CN110367242A - Apoptotic body saves the store method of liquid and apoptotic body - Google Patents

Abstract

The invention provides an apoptotic body cryopreservation solution and an in vitro long-term preservation method of apoptotic bodies. The cryopreservation solution provided by the invention comprises the following components: cryoprotectant, antioxidant and buffer. Through the technical scheme of the present invention, a stable source of apoptotic bodies can be provided for stem cell therapy and cosmetic industry transformation.

Description

Apoptotic body preservation solution and apoptotic body preservation method

technical field

The invention belongs to the technical field of apoptotic bodies, and in particular relates to a preservation solution for long-term preservation of apoptotic bodies of mesenchymal stem cells in vitro and a method for preserving apoptotic bodies.

Background technique

Mesenchymal stem cells are a research branch of stem cells. Stem cells are a type of cells with self-replication and multi-directional differentiation capabilities. They can continuously self-renew and transform into one or more tissues or organs that constitute human body under specific conditions. cells. Stem cells are the origin cells of the body and the progenitor cells that form various tissues and organs of the human body.

Clinical research on mesenchymal stem cells has been carried out in many countries, and more than 60 clinical trials have been approved in the United States. The stage for the research and development of the core technology of stem cells. my country has vigorously strengthened the development of stem cell research. Many authoritative research institutions, including the National Angsai Cell Genetic Engineering Co., Ltd., the National Engineering Research Center for Cell Products, and local umbilical cord blood banks have introduced research technology to the clinic. For the treatment of mesenchymal stem cells for the treatment of more than ten refractory diseases, in addition to promoting the recovery of hematopoiesis, co-transplantation with hematopoietic stem cells to improve leukemia and refractory anemia, etc., it is also used in cardiovascular and cerebrovascular diseases, liver Some of the results of clinical trials that have been encouraging are in the treatment of sclerosis, bone and muscle degenerative diseases, brain and spinal cord nerve damage, Alzheimer's, and autoimmune diseases such as lupus erythematosus and scleroderma.

The chromatin block (nuclear fragment) formed by nuclear fragmentation of apoptotic cells, and then the whole cell forms a spherical protrusion through germination, blistering, etc., and strangulates at its root and falls off to form some different sizes, containing cells. The bodies of cytoplasm, organelles, and nuclear debris are called apoptotic bodies.

The formation of apoptotic bodies can be done in two ways: one is through the budding and shedding mechanism: the aggregated chromatin blocks in apoptotic cells are fragmented to form chromatin blocks of different sizes, and then the whole cell undergoes budding, blistering, etc. It forms a spherical membrane-encapsulated body, containing cytoplasm, organelles and nuclear debris, and falls off to form apoptotic bodies. The second is through the mechanism of autophagosome formation: mitochondria, endoplasmic reticulum and other organelles in apoptotic cells are encapsulated by endoplasmic reticulum membrane together with other cytoplasmic components to form autophagosomes. After fusion with apoptotic cell membrane, autophagosomes are excreted out of cells become apoptotic bodies.

After searching, it is found that the current patent application number CN201580039370.9 for the application of apoptotic bodies describes the medical treatment application of apoptotic bodies and its separation method, but does not describe the preservation and application methods of apoptotic bodies. The prior art also does not disclose the long-term preservation method of apoptotic bodies, which is inconvenient for large-scale industrial application.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide an apoptotic body preservation solution and an in vitro long-term preservation method for apoptotic bodies. Through the technical scheme of the present invention, a stable source of apoptotic bodies can be provided for stem cell therapy and cosmetic industry transformation. .

The purpose of this invention is to realize through the following technical solutions:

A first aspect of the present invention provides a cryopreservation solution for apoptotic bodies, comprising the following components: a cryoprotectant, an antioxidant and a buffer.

Preferably, the cryoprotectant is selected from the group consisting of hydroxyethyl starch, glycerol, human serum albumin and trehalose.

Preferably, the antioxidant is selected from vitamin C.

Preferably, the buffer is selected from one or more of amino acid injection, glucose injection, physiological saline or sodium lactated Ringer's injection.

In a second aspect of the present invention, a cryopreservation solution for apoptotic bodies is provided, which, in parts by weight, comprises: 5-10 parts of hydroxyethyl starch, 5 parts of glycerol, 10-20 parts of human serum albumin, and 5 parts of trehalose , 0.1-1 part of vitamin C injection, 5-10 parts of amino acid injection, 1-5 parts of glucose injection and 45-65 parts of normal saline.

