阅读说明：本技术 一种***梯度分离液及其制备方法和应用 (Sperm gradient separation liquid and preparation method and application thereof ) 是由 何凉 于 2019-10-29 设计创作，主要内容包括：本发明公开了一种精子梯度分离液的制备方法,包括如下步骤：(1)将盐溶液、缓冲剂和螯合剂混合,然后经过高速搅拌,形成基础溶液；(2)将分离剂和水混合,然后经过低速搅拌,形成悬浮液,其中,所述分离剂为经过硅烷涂层处理的二氧化硅颗粒；(3)将基础溶液和悬浮液混合,然后经过中速搅拌,形成精子梯度分离液。本发明还提供了利用上述方法制备的精子梯度分离液和利用该精子梯度分离液进行精子分离的方法。本发明制备的精子梯度分离液中的各个成分均可以得到均匀分布,各个成分分散性好,不易产生沉淀,产品品质高,在精子分离时的使用效果好。(The invention discloses a preparation method of sperm gradient separation liquid, which comprises the following steps: (1) mixing a salt solution, a buffering agent and a chelating agent, and then stirring at a high speed to form a basic solution; (2) mixing a separating agent and water, and then stirring at a low speed to form a suspension, wherein the separating agent is silicon dioxide particles treated by a silane coating; (3) and mixing the base solution and the suspension, and then stirring at medium speed to form the sperm gradient separating medium. The invention also provides the sperm gradient separating medium prepared by the method and a method for separating sperm by using the sperm gradient separating medium. The components in the sperm gradient separating solution prepared by the invention can be uniformly distributed, the dispersibility of the components is good, the precipitation is not easy to generate, the product quality is high, and the using effect is good when sperm is separated.)

1. A preparation method of sperm gradient separation liquid is characterized by comprising the following steps:

(1) mixing a salt solution, a buffering agent and a chelating agent according to a mass ratio of 40-60: 25-35: 10-25, and then stirring at a high speed to form a basic solution, wherein the stirring speed in the high-speed stirring process is 1500-1800 rpm, and the stirring time is 1-5 min;

(2) mixing a separating agent and water in a mass ratio of 40-90: 10-60, and stirring at a low speed to form a suspension, wherein the separating agent is silicon dioxide particles subjected to silane coating treatment, the stirring speed in the low-speed stirring process is 400-600 rpm, and the stirring time is 1-1.5 h;

(3) mixing the basic solution and the suspension according to the mass ratio of 1: 1.5-2.0, and then stirring at a medium speed to form a sperm gradient separation solution, wherein the stirring speed in the medium-speed stirring process is 1000-1500 rpm, and the stirring time is 10-30 min.

2. A process for preparing a gradient sperm cell separator as described in claim 1, wherein the salt solution of step (1) comprises the following concentrations of solutes: 5-10 g/L of sodium chloride, 3-5 g/L of sodium bicarbonate, 1-3 g/L of potassium chloride, 0.1-0.5 g/L of calcium lactate, 0.01-0.05 g/L of sodium pyruvate and 0.01-0.03 g/L of monopotassium phosphate.

3. A process for preparing a sperm gradient separation solution according to claim 1, wherein the buffer in step (1) is one of phosphate buffer, HEPES buffer, Tris-HCl buffer.

4. A process for preparing a gradient sperm separation solution as described in claim 1, wherein the chelating agent used in step (1) is EDTA.

5. A process for preparing a sperm gradient separation solution according to claim 1, wherein in the step (2), if the mass ratio of the separating agent to water is 40:60, the stirring speed is 600rpm, the stirring time is 1 hour, if the mass ratio of the separating agent to water is 80:20, the stirring speed is 500rpm, the stirring time is 1.2 hours, if the mass ratio of the separating agent to water is 90:10, the stirring speed is 400rpm, the stirring time is 1.5 hours.

