CN112056307A - Umbilical cord preservation liquid and umbilical cord preservation method - Google Patents

Abstract

The invention discloses an umbilical cord preservation solution and an umbilical cord preservation method, wherein the umbilical cord preservation solution comprises the following components in terms of 1L of preservation solution: 15-20 mmol of hyperbranched polyglycerol, 99-101 mmol of lactobionic acid, 24-26 mmol of monopotassium phosphate, 4-6 mmol of magnesium sulfate, 4-6 mmol of adenosine, 2-4 mmol of glutathione reduced form, 6-10 mg of dexamethasone, 0.5-1.5 mmol of allopurinol, 50-70 mmol of sucrose, 40-60 g of dextran-4040, 18-22 mg of isobodidine, 4-6 ml of sodium hydroxide and 19-21 ml of potassium hydroxide. When the umbilical cord is stored, the cleaned umbilical cord is cut into small sections with the length of 3-5 cm, then the small sections are immersed into the storage liquid, and the umbilical cord is stored in the environment with the temperature of 3-5 ℃. The preservation solution can effectively solve the problem that the umbilical cord can not be preserved for a long time after being isolated.

Description

A kind of umbilical cord preservation solution and umbilical cord preservation method

technical field

The invention belongs to the technical field of umbilical cord preservation, and in particular relates to an umbilical cord preservation solution and an umbilical cord preservation method.

Background technique

Mesenchymal stem cells (MSCs) are important members of the stem cell family. They are derived from the mesoderm in the early stage of development and belong to pluripotent stem cells. MSCs were originally discovered in bone marrow and are stromal fibroblasts. The characteristics of differentiation potential, hematopoietic support and promotion of stem cell engraftment, immune regulation and self-replication have attracted increasing attention. Previous studies have shown that MSCs are low immunogenic, capable of evading immune detection, and possess immunosuppressive properties and tissue repair/regeneration.

Bone marrow-derived MSCs are the earliest and most in-depth study of mesenchymal stem cells, but the process of isolating bone marrow MSCs is complicated and may lead to trauma to the donor, and the number, proliferation and differentiation ability of the obtained MSCs increase with the age of the donor. While significantly decreased, these factors limit the application of bone marrow-derived MSCs. Therefore, in recent years, obtaining MSCs from discarded fetal tissues (such as placenta, umbilical cord, umbilical cord blood, etc.) has become an important direction of stem cell research. Relevant studies have also confirmed that MSCs derived from the umbilical cord have the same immune properties as MSCs derived from bone marrow, and have similar application prospects. Compared with bone marrow-derived MSCs, umbilical cord-derived MSCs have the advantages of easier isolation, culture and proliferation. It is precisely because the source of umbilical cord MSCs is more abundant that they have high proliferation ability and in vitro passaging ability in vitro, and there are no ethical issues. It has more advantages in clinical application.

However, at present, the isolation of MSCs needs to be carried out in a short time after the umbilical cord leaves the mother. Otherwise, a sufficient amount of primary MSCs cannot be isolated, which will affect the subsequent MSC culture and proliferation. The existing umbilical cords are mostly preserved by soaking in 4° C. physiological saline or lactated Ringer's solution, and the preservation time is short, and the umbilical cords need to be processed in a short time. However, the isolation, culture and proliferation of MSCs all require high-grade sterility and laboratories with relevant conditions to operate, so the umbilical cord transportation time is relatively high. In order to prolong the preservation and transportation time of the umbilical cord and ensure the effect of MSC isolation, culture and proliferation, it is very important to find a new preservation solution.

SUMMARY OF THE INVENTION

In view of the above-mentioned prior art, the present invention provides an umbilical cord preservation solution and an umbilical cord preservation method, so as to solve the problem that the umbilical cord cannot be preserved for a long time after being separated from the body.

