CN107183008A - A kind of placenta mesenchyma stem cell frozen stock solution and its cryopreservation methods - Google Patents

Abstract

The present invention relates to the technical field of stem cells, in particular to a placental mesenchymal stem cell cryopreservation solution, comprising the following components in parts by weight: 40-70 parts of DEME medium, glycerol phosphate 2-6 parts of sodium solution, 1-5 parts of hydroxyethyl starch, 1-4 parts of mannitol solution, 0.3-0.8 parts of ethylene glycol, 0.5-2 parts of polyvinyl alcohol, 0.5-1.8 parts of trehalose and polyvinylpyrrolidone 0.5‑1.5 parts. Utilizing the preservation solution of the present invention to preserve embryonic mesenchymal stem cells can relieve damage to stem cells during cryopreservation and improve the survival rate of stem cells after recovery.

Description

A kind of placental mesenchymal stem cell cryopreservation liquid and cryopreservation method thereof

technical field

The invention relates to the technical field of stem cells, in particular to a cryopreservation solution for placental mesenchymal stem cells and a cryopreservation method thereof.

Background technique

Stem cells are a type of pluripotent cells with self-replicating ability. Under certain conditions, it can differentiate into a variety of functional cells. Stem cells are divided into embryonic stem cells (ES cells) and adult stem cells (somatic stem cells) according to their developmental stages. Stem cells are divided into three categories according to their developmental potential: totipotent stem cells (TSCs), pluripotent stem cells (pluripotent stem cells) and unipotent stem cells (unipotent stem cells). Stem cells are not fully differentiated and immature cells, which have the potential to regenerate various tissues and organs and the human body, and are called "universal cells" in the medical field.

The placenta contains a variety of stem cells, including mesenchymal stem cells, hematopoietic stem cells, endothelial stem cells, and pluripotent stem cells of unknown function. Mesenchymal stem cells, whose English name is mesenchymal stem cells (MSC), also have other names, such as bone marrow stromal cells, adipose stem cells, multipotential stromal cells, etc., which refer to a single or a group of cells that meet international unified standards. Placental mesenchymal stem cells are an important type of stem cells. They have the ability to differentiate into bone, cartilage and adipose tissue in vitro and in vivo, and have functions such as immune regulation, hematopoietic support, and angiogenesis.

Stem cell therapy is to culture stem cells with continuous self-reproduction ability and pluripotency potential in vitro, and then transplant them into damaged or defective tissues of patients, so as to realize the supplement and repair of damaged tissues. At present, stem cells are mainly preserved by cryopreservation, but the process of cryopreservation will cause damage to stem cells, so it is necessary to add a cryopreservation solution to slow down cell damage. The existing cryopreservation solution components include dimethyl sulfoxide, albumin and 1640 medium rich in various nutrients. As a osmotic protectant, dimethyl sulfoxide has strong penetration ability It can lower the freezing point of cells and reduce the damage to cells during freezing, but dimethyl sulfoxide is toxic and has a large proportion in the cryopreservation solution, which can easily cause protein denaturation and ultimately affect the survival rate of stem cells. Therefore, it is necessary to develop a cryopreservation solution and a cryopreservation method for cryopreserving stem cells using the cryopreservation solution.

Contents of the invention

The technical problem to be solved by the present invention is to provide a cryopreservation solution for embryonic mesenchymal stem cells in view of the deficiencies in the prior art, which can slow down the damage to the mesenchymal stem cells during the cryopreservation process, and at the same time solve the problem of The toxic effect of the existing cryopreservation solution on the mesenchymal stem cells improves the cryopreservation effect and increases the survival rate of the mesenchymal stem cells.

In order to solve the problems of the technologies described above, the technical solution of the present invention is:

A cryopreservation solution for embryonic mesenchymal stem cells, comprising the following components in parts by weight: 40-70 parts of DEME medium, 2-6 parts of sodium glycerophosphate solution, 1-5 parts of hydroxyethyl starch, and mannitol solution 1-4 parts, 0.3-0.8 parts of ethylene glycol, 0.5-2 parts of polyvinyl alcohol, 0.5-1.8 parts of trehalose and 0.5-1.5 parts of polyvinylpyrrolidone.

As an improved technical solution, the embryonic mesenchymal stem cell cryopreservation solution includes the following components in parts by weight: 50-70 parts of DEME medium, 4-6 parts of sodium glycerophosphate solution, hydroxyethyl starch 3-5 parts, 2-4 parts of mannitol solution, 0.5-0.8 parts of ethylene glycol, 1-2 parts of polyvinyl alcohol, 0.8-1.5 parts of trehalose and 0.8-1.5 parts of polyvinylpyrrolidone.