6. A sperm gradient separation solution produced by the production method according to any one of claims 1 to 4.

7. Use of a sperm gradient separator of claim 6 in a sperm separation process comprising the steps of:

(1) respectively taking two prepared sperm gradient separation liquids with high densities, adding the sperm gradient separation liquid with high density into a conical centrifugal tube, then adding the sperm gradient separation liquid with low density onto the liquid level of the sperm gradient separation liquid with high density in the conical centrifugal tube, and forming a clear interface between the two layers of sperm gradient separation liquids;

(2) adding the liquefied semen into the conical centrifugal tube, and forming an interface between the semen and the sperm gradient separation liquid with low density;

(3) centrifuging at 300g for 20min at room temperature;

(4) after the centrifugation is finished, a suction pipe is stretched into the conical centrifuge tube, and the sperm particle group at the bottom of the tube is taken out.

8. Use of a sperm gradient separator according to claim 7 in a sperm separation process wherein the density gradient of the sperm gradient separator is divided according to the mass fraction of separating agent in the suspension used in the formulation.

Technical Field

The invention belongs to the technical field of cell biology, and particularly relates to a sperm gradient separation solution, and a preparation method and application thereof.

Background

Density gradient separation of sperm can be effective in removing lymphocytes, epithelial cells, abnormal or immature sperm, cellular debris, bacteria and even viruses. The principle of the density gradient centrifugation method is that normal sperms and teratozoospermia, inactive sperms and other cell components in semen are different in motion capability, motion trail, buoyancy density and the like, and have different running capabilities in a density gradient solution column.

The density gradient separation process of sperm is to add sperm gradient separating liquid into the separating tube to form a gradient layer to realize the separation of sperm. Sperm gradient separations are special liquids that can be used to filter cells. The existing preparation method of the sperm separation solution adopts a direct mixing and stirring mode, colloid particles or other components in the finally obtained sperm gradient separation solution cannot be uniformly distributed in the solution, and the quality of the sperm gradient separation solution is not high, so that the use effect of sperm separation is influenced.

Disclosure of Invention

In order to solve the technical problems, the invention aims to provide a sperm gradient separating medium, a preparation method and application thereof.

In order to achieve the technical purpose and achieve the technical effect, the invention is realized by the following technical scheme:

a preparation method of sperm gradient separation liquid comprises the following steps:

(1) mixing a salt solution, a buffering agent and a chelating agent according to a mass ratio of 40-60: 25-35: 10-25, and then stirring at a high speed to form a basic solution, wherein the stirring speed in the high-speed stirring process is 1500-1800 rpm, and the stirring time is 1-6 min;

(2) mixing a separating agent and water in a mass ratio of 40-90: 10-60, and stirring at a low speed to form a suspension, wherein the separating agent is silicon dioxide particles subjected to silane coating treatment, the stirring speed in the low-speed stirring process is 400-600 rpm, and the stirring time is 1-1.5 h;

(3) mixing the basic solution and the suspension according to the mass ratio of 1: 1.5-2.0, and then stirring at a medium speed to form a sperm gradient separation solution, wherein the stirring speed in the medium-speed stirring process is 1000-1500 rpm, and the stirring time is 10-30 min.

Wherein the salt solution in step (1) comprises the following solutes in concentration: 5-10 g/L of sodium chloride, 3-5 g/L of sodium bicarbonate, 1-3 g/L of potassium chloride, 0.1-0.5 g/L of calcium lactate, 0.01-0.05 g/L of sodium pyruvate and 0.01-0.03 g/L of monopotassium phosphate.

Preferably, the buffer in step (1) is one of phosphate buffer, HEPES buffer, Tris-HCl buffer.

Preferably, the chelating agent in step (1) is EDTA.

Preferably, in the step (2), if the mass ratio of the separating agent to the water is 40:60, the stirring speed is 600rpm, the stirring time is 1h, if the mass ratio of the separating agent to the water is 80:20, the stirring speed is 500 rppm, the stirring time is 1.2h, if the mass ratio of the separating agent to the water is 90:10, the stirring speed is 400rp m, and the stirring time is 1.5 h.

The invention also provides a sperm gradient separating medium which is prepared by the preparation method.