In order to achieve the above object, the technical scheme adopted in the present invention is: a kind of umbilical cord preservation solution is provided, and the preservation solution in the present invention is calculated by 1L volume, including:

Hyperbranched polyglycerol 15～20mmol, lacturonic acid 99～101mmol, potassium dihydrogen phosphate 24～26mmol, magnesium sulfate 4～6mmol, adenosine 4～6mmol, glutathione reduced 2～4mmol, dexamethasone 6～ 10mg, allopurinol 0.5～1.5mmol, sucrose 50～70mmol, dextran-40 40～60g, verapamil 18～22mg, sodium hydroxide 4～6ml and potassium hydroxide 19～21ml.

On the basis of the above technical solutions, the present invention can also be improved as follows.

Further, the preservation solution in the present invention is measured in 1L volume, including:

Hyperbranched polyglycerol 18mmol, lacturonic acid 100mmol, potassium dihydrogen phosphate 25mmol, magnesium sulfate 5mmol, adenosine 5mmol, glutathione reduced 3mmol, dexamethasone 8mg, allopurinol 1mmol, sucrose 60mmol, dextran-4050g, Verapamil 20mg, Sodium Hydroxide 5ml and Potassium Hydroxide 20ml.

Further, the pH of the preservation solution was 7.45±0.10, and the osmotic pressure was 310±10 mOSM/L.

When adopting the preservation solution of the present invention to preserve the umbilical cord, the preservation process comprises the following steps:

S1: Take the umbilical cord of the newborn, wash it with alcohol and PBS solution in turn, and then divide the washed umbilical cord into 3-5 cm sections;

S2: Put the segmented umbilical cord into a preservation container, add the umbilical cord preservation solution according to any one of claims 1 to 3, subject to the umbilical cord being completely submerged, and then place the preservation container in an environment of 3 to 5°C Save it below.

The beneficial effects of the present invention are:

Compared with the common preservation solution, the preservation solution of the present invention can still separate MSCs from the umbilical cord after being stored for 7 days, and the osteogenic differentiation, fibroblast differentiation and chondrogenic differentiation abilities of the MSCs separated from the umbilical cord when the umbilical cord is stored for 7 days are the same as those of MSCs separated from fresh umbilical cords. resemblance. Flow cytometry analysis showed that the cells isolated from the umbilical cord after storage for 1 day and 7 days highly expressed CD73+ and CD90+, but did not express CD34- and CD45-, which was consistent with the immunophenotype of MSC.

Description of drawings

Figure 1 shows the tissue morphology under the microscope 6 to 8 days after seeding;

Figure 2 shows the tissue morphology under the microscope 10 to 12 days after seeding;

Figure 3 shows the results of the fibroblast differentiation investigation of P1 generation umbilical cord mesenchymal stem cells;

Figure 4 shows the results of chondrogenic differentiation of P1 generation umbilical cord mesenchymal stem cells;

Figure 5 shows the results of the osteogenic differentiation investigation of P1 generation umbilical cord mesenchymal stem cells;

Figure 6 is the expression result of cell CD90+;

Fig. 7 is the expression result of cell CD73+;

Fig. 8 is the expression result of cell CD45-;

Figure 9 shows the expression results of CD34- in cells.

Detailed ways

The specific embodiments of the present invention will be described in detail below with reference to the examples.

Example 1

An umbilical cord preservation solution, measured in 1 L of preservation solution, comprising:

Hyperbranched polyglycerol 18mmol, lacturonic acid 100mmol, potassium dihydrogen phosphate 25mmol, magnesium sulfate 5mmol, adenosine 5mmol, glutathione reduced 3mmol, dexamethasone 8mg, allopurinol 1mmol, sucrose 60mmol, dextran-4050g, Verapamil 20mg, sodium hydroxide (50mmol/L) 5ml and potassium hydroxide (50mmol/L) 20ml.

The pH of the preservation solution in this example is about 7.45, and the osmotic pressure is about 310 mOSM/L.

