the Wharton's gel once. Pour out the storage and transport solution and then add physiological saline to wash. Repeat twice. Finally, transfer the Wharton's gel to a new 50ml centrifuge tube and cut it into 1mm3-3mm3 Wharton's gel pieces with scissors. Use a 10ml pipette with the tip removed to draw up the Wharton's gel pieces. Spread them flatly into a T175 culture flask containing 25ml of culture medium. Gently shake to evenly distribute them on the bottom of the flask. Place the flask in a 37°C, 5% CO2 incubator for primary cell culture. S6. Cell passage collection: observe under a microscope on the 6th day of culture, change the medium: use a pipette to discard the original culture medium from the culture bottle, add fresh culture medium, and continue to culture. Digestion and passage on the 12th day: use a pipette to discard the original culture medium in the culture bottle, add 3ml of trypsin substitute to digest the Wharton gel and cells at the bottom of the bottle, let it stand at 37 degrees Celsius for 1min, add the same volume of fresh culture medium to stop digestion, transfer the cells and tissues to a 50ml centrifuge tube and centrifuge, discard the supernatant, add physiological saline to resuspend, filter out the tissue blocks, and centrifuge the remaining cells. This is the P0 generation cell, and perform cell counting and viability detection. According to 1×10 6 / bottle of cells and re-plate the bottle, continue to culture for 2 days, repeat the digestion process on the 3rd day, collect the cells, which are the P1 cells. Take 1.2×10 7 Perform flow cytometry.

[0028] Based on Example 1, the solution in Example 1 is further detailed in combination with the following specific working methods. Figures 1 to 7 As shown, see the following description for details: As a preferred embodiment, the anticoagulant storage and transportation fluid includes PBS buffer, glucose, 20% human albumin, sodium heparin, nattokinase, penicillin and streptomycin, the amount of PBS buffer is 90-110 ml, the amount of glucose is 1 g-15 g, the amount of 20% human albumin is 1 ml-20 ml, the amount of sodium heparin is 5-100 mg, the amount of nattokinase is 2000FU-6000FU, the amount of penicillin is 0.5 mg-2.5 mg, and the amount of streptomycin is 0.5 mg-2.5 mg.

[0029] Specifically, the anticoagulant storage and transportation solution in actual implementation includes PBS buffer, glucose, 20% human albumin, sodium heparin, nattokinase, penicillin and streptomycin. The amount of PBS buffer is 100 ml, and the preparation method is as follows: 8.0 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4 and 0.24 g KH2PO4 are dissolved in 800 ml distilled water, and the solution is adjusted to pH 7.4 with hydrochloric acid. Finally, the volume is adjusted to 1 L with distilled water to maintain the osmotic pressure inside and outside the umbilical cord tissue cells and the normal morphology of the cells; Example 1 of the ratio of anticoagulant storage and transportation solution: The dosage of glucose is 5g, which provides energy for umbilical cord tissue during storage and transportation; the dosage of 20% human serum albumin is 5ml, which provides necessary nutrients for cells; the dosage of heparin sodium is 50mg, which prevents blood coagulation by enhancing the inhibitory effect of antithrombin III; the dosage of nattokinase is 4000FU, which directly decomposes fibrin clots to dissolve blood clots; the dosage of penicillin is 10mg, and the dosage of streptomycin is 10mg, which jointly inhibit bacterial growth to reduce the risk of contamination; the preparation method of the storage and transportation solution is to add the above ingredients in proportion to 1000ml PBS buffer solution and fully dissolve them, filter through a 0.22um filter, and then divide them into umbilical cord storage and transportation bottles, 100ml per bottle, and store at 4°C for future use, thereby effectively reducing coagulation formation and increasing cell viability and quantity during the preparation process.

[0030] Example 2 of the ratio of anticoagulant storage and transportation solution: The amount of glucose is 1g; the amount of 20% human serum albumin is 1ml; the amount of heparin sodium is 10mg; the amount of nattokinase is 1000FU; the amount of penicillin is 5mg; the amount of streptomycin is 5mg; the amount of PBS buffer is 100ml.

[0031] Example 3 of the ratio of anticoagulant storage and transportation solution: The amount of glucose is 10 g; the amount of 20% human serum albumin is 8 ml; the amount of heparin sodium is 70 mg; the amount of nattokinase is 2000 FU; the amount of penicillin is 20 mg; the amount of streptomycin is 20 mg; and the amount of PBS buffer is 100 ml.

