CN112640889A

CN112640889A - Hematopoietic stem cell cryopreservation method

Info

Publication number: CN112640889A
Application number: CN202011615983.1A
Authority: CN
Inventors: 关方霞; 冯娟; 崔南征; 崔必胜
Original assignee: Zhongke Bosheng Bioengineering Co ltd
Current assignee: Zhongke Bosheng Bioengineering Co ltd
Priority date: 2020-12-30
Filing date: 2020-12-30
Publication date: 2021-04-13

Abstract

A hematopoietic stem cell cryopreservation method relates to the field of cell cryopreservation, and comprises the following steps: step S1: mixing sodium alginate, sodium carboxymethyl cellulose solution, trehalose and hematopoietic stem cell suspension, and injecting the mixture as nuclear phase solution into the microfluidic control device from a first inlet; then, sodium alginate solution is used as a middle phase and injected into the micro-fluidic device from the second inlet, peanut oil is used as an outermost layer carrier and injected into the micro-fluidic device from the third inlet, and the outlet is immersed in CaCl₂Standing in the solution; step S2: when the microcapsules are completely crosslinked and sink to the bottom of the container, removing the redundant peanut oil, and sucking the microcapsules from the bottom of the solution for further use at the temperature of 4 ℃; step S3: putting the encapsulated microcapsules into a protective agent for incubation, then putting the microcapsules into a freezing tube, then inserting the microcapsules into a freezing box, wherein the oxygen concentration in the box is 1% -3%, and then putting the freezing box into liquid nitrogen at the temperature of-196 ℃ for freezing and storing. The method can effectively reduce the damage of ice crystals to cells, protect the cells and improve the recovery rate of the cells.

Description

Hematopoietic stem cell cryopreservation method

Technical Field

The invention relates to the field of cell cryopreservation, in particular to a hematopoietic stem cell cryopreservation method.

Background

Hematopoietic stem cells are a group of primitive hematopoietic cells present in hematopoietic tissues, which are the primitive cells of all blood cells. Hematopoietic stem cells are committed to differentiate, proliferate into different blood cell lines, and further generate blood cells. Hematopoietic stem cells have great clinical therapeutic significance, and transplantation of hematopoietic stem cells can be used for treating diseases such as leukemia, tumor and the like, but the hematopoietic stem cells are very rare and can be stored under special storage conditions. The formation of ice crystals in cells is easy to cause the problem of cell damage in the programmed temperature reduction process, and the vitrification rapid freezing usually needs a freezing protection solution with higher concentration and permeability to protect the cells, thus easily causing the problems of cell poisoning and the like. There is therefore a need for a suitable method for cryopreserving hematopoietic stem cells.

Disclosure of Invention

In the process of freezing and storing hematopoietic stem cells, ice crystals generated in cells are easy to damage the cells by utilizing a programmed cooling method, and the hematopoietic stem cells are poisoned by adopting vitrification rapid cooling and needing to add high-concentration osmotic freezing protective solution. Therefore, in order to comprehensively solve the problems of ice crystal formation and use of a proper protective solution in the hematopoietic stem cell cryopreservation, the technical scheme of the invention is to provide a hematopoietic stem cell cryopreservation method, which comprises the following steps:

step S1: mixing 1-2% of sodium alginate, 1-2% of sodium carboxymethylcellulose solution, 0.5-1M of trehalose and hematopoietic stem cell suspension, injecting the mixture serving as an internal nuclear phase solution into the microfluidic device from a first inlet, injecting a 2-3% of sodium alginate solution serving as an intermediate phase into the microfluidic device from a second inlet, injecting peanut oil serving as an outermost layer carrier into the microfluidic device from a third inlet, and immersing an outlet in 0.15-0.3M of CaCl₂Standing the solution for 20-30 minutes;

step S2: when the microcapsules are completely crosslinked and sink to the bottom of the container, absorbing the redundant peanut oil, then flushing the redundant peanut oil by using a 0.9-1.5% NaCl solution of a water phase, absorbing the redundant peanut oil, further absorbing the microcapsules from the bottom of the solution, and placing the collected microcapsules in an environment at 4 ℃ for further use;

step S3: putting the encapsulated microcapsules into a protective agent for incubation for 30 minutes, then filling the hematopoietic stem cells into a low-temperature cryopreservation tube of 1.5ml, putting the low-temperature cryopreservation tube into a freezing box, wherein the oxygen concentration in the box is 1% -3%, and then putting the freezing box into liquid nitrogen of-196 ℃ for freezing and preserving.