Example 2

An umbilical cord preservation solution, measured in 1 L of preservation solution, comprising:

Hyperbranched polyglycerol 15mmol, lacturonic acid 101mmol, potassium dihydrogen phosphate 24mmol, magnesium sulfate 6mmol, adenosine 4mmol, glutathione reduced 4mmol, dexamethasone 6mg, allopurinol 1.5mmol, sucrose 50mmol, dextran-4060g , verapamil 18mg, sodium hydroxide (50mmol/L) 6ml and potassium hydroxide (50mmol/L) 19ml.

The pH of the preservation solution in this example is about 7.35, and the osmotic pressure is about 300 mOSM/L.

Example 3

An umbilical cord preservation solution, measured in 1 L of preservation solution, comprising:

Hyperbranched polyglycerol 20mmol, lacturonic acid 99mmol, potassium dihydrogen phosphate 26mmol, magnesium sulfate 4mmol, adenosine 6mmol, glutathione reduced 2mmol, dexamethasone 10mg, allopurinol 0.5mmol, sucrose 70mmol, dextran-4040g , verapamil 22mg, sodium hydroxide (50mmol/L) 4ml and potassium hydroxide (50mmol/L) 21ml.

The pH of the preservation solution in this example is about 7.55, and the osmotic pressure is about 320 mOSM/L.

Experimental example

The umbilical cords were preserved with the preservation solutions in Examples 1 to 3, respectively, and MSCs were taken out and isolated after 7 days. Since the preservation effect of the preservation solution in Examples 1 to 3 is basically the same, the preservation effect of the preservation solution in the present invention will be described using the preservation solution in Example 1 as an example.

(1) Umbilical cord preservation

In the present invention, when carrying out umbilical cord preservation, the following steps are included:

S1: Take the umbilical cord of the newborn, wash it with alcohol and PBS solution in turn, and then divide the washed umbilical cord into 3-5 cm sections;

S2: Put the segmented umbilical cord into a preservation container, add the umbilical cord preservation solution according to any one of claims 1 to 3, subject to the umbilical cord being completely submerged, and then place the preservation container in an environment of about 4°C Save it.

(2) Separation and culture of umbilical cord stem cells after storage for 7 days

Separation and culture of umbilical cord stem cells in the present invention include the following steps:

S1: Wipe the ultra-clean bench with 75% medical alcohol, and put the sterilized experimental equipment into the ultra-clean bench in turn;

S2: Take out the umbilical cord stored in the umbilical cord preservation solution, cut the umbilical cord about 1 cm long and put it in the culture dish, add about 10 mL of PBS buffer, cut the umbilical cord with sterile scissors, and find one umbilical cord vein and two umbilical cords Arteries, carefully remove blood vessels and blood stains with sterile hemostatic forceps, discard the waste fluid and rinse;

S3: Put the devascularized umbilical cord segment into a new culture dish and wash it three times with PBS buffer until there is no blood or blood vessels in the naked eye, discard the waste liquid, and cut the umbilical cord into small pieces with sterile scissors;

S4: Take out the sterile culture plate, put the cut umbilical cord tissue into the sterile culture plate, gently press the tissue stack to make the umbilical cord tissue adhere to the bottom of the sterile culture plate, and place it at 37°C, 5% CO after inoculation ₂ and in a saturated humidity incubator for 4h;

S5: After 4 hours, take out the sterile culture plate from the incubator, and observe whether the umbilical cord tissue is close to the bottom of the culture plate. After confirming that it is close, add 10 mL of df12+10% FBS complete culture solution with a 10 mL pipette, do not shake , so that the culture medium just covers the small pile of tissue, and the small pile of tissue cannot be separated from the bottom of the culture bottle, and then placed in a 37°C, 5% CO ₂ and saturated humidity incubator to continue culturing;

S6: After 48 hours, the first half of the medium was changed, and then the full amount of the medium was changed every 5 days. During the medium change process, in order to prevent the small pile of tissue from falling off the bottom of the culture bottle, the old culture medium could only be sucked off with a pipette instead of being poured directly. Pay attention when adding liquid. To add liquid along the side of the culture bottle;

S7: Cultured for about 6-8 days, a small amount of adherent cells can be seen crawling out of the small piles of tissue under the microscope, and then the umbilical cord mesenchymal stem cells are gradually expanded. The results are shown in Figure 1; The umbilical cord mesenchymal stem cells will come off the bottom of the culture flask and float in the culture medium, and the umbilical cord mesenchymal stem cells adhere to 80-90% of the cells. Or the first passage can be done when there are more local cell colonies dense.