Preferably, the weight part of the hydroxyethyl starch is 5 parts, 6 parts, 7 parts, 8 parts, 9 parts or 10 parts.

Preferably, the weight parts of the human serum albumin are 10 parts, 11 parts, 12 parts, 13 parts, 14 parts, 15 parts, 16 parts, 17 parts, 18 parts, 19 parts or 20 parts.

Preferably, the parts by weight of the vitamin C injection are 0.1 part, 0.2 part, 0.3 part, 0.4 part, 0.5 part, 0.6 part, 0.7 part, 0.8 part, 0.9 part or 1 part.

Preferably, the parts by weight of the amino acid injection are 5 parts, 6 parts, 7 parts, 8 parts, 9 parts or 10 parts.

Preferably, the parts by weight of the glucose injection are 1 part, 2 parts, 3 parts, 4 parts or 5 parts.

Preferably, the parts by weight of the physiological saline are 45 parts, 46 parts, 47 parts, 48 parts, 49 parts, 50 parts, 51 parts, 52 parts, 53 parts, 54 parts, 55 parts

In a third aspect of the present invention, a cryopreservation solution for apoptotic bodies is provided, which, in parts by weight, comprises: 5-10 parts of hydroxyethyl starch, 5 parts of glycerol, 10-20 parts of human serum albumin, and 5 parts of trehalose , 0.1-1 part of vitamin C injection, 5-10 parts of amino acid injection, 1-5 parts of glucose injection and 45-65 parts of sodium lactated Ringer injection.

Preferably, the weight part of the hydroxyethyl starch is 5 parts, 6 parts, 7 parts, 8 parts, 9 parts or 10 parts.

Preferably, the weight parts of the human serum albumin are 10 parts, 11 parts, 12 parts, 13 parts, 14 parts, 15 parts, 16 parts, 17 parts, 18 parts, 19 parts or 20 parts.

Preferably, the parts by weight of the vitamin C injection are 0.1 part, 0.2 part, 0.3 part, 0.4 part, 0.5 part, 0.6 part, 0.7 part, 0.8 part, 0.9 part or 1 part.

Preferably, the parts by weight of the amino acid injection are 5 parts, 6 parts, 7 parts, 8 parts, 9 parts or 10 parts.

Preferably, the parts by weight of the glucose injection are 1 part, 2 parts, 3 parts, 4 parts or 5 parts.

Preferably, the parts by weight of the sodium lactate Ringer's injection are 45 parts, 46 parts, 47 parts, 48 parts, 49 parts, 50 parts, 51 parts, 52 parts, 53 parts, 54 parts, 55 parts, 56 parts , 57, 58, 59, 60, 61, 62, 63, 64, or 65.

Preferably, the apoptotic body is a mesenchymal stem cell apoptotic body or a bone marrow mesenchymal stem cell apoptotic body.

A fourth aspect of the present invention provides a method for preserving apoptotic bodies, comprising the following steps:

Obtain apoptotic bodies, add the apoptotic bodies to the cryopreservation solution, mix well, and obtain a preserved sample; add the preserved sample to a cryopreservation tube and place it in a programmed cooling box, in a -80 degree refrigerator Store for 24 hours; transfer the stored sample to a gas phase/liquid phase storage tank for storage for later use.

Preferably, the volume ratio of the apoptotic bodies to the cryopreservation solution is 1:(30-100).

Preferably, the volume ratio of the apoptotic bodies to the cryopreservation solution is 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90 or 1:100 .

Preferably, the cryopreservation solution is the above-mentioned cryopreservation solution; the apoptotic bodies of the present invention are prepared by the following methods:

(1) Obtain the umbilical cord tissue, peel off the Wharton's glue, cut it into pieces, and add it to the culture medium for primary cell culture;

(2) Half-volume medium exchange was performed on the cells cultured above on the fifth day after culture;

(3) After that, the cells were changed every 3 days until the cell plating rate was about 80% and then passaged;

(4) Repeat step (3) to extract apoptotic bodies when the cells are passaged to P5 (see Figure 1 for cell morphology);

(5) The apoptotic bodies were obtained by the starvation method of the prior art.

