CN112471138B - Universal preservation solution for human umbilical cord, amnion and placenta samples and preparation method thereof - Google Patents

Abstract

The invention belongs to the technical field of stem cells, and particularly relates to a universal preservation solution for human umbilical cord, amnion and placenta samples and a preparation method thereof. In the universal preservation solution, a DMEM basic culture medium provides the most basic nutrition for mesenchymal stem cells of a sample; the insulin can promote the mesenchymal stem cells of the sample to absorb and utilize sugar in the universal preservation solution, and maintain the activity of the cells; the human serum albumin provides dynamic balance for the mesenchymal stem cells and maintains the osmotic pressure of the cells; the vitamin C can prevent the damage of free radicals generated by long-time oxidation to the mesenchymal stem cells; EGF can promote the proliferation of mesenchymal stem cells and maintain the original form of the cells. Experimental results show that the preservation solution can be used for preserving umbilical cords, amnion and placenta for a long time, the obtained mesenchymal stem cells are large in quantity and high in survival rate, and human serum albumin, insulin, vitamin C and EGF have a synergistic effect in improving the number of the mesenchymal stem cells in the preserved umbilical cords and the survival rate of the mesenchymal stem cells.

Description

A universal preservation solution for human umbilical cord, amniotic membrane and placenta samples and preparation method thereof

technical field

The invention belongs to the technical field of stem cells, and in particular relates to a universal preservation solution for samples of human umbilical cord, amniotic membrane and placenta and a preparation method thereof.

Background technique

Mesenchymal stem cells are a type of cells with self-renewal and multi-directional differentiation potential, also known as pluripotent stromal cells, referred to as MSCs, which are a type of pluripotent stem cells belonging to the mesoderm, mainly present in connective tissues and organ interstitium. Including: bone marrow, umbilical cord, fat, amniotic membrane, dental pulp, placenta, etc. Under suitable conditions, it can differentiate into various tissue cells such as fat, bone and cartilage. Compared with bone marrow-derived MSCs, umbilical cord, amniotic membrane and placenta-derived mesenchymal stem cells are easy to obtain, have no ethical and ethical disputes, can obtain a large number of cells, have a strong proliferation ability, have a strong immune regulation effect, and secrete cell growth factors. The total amount is also very high, which is convenient for expansion and passage, and at the same time, there are no problems such as matching and rejection. It is extremely suitable for clinical research and application, and is an ideal source of mesenchymal stem cells.

However, mesenchymal stem cells need to be prepared and isolated in a GMP laboratory, efficiently purified, and rapidly expanded from samples in order to produce cells that meet the standards. Therefore, the safe preservation of samples has become a basic requirement for the production of mesenchymal stem cells. The sample from the hospital delivery room to the GMP laboratory takes at least a few hours to several days. The sample will be affected by factors such as time, temperature, nutrition, etc., resulting in a small number of cells in the sample and low viability.

Therefore, it has always been an urgent problem for those skilled in the art to seek a universal preservation solution that can preserve the umbilical cord, amniotic membrane and placenta for a long time.

SUMMARY OF THE INVENTION

In view of this, the present invention provides a universal preservation solution for human umbilical cord, amniotic membrane and placenta samples and a preparation method thereof. The universal preservation solution can be used to preserve the umbilical cord, amniotic membrane and placenta for a long time, and the obtained mesenchymal stem cells are large in number, High survival rate.

The concrete technical scheme of the present invention is as follows:

A universal preservation solution for samples of human umbilical cord, amniotic membrane and placenta, the universal preservation solution is composed of DMEM basal medium, insulin, human albumin, vitamin C, EGF, penicillin, streptomycin and heparin sodium;

The pH value of the universal preservation solution is 7.5-7.8.

Preferably, the concentration of the insulin in the universal preservation solution is 10-20 mg/L;

The concentration of the human albumin in the general preservation solution is 10-15g/L;

The concentration of the vitamin C in the general preservation solution is 100-200 mg/L;

The concentration of the EGF in the general preservation solution is 10-50 μg/L.