The fresh umbilical cord was isolated and cultured by the above method, and compared with the stem cells isolated from the umbilical cord preserved by the preservation solution of the present invention. The results are listed in Table 1.

Table 1 Characteristics of umbilical cord isolated stem cells

In addition, the osteogenic differentiation, fibroblast differentiation, and chondrogenic differentiation abilities of the P1 generation of MSCs isolated from the preserved umbilical cord were examined, and the results are shown in FIG. 3 , FIG. 4 , and FIG. 5 , respectively.

As can be seen from Table 1 and Figures 3 to 5, after the umbilical cord preservation solution of the present invention is used to preserve the umbilical cord for 7 days, a sufficient amount of MSCs can still be isolated, and the osteogenic differentiation, fibroblastic differentiation, and formation of the isolated MSCs can be isolated. The cartilage differentiation ability is similar to that of MSCs obtained from fresh umbilical cord; the preserved umbilical cord is similar to fresh umbilical cord, indicating that the preservation solution of the present invention has a good preservation effect.

In order to prove that the cells isolated from the preserved umbilical cord are indeed MSCs, the expression of CD73+, CD90+, CD34-, CD45-, etc. in the cells was analyzed by flow cytometry, and the results are shown in Figures 6-9. As can be seen from the figure, after 1 day and 7 days of storage, the isolated cells highly expressed CD73+ and CD90+, but did not express CD34- and CD45-, which was in line with the immunophenotype of MSC, and the isolated cells were MSCs.

Although the specific embodiments of the present invention have been described in detail with reference to the examples, they should not be construed as limiting the protection scope of the present patent. Within the scope described in the claims, various modifications and variations that can be made by those skilled in the art without creative efforts still belong to the protection scope of this patent.

Claims (5)

1. An umbilical cord preservation solution characterized by comprising, in 1L of preservation solution:

15-20 mmol of hyperbranched polyglycerol, 99-101 mmol of lactobionic acid, 24-26 mmol of monopotassium phosphate, 4-6 mmol of magnesium sulfate, 4-6 mmol of adenosine, 2-4 mmol of glutathione reduced form, 6-10 mg of dexamethasone, 0.5-1.5 mmol of allopurinol, 50-70 mmol of sucrose, 40-60 g of dextran-4040, 18-22 mg of isobodidine, 4-6 ml of sodium hydroxide and 19-21 ml of potassium hydroxide.

2. The umbilical cord preservation solution according to claim 1, comprising, in terms of 1L preservation solution:

18mmol of hyperbranched polyglycerol, 100mmol of lactobionic acid, 25mmol of monopotassium phosphate, 5mmol of magnesium sulfate, 5mmol of adenosine, 3mmol of glutathione reduced form, 8mg of dexamethasone, 1mmol of allopurinol, 60mmol of sucrose, 4050 g of dextran, 20mg of isobinder, 5ml of sodium hydroxide and 20ml of potassium hydroxide.

3. The umbilical cord preservation solution according to claim 1, wherein: the pH value of the preservation solution is 7.45 +/-0.10, and the osmotic pressure is 310 +/-10 mOSM/L.

4. A method for preserving an umbilical cord, comprising the steps of:

s1: taking a newborn umbilical cord, sequentially cleaning the newborn umbilical cord with alcohol and a PBS solution, and dividing the cleaned umbilical cord into small sections with the length of 3-5 cm;

s2: putting the segmented umbilical cord into a storage container, adding the umbilical cord storage solution according to any one of claims 1 to 3 until the umbilical cord is completely submerged, and then placing the storage container in an environment at 3-5 ℃ for storage.

5. The saving method according to claim 4, comprising the steps of: the storage temperature in S2 was 4 ℃.

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