Compared with the prior art, the beneficial effects of the present invention are:

The cryopreservation solution provided by the present invention is compounded by cryoprotectant, antioxidant and buffer, and is used for in vitro preservation of apoptotic bodies, and the preservation rate of the structure and activity of apoptotic bodies after preservation is 95% Above, the industrial application of apoptotic bodies is greatly facilitated.

Further, the cryopreservation solution provided by the present invention specifically adopts 5-10 parts of hydroxyethyl starch, 5 parts of glycerol, 10-20 parts of human serum albumin, 5 parts of trehalose, 0.1-1 part of vitamin C injection, amino acid 5-10 parts of injection, 1-5 parts of glucose injection and 45-65 parts of sodium lactate Ringer's injection are compounded. The above components are all clinically available products. It meets the needs of cryopreservation and recovery of apoptotic bodies, and has high safety. It reduces the removal of impurities during later application, which is conducive to the efficient and fast application of products. It can be used in biological preparations for disease treatment, beauty products, Health Care Products.

Hydroxyethyl starch (Hetastarch) is a synthetic colloidal solution widely used in clinical practice, and it is also a natural polysaccharide. Hydroxyethyl starch (HES) is a polymer complex formed by hydroxyethylation of the glucose ring of amylopectin in corn or potato. Native starch cannot be used as a plasma substitute because native starch is unstable and susceptible to hydrolysis by endogenous amylases. After starch is hydroxyethylated, its decomposition and elimination in blood can be delayed, and its residence time in blood vessels can be significantly prolonged. HES has the effect of expanding blood volume. 10 minutes after infusion of HES 1000ml in healthy volunteers, the average blood volume increased by 900ml compared with that before infusion, decreased to 415ml after 6 hours, and remained at 285ml after 24 hours.

As a good humectant, glycerol has a good shrinkage effect, which can avoid the destruction of apoptotic bodies due to water crystallization under freezing conditions.

Human serum albumin can transport fatty acids, bile pigments, amino acids, steroid hormones, metal ions, and many therapeutic molecules in body fluids, while maintaining normal blood osmotic pressure. Clinically, human serum albumin can be used to treat shock and burns, to supplement blood loss caused by surgery, accidents or hemorrhage, and as a plasma expander. In the present invention, human serum albumin can maintain the osmotic pressure of apoptotic bodies.

Trehalose has a magical protective effect on organisms, because trehalose can form a unique protective film on the cell surface under harsh environmental conditions such as high temperature, high cold, high osmotic pressure and drying and dehydration, effectively protecting protein molecules from denaturation and inactivation. , so as to maintain the life processes and biological characteristics of living organisms.

Vitamin C has antioxidant and anti-free radical properties.

Amino acid injection is used to supplement amino acids for apoptotic bodies and maintain the nutritional needs of apoptotic bodies.

Glucose injection is used to supplement the energy requirements of apoptotic bodies.

Lactated Ringer's Injection is used to adjust the electrolytes and acid-base balance of the culture medium.

The present invention proposes for the first time that apoptotic bodies are preserved in vitro by freezing, and the preservation rate of the structure and activity of apoptotic bodies after preservation is over 95%, which greatly facilitates the industrial application of apoptotic bodies. .

Description of drawings

Figure 1. Morphological photographs of umbilical cord mesenchymal stem cells;

Figure 2 shows the effect of umbilical cord mesenchymal stem cell-derived apoptotic bodies on cell proliferation rate under injury conditions;

Figure 3 is a morphological photograph of bone marrow mesenchymal stem cells;

Figure 4 shows the effect of bone marrow mesenchymal stem cell-derived apoptotic bodies on cell proliferation rate under injury conditions.

Detailed ways

The content of the present invention will be described in further detail below with reference to the accompanying drawings and embodiments.

Example 1

This embodiment provides a cryopreservation solution for apoptotic bodies. According to the parts by weight, the cryopreservation solution includes the following components: 5 parts of hydroxyethyl starch, 5 parts of glycerol, 20 parts of human serum albumin, 5 parts of trehalose 1 part of vc injection, 10 parts of amino acid injection, 5 parts of glucose injection and 49 parts of normal saline.

Example 2

This embodiment provides a cryopreservation solution for apoptotic bodies. According to the parts by weight, the cryopreservation solution includes the following components: 6 parts of hydroxyethyl starch, 5 parts of glycerol, 10 parts of human serum albumin, and 5 parts of trehalose. 0.1 part of vitamin C injection, 5 parts of amino acid injection, 1 part of glucose injection and 45 parts of lactated Ringer's injection.