Preferably, the concentration of the insulin in the universal preservation solution is 10 mg/L;

The concentration of the human albumin in the universal preservation solution is 10 g/L;

The concentration of the vitamin C in the general preservation solution is 100 mg/L;

The concentration of the EGF in the general preservation solution is 10 μg/L.

Preferably, the concentration of the penicillin in the universal preservation solution is 50,000-100,000 U/L;

The concentration of the streptomycin in the general preservation solution is 50-100 mg/L;

The concentration of the heparin sodium in the universal preservation solution is 6250-12500 U/L.

Preferably, the concentration of the penicillin in the universal preservation solution is 100,000 U/L;

The concentration of the streptomycin in the general preservation solution is 100 mg/L;

The concentration of the heparin sodium in the universal preservation solution is 6250 U/L.

The present invention also provides the preparation method of the general preservation solution described in the above technical solution, comprising the following steps:

Human serum albumin and heparin sodium were added to the DMEM basal medium, and then penicillin, streptomycin, insulin, vitamin C and EGF were added in sequence, and then filtered through a membrane to obtain the universal preservation solution.

The present invention also provides a method for preserving the sample, comprising the following steps:

The sample is preserved by adopting the universal preservation solution described in the above technical solution;

The sample is human umbilical cord, amniotic membrane and/or placenta.

Preferably, the target cells of the sample are mesenchymal stem cells.

Preferably, the storage time is less than 72h.

To sum up, the present invention provides a general preservation solution, which is composed of DMEM basal medium, insulin, human albumin, vitamin C, EGF, penicillin, streptomycin and heparin sodium; the The pH of the general preservation solution is 7.5 to 7.8. In the present invention, the DMEM basal medium provides the most basic nutrition for the mesenchymal stem cells of the sample; insulin can promote the mesenchymal stem cells of the sample to absorb and utilize the sugar in the universal preservation solution to maintain cell activity; human albumin is Mesenchymal stem cells provide dynamic balance and maintain cell osmotic pressure; vitamin C can prevent free radicals generated by long-term oxidation from causing damage to mesenchymal stem cells; EGF can promote the proliferation of mesenchymal stem cells and maintain the original shape of cells; penicillin and Streptomycin can prevent sample contamination caused by bacterial growth; heparin sodium can prevent blood from coagulating into blood clots and reduce metabolism; the pH of the universal preservation solution is 7.5 to 7.8, which provides a suitable acid-base environment for mesenchymal stem cells. The experimental results show that the universal preservation solution can be used to preserve the umbilical cord, amniotic membrane and placenta for a long time, and the obtained mesenchymal stem cells have a large number and high viability. The number of mesenchymal stem cells and the viability of mesenchymal stem cells have a synergistic effect. In addition, the shelf life of the universal preservation solution can be as long as half a year, and can be prepared in large quantities at one time to control costs.

Description of drawings

In order to illustrate the embodiments of the present invention or the technical solutions in the prior art more clearly, the following briefly introduces the accompanying drawings that are required to be used in the description of the embodiments or the prior art.

FIG. 1 is a graph showing the viability of mesenchymal stem cells after umbilical cord was preserved for 24h, 48h and 72h by different preservation methods in Example 2 of the present invention.

Detailed ways

The invention provides a universal preservation solution for human umbilical cord, amniotic membrane and placenta samples and a preparation method thereof. The universal preservation solution can be used to preserve the umbilical cord, amniotic membrane and placenta for a long time, and the obtained mesenchymal stem cells have a large number and high viability.

The technical solutions in the embodiments of the present invention will be described clearly and completely below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

Example 1

The present embodiment carries out the preparation of general preservation solution, comprising the following steps:

Add 50mL of 20% human albumin (the concentration of human albumin is 10g/50mL) and 1mL of heparin sodium injection in 950mL DMEM basal medium, mix well, and then add 100000U penicillin, 100mg streptomycin, 10mg insulin, 100mg Vitamin C and 10 μg EGF were dissolved by stirring, and then filtered through a 0.22 μm sterile filter to remove bacteria to obtain a general preservation solution, marked with the production date, and stored at 4°C.