[0032] By comparing Examples 1, 2, and 3, it can be found that the ratio of heparin sodium to nattokinase strictly complies with 1:80-120 (50 mg:4000 FU), achieving complete clearance of blood clots, and the number of viable cells in the PO generation was 4.37×10 6 , viability 95.35%; by comparison, it can be seen that the synergistic ratio of heparin sodium and nattokinase (1:80-120) is the key to improving cell quantity and quality. If this ratio is deviated from, even if other ingredients are adjusted, the viability and cell number will still be significantly reduced.

[0033] As a preferred embodiment, the preparation method of PBS buffer is: A1. Dissolve 8.0 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4, and 0.24 g KH2PO4 in 800 ml of distilled water. A2. Use hydrochloric acid to adjust the solution to pH 7.4; A3. Finally, dilute the volume to 1 L with distilled water.

[0034] As a preferred embodiment, the preparation method of the anticoagulant storage and transportation solution is as follows: B1. Add glucose, human serum albumin, heparin sodium, nattokinase, penicillin, and streptomycin in 1000 ml PBS buffer in appropriate proportions and fully dissolve. B2. Filter the prepared storage and transport solution through a 0.22 μm filter and dispense it into umbilical cord storage and transport bottles, 100 ml per bottle, and store at 4°C until use.

[0035] Specifically, in actual implementation, 8.0 g of NaCl, 0.2 g of KCl, 1.44 g of Na2HPO4, and 0.24 g of KH2PO4 were weighed and dissolved in 800 ml of distilled water to form an ionic base solution, ensuring the supply of key electrolytes such as sodium, potassium, and phosphate to maintain the balance of osmotic pressure inside and outside the cells; hydrochloric acid was then used to finely adjust the pH of the solution to 7.4. This pH value simulates the physiological environment and prevents the cells from being damaged in morphology due to acid-base fluctuations during storage and transportation, thereby ensuring the activity of umbilical cord tissue cells; finally, the volume was adjusted to 1 L with distilled water to obtain a standardized PBS buffer. This operation ensures a consistent buffer concentration, which facilitates subsequent synergistic action with other components of the anticoagulant storage and transportation solution (such as sodium heparin and nattokinase), effectively preventing coagulation and maintaining tissue integrity during storage, transportation, and separation. As shown in the examples in the briefing document, this buffer significantly increases the cell number and viability of umbilical cord mesenchymal stem cells by maintaining osmotic pressure and cell stability, thereby enhancing overall preparation efficiency.

[0036] As a preferred embodiment, in step S4, the washing operation includes: First, use anticoagulant storage and transportation solution to stir and wash the Wharton's glue once; Second, wash twice with saline.

[0037] Specifically, the torn Wharton's jelly was transferred to a 50 ml centrifuge tube filled with anticoagulant storage and transport solution, and fully stirred using tissue forceps to achieve a thorough washing, thereby effectively removing residual blood clots and dissolving fibrin clots; Next, after discarding the storage and transport solution, saline was added for washing, and this process was repeated twice to ensure that components such as sodium heparin and nattokinase in the storage and transport solution were completely removed to prevent interference with subsequent culture. Finally, the washed Wharton's gel was transferred to a new 50ml centrifuge tube and prepared for shredding. This washing method significantly reduces the risk of coagulation formation through the anticoagulant and thrombolytic effects of the storage and transport solution. Simultaneously, two washes with saline help maintain cellular osmotic pressure balance, reduce the possibility of bacterial contamination, and ultimately improve the purity and viability of umbilical cord mesenchymal stem cells. As observed in the examples, no blood clots remained in the Wharton's gel after washing, and the number of cells that crawled out increased, with a viability of over 95.35%.

[0038] As a preferred embodiment, in step S4, after flushing three times, the umbilical cord tissue is transferred to a new culture dish, half of the anticoagulant storage and transport solution is added to the culture dish, and one vein and two arteries are dissected, the amniotic membrane is removed with tissue forceps, and Wharton's jelly is torn off.

[0039] Specifically, the operator needs to transfer the flushed umbilical cord tissue to a sterile culture dish, which is pre-added with half of the anticoagulant storage and transportation solution, with a volume of about 50 ml, to maintain tissue activity and prevent cell damage; then use tissue forceps to accurately dissect one vein and two arteries in the umbilical cord. During this process, excessive pulling of the tissue should be avoided. After dissection, the wrapped amniotic membrane is quickly removed with tissue forceps, and then the gelatin is gently torn off along the texture of the Wharton's jelly. This operation can reduce mechanical damage and retain the activity of mesenchymal stem cells. At the same time, the sodium heparin and nattokinase in the storage and transportation solution work synergistically to effectively dissolve residual fibrin clots, ensuring that there is no blood clot residue in the subsequent shredded Wharton's jelly, thereby improving the primary cell crawling efficiency and the final cell viability.