As an improved technical solution, the embryonic mesenchymal stem cell cryopreservation solution also includes 1-3 parts of collagen.

As an improved technical solution, the mass concentration of the sodium glycerophosphate solution is 0.5g/L-1.8g/L.

As an improved technical solution, the mass concentration of the mannitol solution is 0.2-1.5 g/ml.

Another technical problem to be solved by the present invention is to provide a cryopreservation method for embryonic mesenchymal stem cells in view of the deficiencies in the prior art.

In order to solve the problems of the technologies described above, the technical solution of the present invention is:

A cryopreservation method for embryonic mesenchymal stem cells, the cryopreservation method comprising the following steps:

(1) Preparation of cryopreservation solution: fully mix the weighed mannitol solution, sodium glycerophosphate solution, ethylene glycol and polyvinyl alcohol, then add DEME medium, trehalose, collagen and polyvinylpyrrolidone in sequence, Mix to obtain a frozen storage solution;

(2) Placental tissue pretreatment: the obtained fresh placental tissue was rinsed repeatedly with PBS to remove blood stains, the surface of the placenta was disinfected with alcohol cotton balls, and then rinsed with normal saline for 2-3 times for later use;

(3) Isolation of placental mesenchymal stem cells: take the placental tissue in step (2), cut it into pieces and place it in a centrifuge tube, then add an equal volume of digestive enzymes, and digest it at 30-37°C. After the end, the digestive juice was collected, centrifuged, and the cell pellet was collected to obtain placental mesenchymal stem cells;

(4) Culture of placental mesenchymal stem cells: the placental mesenchymal stem cells in step (3) are inoculated and cultured, and the placental mesenchymal stem cells in logarithmic growth phase are taken, washed and placed into the frozen cells of step (1). Store in the solution, mix thoroughly to obtain a mixture of placental mesenchymal stem cells and cryopreservation solution;

(5) Cryopreservation of placental mesenchymal stem cells: take the mixture in step (4) and place it on an ice bath for 10-30 minutes, then cool the mixture in stages, and finally place the mixture in liquid nitrogen to freeze save.

As an improved technical solution, the digestive enzymes include trypsin, collagenase and hyaluronidase, and the trypsin, collagenase and hyaluronidase are according to the ratio of 0.1～0.7:0.3～0.8:0.2～0.5 Composition by volume.

As an improved technical solution, the cooling method in stages is specifically that the first stage is cooling at -10°C to -20°C, the second stage is cooling at -25°C to -40°C, and the third stage is at -80°C cool down.

After adopting above-mentioned technical scheme, the beneficial effect of the present invention is:

(1) DEME medium is used to provide amino acids and nutrients needed for the cryopreservation and recovery of embryonic mesenchymal stem cells; mannitol solution can effectively remove the water in the cells and reduce the formation of ice crystals; sodium glycerophosphate solution, hydroxyl Ethyl starch, ethylene glycol, polyvinyl alcohol, trehalose, polyvinylpyrrolidone and collagen cooperate to form a stable liquid system, which forms a membrane-like substance, and the embryonic mesenchymal stem cells are covered by the membrane-like substance , The cryopreservation solution not only infiltrates the embryonic mesenchymal stem cells, but also plays a protective role, reducing the damage to the embryonic mesenchymal stem cells during the cryopreservation process, and greatly improving the survival rate of the embryonic mesenchymal stem cells;

(2) Use the above-mentioned cryopreservation solution to freeze the cells, and when the cells are frozen, use an ice bath to cool them first, and then use a staged cooling method to freeze the cells, which can effectively reduce the formation of ice crystals inside the cells and further reduce Possibility of cell damage.

detailed description

In order to make the object, technical solution and advantages of the present invention more clear, the present invention will be further described in detail below in conjunction with the examples. It should be understood that the specific embodiments described here are only used to explain the present invention, not to limit the present invention.

Example 1

A cryopreservation solution for embryonic mesenchymal stem cells, comprising the following components in parts by weight: 45 parts of DEME medium, 3 parts of sodium glycerophosphate solution (mass concentration 0.5g/L), 2 parts of hydroxyethyl starch, manna 2 parts of alcohol solution (mass concentration 0.5g/L), 0.3 part of ethylene glycol, 0.5 part of polyvinyl alcohol, 0.5 part of trehalose and 0.5 part of polyvinylpyrrolidone.

Example 2

A cryopreservation solution for embryonic mesenchymal stem cells, comprising the following components in parts by weight: 55 parts of DEME medium, 4 parts of sodium glycerophosphate solution (mass concentration 1g/L), 3 parts of hydroxyethyl starch, mannitol 3 parts of solution (mass concentration 0.8g/L), 0.5 part of ethylene glycol, 1 part of polyvinyl alcohol, 0.8 part of trehalose, 2 parts of collagen and 0.8 part of polyvinylpyrrolidone.