Example 3

This embodiment provides a cryopreservation solution for apoptotic bodies. According to the parts by weight, the cryopreservation solution includes the following components: 7 parts of hydroxyethyl starch, 5 parts of glycerol, 12 parts of human serum albumin, and 5 parts of trehalose. 0.2 parts of vitamin C injection, 6 parts of amino acid injection, 2 parts of glucose injection and 46 parts of lactated Ringer's injection.

Example 4

This embodiment provides a cryopreservation solution for apoptotic bodies. According to the parts by weight, the cryopreservation solution includes the following components: 8 parts of hydroxyethyl starch, 5 parts of glycerol, 12 parts of human serum albumin, and 5 parts of trehalose. 0.3 parts of vitamin C injection, 7 parts of amino acid injection, 3 parts of glucose injection and 50 parts of normal saline.

Example 5

This embodiment provides a cryopreservation solution for apoptotic bodies. According to the parts by weight, the cryopreservation solution includes the following components: 9 parts of hydroxyethyl starch, 5 parts of glycerol, 12 parts of human serum albumin, and 5 parts of trehalose. 0.5 parts of vitamin C injection, 8 parts of amino acid injection, 4 parts of glucose injection and 55 parts of lactated Ringer's injection.

Example 6

This embodiment provides a cryopreservation solution for apoptotic bodies. According to the parts by weight, the cryopreservation solution includes the following components: 10 parts of hydroxyethyl starch, 5 parts of glycerol, 15 parts of human serum albumin, and 5 parts of trehalose. 0.7 parts of vitamin C injection, 9 parts of amino acid injection, 4 parts of glucose injection and 60 parts of lactated Ringer's injection.

Example 7

This embodiment provides a cryopreservation solution for apoptotic bodies. According to the parts by weight, the cryopreservation solution includes the following components: 10 parts of hydroxyethyl starch, 5 parts of glycerol, 18 parts of human serum albumin, and 5 parts of trehalose. 0.9 parts of vitamin C injection, 10 parts of amino acid injection, 5 parts of glucose injection and 65 parts of lactated Ringer's injection.

Example 8

The present embodiment provides a method for cryopreserving apoptotic bodies of umbilical cord mesenchymal stem cells, comprising the following steps:

Obtain the umbilical cord mesenchymal stem cell apoptotic body, add the umbilical cord mesenchymal stem cell apoptotic body into the cryopreservation solution described in Example 1, mix well, and obtain a preservation sample; add the preservation sample to a cryopreservation tube Afterwards, it was placed in a programmed cooling box, and stored in a -80 degree refrigerator for 24 hours; the preserved samples were transferred to a gas/liquid phase storage tank for preservation for later use.

The umbilical cord mesenchymal stem cell apoptotic body described in this example is prepared by the following method:

(1) Obtain the umbilical cord tissue, peel off Wharton's glue, cut it into pieces, and add it to the medium for culture;

(2) Half-volume medium exchange was performed on the cells cultured above on the fifth day after culture;

(3) After that, the cells were changed every 3 days until the cell plating rate was about 80% and then passaged;

(4) Repeat step (3) to extract apoptotic bodies when the cells are passaged to P5. The cell morphology of P5 passage is shown in Figure 3;

(5) The apoptotic bodies were obtained by the starvation method of the prior art.

Example 9

The present embodiment provides a method for cryopreserving apoptotic bodies of bone marrow mesenchymal stem cells, comprising the following steps:

Obtain bone marrow mesenchymal stem cell apoptotic bodies, add the bone marrow mesenchymal stem cell apoptotic bodies to the cryopreservation solution described in Example 7, and mix well to obtain a preserved sample; add the preserved sample to a cryopreservation tube Afterwards, it was placed in a programmed cooling box, and stored in a -80 degree refrigerator for 24 hours; the preserved samples were transferred to a gas/liquid phase storage tank for preservation for later use.

The bone marrow mesenchymal stem cell apoptotic bodies described in this example are prepared by the following method:

(1) Rat bone marrow was extracted with a syringe, resuspended and washed with PBS for 3 times, and then added to the culture medium for resuspending and then added to the culture flask.