The invention also provides application of the sperm gradient separating medium in a sperm separating method, and particularly the sperm separating method comprises the following steps:

(1) respectively taking two prepared sperm gradient separation liquids with high densities, adding the sperm gradient separation liquid with high density into a conical centrifugal tube, then adding the sperm gradient separation liquid with low density onto the liquid level of the sperm gradient separation liquid with high density in the conical centrifugal tube, and forming a clear interface between the two layers of sperm gradient separation liquids;

(2) adding liquefied semen into the conical centrifugal tube, and forming an interface between the semen and the sperm gradient separation liquid with low density;

(3) centrifuging at 300g for 20min at room temperature;

(4) after the centrifugation is finished, a suction pipe is stretched into the conical centrifuge tube, and the sperm particle group at the bottom of the tube is taken out.

Wherein, the centrifugal speed in the step (3) needs to be according to the following formula:

in the formula, G is the centrifugal force, and r is the radius of rotation, i.e., the distance (mm) from the center of the rotor to the bottom of the centrifuge tube when the bucket of the centrifuge is lifted to the horizontal position.

Wherein, the density gradient division standard of the sperm gradient separation liquid is divided according to the mass fraction of the separating agent in the suspension used in the preparation.

The invention has the beneficial effects that: in the preparation method, high-speed stirring and short stirring time are adopted to prepare the basic solution, low-speed stirring and long stirring time are adopted to prepare the suspension, and then medium-speed stirring and reasonable stirring time are adopted to mix the basic solution and the suspension to prepare the sperm gradient separation solution; according to the invention, the targeted stirring speed and stirring time are adopted for respectively stirring and mixing stirring, so that silicon dioxide particles and other components in the prepared sperm gradient separation liquid can be uniformly distributed in the separation liquid;

the sperm gradient separation solution adopts a buffering agent to adjust the pH value of the separation solution, so that the pH value of the separation solution reaches the required requirement (lower than the pH value of semen and between about 7.35 and 7.8); in addition, the chelating agent in the sperm gradient separating medium can play a certain role in dispersion and can prevent the effective components in the separating medium from precipitating; the sperm gradient separating liquid adopts silicon dioxide colloidal particles coated by silane as a separating agent, the colloidal particles are very small, so that the mutual repulsion of the electric potentials of atoms forming the particles can be larger than the suspension gravity of a single particle, the repulsion can ensure that all the particles are uniformly distributed in the solution and cannot fall off or precipitate, and therefore, the flow of cells can be hindered in the centrifugal process, and the purpose of separating sperms is achieved.

In the sperm gradient separation solution, the salt solution contains various salt components, can provide proper ion level for sperm cells, ensures the survival of normal sperm cells in the separation process, and maintains the osmotic pressure of the sperm gradient separation solution between 290 and 340mOsmol/kg by matching the components.

Detailed Description

The following detailed description of the preferred embodiments of the present invention is provided to enable those skilled in the art to more readily understand the advantages and features of the present invention, and to clearly and unequivocally define the scope of the present invention.

A preparation method of sperm gradient separation liquid comprises the following steps:

(1) mixing a salt solution, a buffering agent and a chelating agent according to a mass ratio of 40-60: 25-35: 10-25, and then stirring at a high speed to form a basic solution, wherein the stirring speed in the high-speed stirring process is 1500-1800 rpm, and the stirring time is 1-5 min; wherein the salt solution comprises the following solutes in concentration: 5-10 g/L of sodium chloride, 3-5 g/L of sodium bicarbonate, 1-3 g/L of potassium chloride, 0.1-0.5 g/L of calcium lactate, 0.01-0.05 g/L of sodium pyruvate and 0.01-0.03 g/L of monopotassium phosphate;

(2) mixing a separating agent and water in a mass ratio of 40-90: 10-60, and stirring at a low speed to form a suspension, wherein the separating agent is silicon dioxide particles subjected to silane coating treatment, the stirring speed in the low-speed stirring process is 400-600 rpm, and the stirring time is 1-1.5 h;

(3) mixing the basic solution and the suspension according to the mass ratio of 1: 1.5-2.0, and then stirring at a medium speed to form a sperm gradient separation solution, wherein the stirring speed in the medium-speed stirring process is 1000-1500 rpm, and the stirring time is 10-30 min.

Wherein the buffer in the step (1) is one of phosphate buffer, HEPES (4-hydroxyethylpiperazine ethanesulfonic acid) buffer and Tris-HCl buffer.

Wherein, the chelating agent in the step (1) is EDTA.