Example 3

A cryopreservation solution for embryonic mesenchymal stem cells, 68 parts of DEME medium, 6 parts of sodium glycerophosphate solution (mass concentration 1.5g/L), 5 parts of hydroxyethyl starch, 4 parts of mannitol solution (mass concentration 1.2g/L) L), 0.8 parts of ethylene glycol, 2 parts of polyvinyl alcohol, 1.5 parts of trehalose, 2.5 parts of collagen and 1.5 parts of polyvinylpyrrolidone.

Example 4

A cryopreservation method for embryonic mesenchymal stem cells, the cryopreservation method comprising the following steps:

(1) Preparation of cryopreservation solution: fully mix the weighed mannitol solution, ethylene glycol and polyvinyl alcohol, then add DEME, trehalose, collagen and polyvinylpyrrolidone in sequence, and fully mix to obtain a cryopreservation solution;

(2) Placental tissue pretreatment: the obtained fresh placental tissue was rinsed repeatedly with PBS to remove blood stains, the surface of the placenta was disinfected with alcohol cotton balls, and then rinsed with normal saline for 2-3 times for later use;

(3) Isolation of placental mesenchymal stem cells: take the placental tissue in step (2), cut it into pieces and place it in a centrifuge tube, then add an equal volume of digestive enzymes (trypsin, collagenase and hyaluronidase according to 0.4:0.3:0.2 volume ratio), digest at 30-37°C, collect the digestive juice after digestion, centrifuge, collect cell pellets, and obtain placental mesenchymal stem cells;

(4) Culture of placental mesenchymal stem cells: the placental mesenchymal stem cells in step (3) are inoculated and cultured, and the placental mesenchymal stem cells in logarithmic growth phase are taken, washed and placed into the frozen cells of step (1). Store in the solution, mix thoroughly to obtain a mixture of placental mesenchymal stem cells and cryopreservation solution;

(5) Cryopreservation of placental mesenchymal stem cells: take the mixture in step (4) and place it on an ice bath for 10-30 minutes, and then freeze the mixture in a staged cooling method, in which the first stage is at -10°C~ The temperature is lowered at -20°C, the temperature is lowered at -25°C to -40°C in the second stage, and the temperature is lowered at -80°C in the third stage.

Comparative example 1

A cryopreservation solution for embryonic mesenchymal stem cells. Compared with Example 3, the only difference is that the cryopreservation solution does not contain sodium glycerophosphate solution.

Comparative example 2

A cryopreservation solution for embryonic mesenchymal stem cells. Compared with Example 3, the difference is that the cryopreservation solution does not contain mannitol solution.

Comparative example 3

A cryopreservation solution for embryonic mesenchymal stem cells. Compared with Example 3, the difference is that the cryopreservation solution does not contain collagen.

Comparative example 4

A cryopreservation solution for embryonic mesenchymal stem cells. Compared with Example 3, the difference is that the cryopreservation solution does not contain polyvinylpyrrolidone.

Comparative example 5

A cryopreservation solution for embryonic mesenchymal stem cells. Compared with Example 3, the difference is that the cryopreservation solution does not contain polyvinyl alcohol.

The embryonic mesenchymal stem cells obtained in the same batch were divided into 6 groups on average, with 20 samples in each group, and the components in Examples 1-3 and Comparative Examples 1-3 were used to configure the cryopreservation solution, and cryopreserved In the experiment, the frozen storage time was 6 months. After the frozen storage, it was recovered in a 37°C water bath, the precipitation of the samples was observed, and the survival rate of the samples in each group of examples was determined by the trypan blue exclusion method, and the data were recorded and carried out. The average treatment, the test results are shown in Table 1.

Table 1 is the trypan blue dye rejection rate and precipitation situation of embodiment 1-3 and comparative example 1-3 sample

Test case Trypan blue rejection rate Precipitation Example 1 96.2±0.5 none Example 2 97.3±1.2 none Example 3 97.6±1.5 none Comparative example 1 42.3±1.2 less precipitation Comparative example 2 40.6±1.5 less precipitation Comparative example 3 56.8±1.5 less precipitation Comparative example 4 43.6±1.3 less precipitation

It can be seen from Table 1 that the cryopreservation solution prepared in Examples 1-3 cryopreserved mesenchymal stem cells, and the trypan blue rejection rate of the resuscitated mesenchymal stem cells was high, indicating that the mesenchymal stem cells died less after resuscitation , indicating that the interaction of sodium glycerophosphate solution, hydroxyethyl starch, mannitol solution, ethylene glycol, polyvinyl alcohol, trehalose and polyvinylpyrrolidone forms a stable liquid system. Stem cell survival plays a role. While the cryopreservation solution configured in Comparative Example 1-4 was used for cryopreservation of mesenchymal stem cells, the trypan blue exclusion rate of the resuscitated mesenchymal stem cells was low, indicating that the mesenchymal stem cells died more after resuscitation, indicating that they did not contain glycerol Sodium phosphate solution, mannitol solution, polyvinyl alcohol, hydroxyethyl starch, collagen, and polyvinylpyrrolidone cryopreservation solution to cryopreserve mesenchymal stem cells will reduce the survival rate of mesenchymal stem cells.