[0040] As a preferred embodiment, in step S5, the tissue blocks are pipetted using a 10 ml pipette with a head removed and evenly spread on the bottom of a culture flask containing 25 ml of culture medium, wherein the culture flask is a T175 culture flask.

[0041] Specifically, a 10ml pipette with a removed head is used to absorb the Wharton gel block, which has been cut into pieces of 1mm³~3mm³ in size in step S4. During the specific operation, the head of the pipette is removed to facilitate the absorption of the gel block, and then it is evenly spread on the bottom of a T175 culture flask, into which 25ml of culture medium has been added in advance. The culture flask is then gently shaken to evenly distribute the gel block on the bottom of the flask to avoid aggregation. This even distribution method helps promote the efficient crawling of cells out of the tissue block, reduces local hypoxia or malnutrition, thereby improving the efficiency of cell adhesion and proliferation, and ultimately increasing the number and viability of primary cultured cells.

[0042] As a preferred embodiment, in step S6, the digestion is performed using a pancreatic enzyme substitute, and the digestion time is 1 minute. The digestion is terminated with an equal volume of fresh culture medium, and the synergistic ratio of sodium heparin and nattokinase is: 800-1200FU of nattokinase per 10 mg of sodium heparin.

[0043] Specifically, the digestion process of step S6 uses a pancreatic enzyme substitute as a digestive agent. In actual implementation, 3 ml of pancreatic enzyme substitute is added to the culture flask and allowed to stand at 37°C for 1 minute to fully digest the Wharton's jelly and cells at the bottom of the flask. Then, an equal volume of fresh culture medium is immediately added to terminate the digestion reaction to ensure that cell damage is minimized and the cell recovery efficiency is improved. At the same time, the synergistic ratio of sodium heparin and nattokinase in the anticoagulant storage and transportation solution is strictly controlled at 800-1200 FU of nattokinase per 10 mg of sodium heparin. In actual implementation, 50 mg of sodium heparin is preferably combined with 4000 FU of nattokinase (i.e., 800 FU per 10 mg). This ratio effectively reduces blood clot formation and promotes the removal of blood congestion in umbilical cord tissue through the synergistic effect of sodium heparin anticoagulation and nattokinase dissolving fibrin, thereby significantly improving the viability of PO generation cells during digestion and passage.

[0044] The present invention works as follows: After collection, the umbilical cord tissue is immediately placed in a storage bottle containing anticoagulant storage solution and stored at 2-8°C (preferably 4°C). The storage solution contains sodium heparin (5-100 mg) to inhibit blood coagulation, nattokinase (2000-6000 FU) to dissolve formed fibrin clots, penicillin and streptomycin (0.5-2.5 mg each) to prevent microbial contamination, PBS buffer to maintain osmotic pressure, and glucose (1-15 g) and human albumin (1-20 ml) to provide energy and nutritional support. After removing the umbilical cord, rinse the outer surface repeatedly with the same storage and transport solution. Cut off 1 cm of the potentially contaminated area at both ends until no blood clots are visible. Cut the umbilical cord into 1-3 cm segments and hang them vertically in a culture dish. Use a Pasteur pipette to draw up the storage and transport solution and rinse the inside of the blood vessels three times to use the thrombolytic effect of the storage and transport solution to remove any residual blood clots. The umbilical cord vessels (1 vein + 2 arteries) were dissected, the amniotic membrane was removed, and the Wharton's jelly was removed. The Wharton's jelly was washed once with anticoagulant storage and transport solution to dissolve residual fibrin. The Wharton's jelly was then washed twice with normal saline to remove the storage and transport solution components and minced into 1-3 mm³ tissue blocks. Use a 10ml pipette with a cut end to draw up the tissue fragments and evenly spread them on the bottom of a T175 culture flask containing 25ml of culture medium. Incubate at 37°C with 5% CO2. Pre-treatment with the storage and transport solution ensures that the tissue fragments are free of residual blood clots and promotes efficient cell migration. Replace the culture medium with fresh one to maintain cell growth, discard the old culture medium, add 3 ml of trypsin substitute and digest for 1 minute, immediately stop the digestion with an equal volume of fresh culture medium, collect cells and tissues, discard the supernatant after centrifugation, resuspend with physiological saline and filter to remove tissue chunks to obtain P0 generation cells, repeat the digestion steps to collect P1 generation cells for flow cytometry detection.

[0045] The above is the working principle of a method for preparing umbilical cord mesenchymal stem cells using anticoagulant blood storage and transportation fluid.