Specifically, 1% sodium alginate, 1% sodium carboxymethylcellulose solution, 0.5M trehalose, hematopoietic stem cell suspension are in a ratio of 1: 1: 2: 1 mixing to form the nuclear phase solution.

Specifically, the flow rate at the first inlet was 500. mu.L/min, the flow rate at the second inlet was 250. mu.L/min, and the flow rate at the third inlet was 100. mu.L/min.

Specifically, the microcapsule is dripped with CaCl₂When in solution, the CaCl was adjusted with 10mM HEPES₂The pH of the solution was 7.2.

Specifically, the protective agent comprises the following components: 1-1.5M 1, 2-propylene glycol, 1-1.5M ethylene glycol, 10-15% dextran T50, 1-2M trehalose, and 1% polyvinyl alcohol.

The advantages of this method can be derived from the above description: the sodium alginate capsule can effectively inhibit the generation of ice crystals and reduce the damage of the ice crystals of cells to the damage of cell structures caused by the ice crystals generation of the cells. The protective agent is selected to have no high permeability component, so that the toxic influence on the freezing storage process of the hematopoietic stem cells is reduced.

Drawings

FIG. 1 is a flow chart of a method for cryopreserving hematopoietic stem cells.

Detailed Description

In order to achieve the technical purpose, the technical scheme of the invention provides a hematopoietic stem cell cryopreservation method, which comprises the following steps:

step S1: firstly, the hematopoietic stem cells are encapsulated by a microfluidic device, and ice crystals are generated in the process of low-vitrification cryopreservation of the hematopoietic stem cells, so that the encapsulated hematopoietic stem cells can be reduced by utilizing the sodium alginate microcapsules. Firstly, 1% of sodium alginate, 1% of sodium carboxymethylcellulose solution, 0.5M of trehalose and hematopoietic stem cell suspension are mixed according to the proportion of 1: 1: 2: 1, and then injected as an internal nuclear phase solution into the microfluidic device from a first inlet, which is a capillary glass tube having a diameter of 175 μm, and the inflow rate is controlled to be 500 μ L/min. Injecting a 2% sodium alginate solution serving as an intermediate phase into the microfluidic device from a second inlet, wherein the second inlet is a capillary glass tube with the diameter of 350 mu m, and the inflow speed is controlled to be 250 mu L/min; injecting peanut oil serving as an outermost layer carrier into the microfluidic device from a third inlet, wherein the third inlet is a capillary glass tube with the diameter of 500 mu m, and the inflow speed is controlled to be 100 mu L/min; immersing the outlet in 0.15-0.3M CaCl₂In solution, and adjusting the CaCl with 10mM HEPES₂The solution was allowed to stand at pH 7.2 for 30 minutes.

Step S2: and standing for 30 minutes, and then finishing the encapsulation of the hematopoietic stem cells by the microcapsules, wherein the microcapsules sink to the bottom of the container after the crosslinking is finished. Sucking away the excessive peanut oil on the surface of the solution, then flushing the excessive peanut oil with a 0.9% NaCl solution of a water phase, sucking away the excessive peanut oil, then sucking away the microcapsules from the bottom of the solution, and placing the collected microcapsules in an environment of 4 ℃ for further use;