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements and improvements made within the spirit and principles of the present invention should be included in the protection of the present invention. within range.

Claims (8)

1. a kind of placenta mesenchyma stem cell frozen stock solution, it is characterised in that：Include following component in parts by weight：DEME is trained Support 40-70 parts of base, 2-6 parts of phosphoglycerol sodium solution, 1-5 parts of HES, 1-4 parts of mannitol solution, ethylene glycol 0.3-0.8 Part, 0.5-2 parts of polyvinyl alcohol, 0.5-1.8 parts of trehalose and 0.5-1.5 parts of polyvinylpyrrolidone.

2. a kind of placenta mesenchyma stem cell frozen stock solution according to claim 1, it is characterised in that:In parts by weight Including following component：50-70 parts of DEME culture mediums, 4-6 parts of sodium glycero-phosphate, 3-5 parts of HES, 2-4 parts of mannitol, second 0.8-1.5 parts of 0.5-0.8 parts of glycol, 1-2 parts of polyvinyl alcohol, 0.8-1.5 parts of trehalose and polyvinylpyrrolidone.

3. a kind of placenta mesenchyma stem cell frozen stock solution according to claim 1, it is characterised in that:The placenta mesenchyma Stem cell cryopreserving liquid also includes 1-3 parts of collagen.

4. a kind of placenta mesenchyma stem cell frozen stock solution according to claim 1, it is characterised in that:The sodium glycero-phosphate The mass concentration of solution is 0.5g/L-1.8g/L.

5. a kind of placenta mesenchyma stem cell frozen stock solution according to claim 1, it is characterised in that:The mannitol solution Mass concentration be 0.2-1.5g/ml.

6. a kind of frozen stock solution using described in claim 1 freezes the cryopreservation methods of placenta mesenchyma stem cell, its feature exists Comprise the following steps in the cryopreservation methods：

(1) preparation of frozen stock solution：It is fully mixed by the mannitol solution weighed, phosphoglycerol sodium solution, ethylene glycol and polyvinyl alcohol Close uniform, sequentially add DEME culture mediums, trehalose, collagen and polyvinylpyrrolidone, be sufficiently mixed, obtain frozen stock solution；

(2) placenta tissue is pre-processed：By the fresh human placenta tissue of acquisition, rinse removing bloodstain repeatedly with PBS, disappeared with cotton ball soaked in alcohol Malicious placenta surface, then with normal saline flushing 2-3 times, it is standby；

(3) separation of placenta mesenchyma stem cell：The placenta tissue in step (2) is taken, fragment is cut into and is placed in centrifuge tube, then Isometric digestive ferment is added, is digested under the conditions of 30-37 DEG C, digestion collects digestive juice after terminating, is centrifuged, cell is collected Precipitation, obtains placenta mesenchyma stem cell；

(4) culture of placenta mesenchyma stem cell：Placenta mesenchyma stem cell in step (3) is inoculated with and cultivated, is taken In the frozen stock solution that step (1) is inserted after the placenta mesenchyma stem cell of exponential phase, cleaning, fully mix, obtain between placenta The mixture of mesenchymal stem cells and frozen stock solution；

(5) placenta mesenchyma stem cell freezes：The mixture in step (4) is taken to place 10-30min on ice bath, then will be mixed Compound is frozen using cooling method stage by stage, and the mixture after cooling stage by stage finally is inserted into freezen protective in liquid nitrogen.

7. a kind of cryopreservation methods that placenta mesenchyma stem cell is frozen using frozen stock solution according to claim 6, it is special Levy and be:The digestive ferment includes trypsase, clostridiopetidase A and hyaluronidase, and the trypsase, clostridiopetidase A and transparent Matter acid enzyme is according to 0.1~0.7:0.3~0.8:0.2~0.5 volume ratio composition.

8. a kind of cryopreservation methods that placenta mesenchyma stem cell is frozen using frozen stock solution according to claim 6, it is special Levy and be:Cooling method is specially and cooled at -10 DEG C~-20 DEG C the first stage stage by stage, second stage -25 DEG C~- 40 DEG C are cooled, and the phase III is cooled at -80 DEG C.