Claims

1. A method for preparing umbilical cord mesenchymal stem cells using anticoagulant blood storage and transportation fluid, characterized in that: The following steps are involved: S1. Place the collected umbilical cord tissue in a storage bottle containing anticoagulant storage solution and store at 2-8°C; S2. Remove the umbilical cord tissue, rinse the outer surface with the anticoagulant blood storage and transportation solution, cut off the two ends, and then rinse until there is no blood clot; S3. Cut the umbilical cord into 1-3 cm segments and vertically flush the blood vessels three times with anticoagulant storage and transport solution. S4. Dissect the blood vessels and remove the amniotic membrane. Tear off the Wharton's jelly, wash it with anticoagulant storage and transport solution, and then cut it into 1-3 mm³ Wharton's jelly pieces. S5. Spread the tissue pieces in a culture flask containing culture medium and culture them in primary culture at 37°C and 5% CO2. S6. On the 6th day of culture, the culture medium was replaced. On the 12th day, the P0 cells were digested and subcultured to collect the cells for cell counting and viability detection. The culture medium was 1×10 6 The cells were re-plated at a density of 1 / 4 cell / bottle and cultured for 2 more days. The P0 cells were subcultured to the P1 cells.

2. The method for preparing umbilical cord mesenchymal stem cells using an anticoagulant blood storage and transportation solution according to claim 1, characterized in that: The anticoagulant blood storage and transportation solution comprises PBS buffer, glucose, 20% human serum albumin, heparin sodium, nattokinase, penicillin and streptomycin.

3. The method for preparing umbilical cord mesenchymal stem cells using an anticoagulant blood storage and transportation solution according to claim 2, wherein: The amount of PBS buffer is 90-110 ml, the amount of glucose is 1 g-15 g, the amount of 20% human serum albumin is 1 ml-20 ml, the amount of heparin sodium is 5-100 mg, the amount of nattokinase is 2000 FU-6000 FU, the amount of penicillin is 0.5 mg-2.5 mg, and the amount of streptomycin is 0.5 mg-2.5 mg.

4. The method for preparing umbilical cord mesenchymal stem cells using an anticoagulant blood storage and transportation solution according to claim 2, wherein: The preparation method of the PBS buffer solution is: A1. Dissolve 8.0 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4, and 0.24 g KH2PO4 in 800 ml of distilled water. A2. Use hydrochloric acid to adjust the solution to pH 7.4; A3. Finally, dilute the volume to 1 L with distilled water.

5. The method for preparing umbilical cord mesenchymal stem cells using an anticoagulant blood storage and transportation solution according to claim 2, wherein: The preparation method of the anticoagulant blood storage and transportation solution is as follows: B1. Add glucose, human serum albumin, heparin sodium, nattokinase, penicillin, and streptomycin in 1000 ml PBS buffer in appropriate proportions and fully dissolve. B2. Filter the prepared storage and transport solution through a 0.22 μm filter and dispense it into umbilical cord storage and transport bottles, 100 ml per bottle, and store at 4°C until use.

6. The method for preparing umbilical cord mesenchymal stem cells using an anticoagulant blood storage and transportation solution according to claim 1, characterized in that: In step S4, the washing operation includes: First, using the anticoagulant storage and transportation solution to stir and wash the Wharton's gel once; Second, wash twice with saline.

7. The method for preparing umbilical cord mesenchymal stem cells using an anticoagulant blood storage and transportation solution according to claim 1, characterized in that: In step S4, after rinsing three times, the umbilical cord tissue is transferred to a new culture dish, half of the anticoagulant storage and transport solution is added to the culture dish, and one vein and two arteries are dissected, the amniotic membrane is removed with tissue forceps, and Wharton's jelly is torn off.

8. The method for preparing umbilical cord mesenchymal stem cells using an anticoagulant blood storage and transportation solution according to claim 1, characterized in that: In step S5, the tissue blocks are pipetted using a 10 ml pipette with a head removed and evenly spread on the bottom of a culture flask containing 25 ml of culture medium. The culture flask is a T175 culture flask.

9. The method for preparing umbilical cord mesenchymal stem cells using an anticoagulant blood storage and transportation solution according to claim 1, characterized in that: In step S6, the digestion is performed using a trypsin substitute for 1 minute, and the digestion is terminated by adding an equal volume of fresh culture medium.

10. The method for preparing umbilical cord mesenchymal stem cells using an anticoagulant blood storage and transportation solution according to claim 1, characterized in that: In step S6, the synergistic ratio of heparin sodium and nattokinase is: 800-1200 FU of nattokinase per 10 mg of heparin sodium.

Images