Among them, the concentration of heparin sodium injection is 6250U/mL.

Comparative Example 1

In this comparative example, the control universal preservation solution 1 was prepared, and the preparation method was the same as that of Example 1, but the control universal preservation solution 1 replaced 50 mL of 20% human albumin with an equal volume of PBS.

Comparative Example 2

In this comparative example, the preparation of reference universal preservation solution 2 is carried out, and the preparation method is the same as that of Example 1, but the comparison universal preservation solution 2 is replaced with PBS of the same quality as insulin.

Comparative Example 3

In this comparative example, the reference universal preservation solution 3 was prepared, and the preparation method was the same as that of Example 1, but the reference universal preservation solution 3 replaced vitamin C with PBS of the same quality.

Comparative Example 4

In this comparative example, the preparation of the reference universal preservation solution 4 is carried out, and the preparation method is the same as that of Example 1, but the reference universal preservation solution 4 is replaced with PBS of the same quality as EGF.

Example 2

Under aseptic conditions, take 24cm of umbilical cords of neonates with healthy cesarean section and no infectious diseases, evenly divide them into 8 parts, and store them in the general preservation solution of Example 1, physiological saline, DMEM medium, sterile container, and control universal respectively. Preservation Solution 1, Control Universal Preservation Solution 2, Control Universal Preservation Solution 3, and Control Universal Preservation Solution 4 were stored at 4°C for 24h, 48h, and 72h, and then the same length of umbilical cord tissue was taken to separate umbilical cord mesenchyme by collagenase digestion. Stem cells, filtered and centrifuged, were counted using trypan blue, and cell viability was calculated.

Please refer to Fig. 1, Table 1 and Table 2. Fig. 1 is a graph showing the viability of mesenchymal stem cells after the umbilical cord is preserved for 24h, 48h and 72h by different preservation methods in Example 2 of the present invention. , the number of mesenchymal stem cells after 48h and 72h, and Table 2 shows the viability of mesenchymal stem cells after 24h, 48h and 72h of umbilical cord preservation by different preservation methods. The results show that the universal preservation solution of Example 1 can preserve the umbilical cord within 72 hours, can ensure that the isolated mesenchymal stem cells have a viability of more than 90% within 72 hours, and can maximize the maintenance of the umbilical cord mesenchymal stem cells. It can improve the extraction degree of umbilical cord mesenchymal stem cells, greatly improve the post-cultivation of mesenchymal stem cells and save costs. Physiological saline and DMEM basal medium can preserve the umbilical cord within 24 hours, but with the extension of time, the number and viability of the obtained umbilical cord mesenchymal stem cells gradually decrease; the umbilical cord stored in the sterile container loses the liquid environment, and the cells There was a large amount of water shortage during the period, which led to the rapid apoptosis of umbilical cord mesenchymal stem cells, and the viable umbilical cord mesenchymal stem cells could not be isolated at 48 h. And, compared with the control universal preservation solution 1, the control universal preservation solution 2, the control universal preservation solution 3 and the control universal preservation solution 4, the number of umbilical cord mesenchymal stem cells and the viability of mesenchymal stem cells preserved by the universal preservation solution of Example 1 were compared. The rate was significantly higher, indicating that human serum albumin, insulin, vitamin C and EGF have a synergistic effect in increasing the number of mesenchymal stem cells in the preserved umbilical cord and the viability of mesenchymal stem cells.