step S3: in order to reduce the toxic effect of the cryoprotective agent on the cells, the high-permeability components in the protective liquid in the refrigerating liquid are reduced. Putting the encapsulated microcapsules into a protective agent and incubating for 30 minutes, wherein the protective agent comprises the following components: 1M 1, 2-propylene glycol, 1M ethylene glycol, 10% dextran T50, 2M trehalose and 1% polyvinyl alcohol, then filling the mixture of the incubated microcapsules and the protective agent into 1.5ml low-temperature freezing tubes, and putting the low-temperature freezing tubes into a freezing box, wherein 9 freezing tubes are used in total. In order to reduce the uncontrolled self-differentiation of the hematopoietic stem cells, the oxygen concentration in the box is controlled to be 1%, and then the freezing box is placed in an environment of-196 ℃ for freezing and preservation.

And (3) performing cell rewarming treatment on the hematopoietic stem cells after being frozen and stored for 1 month and 6 months respectively, putting the low-temperature freezing tube in a water bath at 37 ℃ for rewarming until the hematopoietic stem cells are completely melted and uniformly stirred, and detecting the activity of the revived cells by trypan blue staining after rewarming is completed. The detection results are shown in table 1, and it can be seen from the table that the cell survival rate is higher after 3 months, 6 months and 12 months of cryopreservation, and is higher than that of the traditional cryopreservation mode.

TABLE 1 survival of hematopoietic stem cells after 3, 6 and 12 months of cryopreservation

In summary, the method has the following advantages: the sodium alginate capsule can effectively inhibit the generation of ice crystals and reduce the damage of the ice crystals of cells to the damage of cell structures caused by the ice crystals generation of the cells. The protective agent is selected to have no high permeability component, so that the toxic influence on the freezing storage process of the hematopoietic stem cells is reduced.

It should be understood that the detailed description of the invention is merely illustrative of the invention and is not intended to limit the invention to the specific embodiments described. It will be appreciated by those skilled in the art that the present invention may be modified or substituted equally as well to achieve the same technical result; as long as the use requirements are met, the method is within the protection scope of the invention.

Claims

1. A method for cryopreserving hematopoietic stem cells, comprising the steps of:

step S1: mixing 1-2% of sodium alginate, 1-2% of sodium carboxymethylcellulose solution, 0.5-1M of trehalose and hematopoietic stem cell suspensionInjecting a nuclear phase solution serving as an inner part into the micro-fluidic device from a first inlet, injecting a 2-3% sodium alginate solution serving as an intermediate phase into the micro-fluidic device from a second inlet, injecting peanut oil serving as an outermost layer carrier into the micro-fluidic device from a third inlet, and immersing an outlet in 0.15-0.3M CaCl₂Standing the solution for 20-30 minutes;

step S2: when the microcapsules are completely crosslinked and sink to the bottom of the container, sucking away the redundant peanut oil, then flushing the redundant peanut oil with a 0.9-1.5% NaCl solution of a water phase, sucking away the redundant peanut oil, further sucking away the microcapsules from the bottom of the solution, and placing the collected microcapsules in an environment at 4 ℃ for further use;

2. The method for cryopreserving hematopoietic stem cells according to claim 1, wherein the 1-2% sodium alginate, the 1-2% sodium carboxymethyl cellulose solution, the 0.5-1M trehalose and the hematopoietic stem cell suspension are mixed according to a ratio of 1: 1: 1-2: 1: 1 mixing to form the nuclear phase solution.

3. The method according to claim 1, wherein the flow rate of the first inlet is 500 μ L/min, the flow rate of the second inlet is 250 μ L/min, and the flow rate of the third inlet is 100 μ L/min.

4. The method for cryopreserving hematopoietic stem cells according to claim 1, wherein the CaCl is dripped into the microcapsules₂When in solution, the CaCl was adjusted with 10mM HEPES₂The pH of the solution was 7.2.

5. The method of claim 1, wherein the protectant composition comprises: 1-1.5M 1, 2-propylene glycol, 1-1.5M ethylene glycol, 10-15% dextran T50, 1-2M trehalose, and 1% polyvinyl alcohol.