(2) The apoptotic bodies were isolated when the cells were passaged to the P5 generation, and the cell morphology of the P5 generation was shown in Figure 3;

(3) After that, the cells were changed every 3 days until the cell plating rate was about 80% and then passaged;

(4) The thermal stress method in the prior art is used to obtain apoptotic bodies, and some apoptotic bodies are directly frozen in a -80 degree refrigerator.

Example 10 Effect verification

The freshly extracted apoptotic bodies of umbilical cord mesenchymal stem cells were taken as group A; the apoptotic bodies of umbilical cord mesenchymal stem cells cryopreserved in Example 8 of the present invention were recovered, and the resuscitated apoptotic bodies were obtained as group B Group. Cell culture was carried out after adding cell culture medium to group A and group B respectively. The cells were cultured to about 60%. After 5 minutes of UVA irradiation, the cell culture was continued, and the cells were counted after 2 days of culture. The counting results are shown in Figure 2. shown. It can be seen that when the apoptotic bodies are cryopreserved using the cryopreservation solution and the freezing method provided in the embodiments of the present invention, the preservation rate of the structure and activity of the apoptotic bodies after preservation is over 95%, which is called apoptosis. The industrial application of the corpuscle provides convenience.

Example 11 Effect verification

The freshly extracted apoptotic bodies of bone marrow mesenchymal stem cells were taken as group C; the cryopreserved apoptotic bodies in Example 9 of the present invention were recovered, and the resuscitated apoptotic bodies were obtained as group D. Cell culture was carried out after adding cell culture medium to group C and group D respectively. The cells were cultured to about 60%. After irradiating with UVA for 5 minutes, the cell culture was continued. After culturing for 2 days, the cells were counted. shown. It can be seen that, using the cryopreservation solution and the freezing method provided in the embodiment of the present invention to cryopreserve the apoptotic bodies of bone marrow mesenchymal stem cells, the structure and activity of the apoptotic bodies of bone marrow mesenchymal stem cells are preserved after preservation. The rate is above 95%, which provides convenience for the industrial application of apoptotic bodies.

Claims (10)

1. apoptotic body freezen protective liquid, which is characterized in that include following components: cryoprotector, antioxidant and buffer.

2. freezen protective liquid according to claim 1, which is characterized in that the cryoprotector be selected from hydroxyethyl starch, Glycerol, human serum albumins and trehalose.

3. freezen protective liquid according to claim 1, which is characterized in that the antioxidant is selected from vitamin C.

4. freezen protective liquid according to claim 1, which is characterized in that the buffer is selected from amino acid injection, Portugal One of grape sugar injection, physiological saline or sodium lactate ringer's injection are a variety of.

5. apoptotic body freezen protective liquid, which is characterized in that according to parts by weight, include: 5-10 parts of hydroxyethyl starch, glycerol 5 Part, 10-20 parts of human serum albumins, 5 parts of trehalose, 0.1-1 parts of vitamin C injection, 5-10 parts of amino acid injection, grape Sugared injection 1-5 parts and physiological saline 45-65 parts.

6. apoptotic body freezen protective liquid, which is characterized in that according to parts by weight, include: 5-10 parts of hydroxyethyl starch, glycerol 5 Part, 10-20 parts of human serum albumins, 5 parts of trehalose, 0.1-1 parts of vitamin C injection, 5-10 parts of amino acid injection, grape Sugared injection 1-5 parts and sodium lactate ringer's injection 45-65 parts.

7. freezen protective liquid according to claim 5 or 6, which is characterized in that the apoptotic body is mescenchymal stem cell Apoptotic body or apoptosis of mesenchymal stem cell corpusculum.

8. a kind of store method of apoptotic body, which comprises the following steps:

Apoptotic body is obtained, the apoptotic body is added in the freezen protective liquid, is mixed, obtains and saves sample；It will be described It saves sample addition cryopreservation tube to be placed in Programmed cryopreservation box, be saved 24 hours in -80 degree refrigerators；The preservation sample is turned It moves in gas phase and liquid phase holding vessel and saves, it is spare.

9. according to the method described in claim 8, it is characterized in that, the volume ratio of the apoptotic body and the freezen protective liquid For 1:(30 ~ 100).

10. according to the method described in claim 8, it is characterized in that, the preservation liquid is that claim 1-4 is described in any item Freezen protective liquid.