Table 1 The number of mesenchymal stem cells after preservation of umbilical cord for 24h, 48h and 72h by different preservation methods

preservation method 24h 48h 72h Example 1 Universal preservation solution 7.17×10⁶ 6.78×10⁶ 6.36×10⁶ normal saline 8.02×10⁵ 7.88×10⁵ 5.65×10⁵ DMEM medium 4.32×10⁶ 3.01×10⁶ 1.94×10⁶ sterile container 3.86×10³ 0 0 Control Universal Preservation Solution 1 4.77×10⁶ 4.02×10⁶ 3.29×10⁶ Control Universal Preservation Solution 2 4.02×10⁶ 3.76×10⁶ 3.17×10⁶ Control Universal Preservation Solution 3 4.57×10⁶ 2.99×10⁶ 1.88×10⁶ Control Universal Preservation Solution 4 4.98×10⁶ 4.19×10⁶ 3.67×10⁶

Table 2 The viability of mesenchymal stem cells after preservation of umbilical cord for 24h, 48h and 72h by different preservation methods

Example 3

Under sterile conditions, a healthy, non-infectious neonatal placenta that underwent cesarean section was taken, the placental amniotic membrane was peeled off, divided into 8 parts, and stored in the general preservation solution of Example 1, normal saline, DMEM medium, Sterile container, control universal preservation solution 1, control universal preservation solution 2, control universal preservation solution 3 and control universal preservation solution 4, store at 4°C for 24h, 48h, and 72h at a constant temperature, take the same quality of amniotic membrane tissue and digest it with collagenase Amniotic membrane-derived mesenchymal stem cells were isolated by filtration, centrifuged, and counted using trypan blue.

The results are shown in Table 3. The results show that the general preservation solution of Example 1 has a good preservation effect on the amniotic membrane, and a large number of mesenchymal stem cells can still be isolated within 72 hours, while the amniotic membrane in the normal saline and DMEM basal medium will change with time. The number of extended cells is reduced; because the amniotic membrane contains a lot of water, it can still be stored in a sterile container for 72 hours, but the number of mesenchymal stem cells will be greatly reduced. Compared with the control universal preservation solution 1, the control universal preservation solution 2, the control universal preservation solution 3 and the control universal preservation solution 4, the number of amniotic mesenchymal stem cells preserved by the universal preservation solution of Example 1 was significantly larger, indicating that human blood white Protein, insulin, vitamin C and EGF have a synergistic effect in increasing the number of preserved amniotic mesenchymal stem cells.

Table 3 The number of mesenchymal stem cells after the amniotic membrane was preserved for 24h, 48h and 72h by different preservation methods

preservation method 24h 48h 72h Example 1 Universal preservation solution 6.66×10⁶ 6.17×10⁶ 5.83×10⁶ normal saline 4.12×10⁶ 1.59×10⁶ 7.98×10⁵ DMEM medium 5.52×10⁶ 3.07×10⁶ 1.50×10⁶ sterile container 3.23×10⁵ 1.14×10⁴ 3.04×10³ Control Universal Preservation Solution 1 5.12×10⁶ 4.73×10⁶ 2.01×10⁶ Control Universal Preservation Solution 2 4.98×10⁶ 3.33×10⁶ 1.45×10⁶ Control Universal Preservation Solution 3 5.56×10⁶ 4.90×10⁶ 1.99×10⁶ Control Universal Preservation Solution 4 5.88×10⁶ 4.91×10⁶ 2.49×10⁶

Example 4

Under aseptic conditions, a healthy neonatal placenta without infectious disease was taken by cesarean section, and the placenta was evenly divided into 8 parts, which were respectively stored in the general preservation solution of Example 1, physiological saline, DMEM medium, sterile container, In the control universal preservation solution 1, the control universal preservation solution 2, the control universal preservation solution 3 and the control universal preservation solution 4, the same quality of placental tissue was taken after being stored at a constant temperature of 4 ℃ for 24h, 48h, 72h, and the placental tissue was separated by collagenase digestion method. Mesenchymal stem cells, filtered and centrifuged, were counted using trypan blue.

The results are shown in Table 4. The results show that the general preservation solution of Example 1 has a better preservation effect on the placenta, and a large number of mesenchymal stem cells can still be isolated at 72h, while the placenta in the physiological saline, DMEM basal medium and sterile containers can still be isolated. There was a large loss of mesenchymal stem cells. Compared with the control universal preservation solution 1, the control universal preservation solution 2, the control universal preservation solution 3 and the control universal preservation solution 4, the number of placental mesenchymal stem cells preserved by the universal preservation solution of Example 1 was significantly larger, indicating that human blood white Protein, insulin, vitamin C and EGF have synergistic effects in increasing the number of preserved placental mesenchymal stem cells.

Table 4 The number of mesenchymal stem cells after the placenta was preserved by different preservation methods for 24h, 48h and 72h

preservation method 24h 48h 72h Example 1 Universal preservation solution 9.88×10⁶ 9.12×10⁶ 8.87×10⁶ normal saline 5.98×10⁵ 1.26×10⁵ 8.44×10⁴ DMEM medium 7.32×10⁶ 4.01×10⁶ 2.45×10⁶ sterile container 4.22×10⁵ 8.17×10⁴ 2.01×10³ Control Universal Preservation Solution 1 7.08×10⁶ 5.07×10⁶ 3.81×10⁶ Control Universal Preservation Solution 2 6.89×10⁶ 4.15×10⁶ 2.74×10⁶ Control Universal Preservation Solution 3 7.88×10⁶ 5.48×10⁶ 3.91×10⁶ Control Universal Preservation Solution 4 7.46×10⁶ 4.22×10⁶ 2.31×10⁶

The above are only the preferred embodiments of the present invention. It should be pointed out that for those skilled in the art, without departing from the principles of the present invention, several improvements and modifications can be made. It should be regarded as the protection scope of the present invention.

Claims (8)

1. The universal preservation solution for the human umbilical cord, amnion and placenta samples is characterized by comprising a DMEM basic culture medium, insulin, human serum albumin, vitamin C, EGF, penicillin, streptomycin and heparin sodium;

the pH value of the universal preservation solution is 7.5-7.8; the concentration of the insulin in the universal preservation solution is 10-20 mg/L;

the concentration of the human serum albumin in the universal preservation solution is 10-15 g/L;

the concentration of the vitamin C in the universal preservation solution is 100-200 mg/L;

the concentration of the EGF in the universal preservation solution is 10-50 mu g/L.

2. The universal preservation solution according to claim 1, wherein the concentration of insulin in the universal preservation solution is 10 mg/L;

the concentration of the human serum albumin in the universal preservation solution is 10 g/L;

the concentration of the vitamin C in the universal preservation solution is 100 mg/L;

the concentration of the EGF in the universal preservation solution is 10 mug/L.

3. The universal preservation solution according to claim 1, wherein the concentration of the penicillin in the universal preservation solution is 50000-100000U/L;

the concentration of the streptomycin in the universal preservation solution is 50-100 mg/L;

the concentration of the heparin sodium in the universal preservation solution is 6250-12500U/L.

4. The universal preservation solution according to claim 3, wherein the concentration of said penicillin in said universal preservation solution is 100000U/L;

the concentration of the streptomycin in the universal preservation solution is 100 mg/L;

the concentration of the heparin sodium in the universal preservation solution is 6250U/L.

5. The method for preparing a universal preservation solution for human umbilical cord, amniotic membrane and placental sample according to any one of claims 1 to 4, comprising the steps of:

adding human serum albumin and heparin sodium into a DMEM basic culture medium, sequentially and respectively adding penicillin, streptomycin, insulin, vitamin C and EGF, and filtering by adopting a filter membrane to obtain the universal preservation solution.

6. A method of preserving a sample, comprising the steps of:

preserving the sample by using the universal preserving fluid of any one of claims 1 to 4;

the sample is human umbilical cord, amnion and/or placenta.

7. The method of claim 6, wherein the cells of interest of the sample are mesenchymal stem cells.

8. The method of claim 6, wherein the holding time is 72 hours or less.

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