CN111713488B

CN111713488B - Cryopreservation agent, preparation method thereof and application thereof in coccidian oocysts

Info

Publication number: CN111713488B
Application number: CN202010687786.4A
Authority: CN
Inventors: 王健君; 赵立山; 吕健勇
Original assignee: Songshan Lake Materials Laboratory
Current assignee: Songshan Lake Materials Laboratory
Priority date: 2020-07-16
Filing date: 2020-07-16
Publication date: 2022-01-18
Anticipated expiration: 2040-07-16
Also published as: CN111713488A

Abstract

The invention discloses a cryopreservation agent and a preparation method thereof and application in coccidioid oocysts. The cryopreservation agent of the invention comprises lauric acid, hydrogenated phosphatidylcholine, sodium fluoride, ammonium chloride, aspartate amino acid, proline, sucrose, sodium dihydrogen phosphate, disodium hydrogen phosphate, PBS buffer and water for injection. The cryopreservation agent of the invention has a reasonable formula, can effectively inhibit the recrystallization of ice crystals, regulate the shape of ice crystals, reduce the growth rate of ice crystals, and reduce the damage caused by ice crystals to coccidian oocysts in the process of cryopreservation. On the wall of the oocysts, the ice damage resistance is enhanced, so that the coccidial oocysts can be stored in liquid nitrogen for a long time. Moreover, compared with traditional cryopreservants, it does not contain toxic molecules such as DMSO and expensive serum and other components, which not only avoids the toxic damage of DMSO to coccidial oocysts, but also reduces production costs and effectively enhances its use in the field of coccidial vaccines. The applicability is conducive to wide popularization and application.

Description

Cryopreservation agent, preparation method thereof and application thereof in coccidian oocysts

Technical Field

The invention relates to the technical field of low-temperature cryopreservation, in particular to a cryopreservation agent, a preparation method thereof and application thereof in coccidian oocysts.

Background

In recent years, the cryopreservation of cells, tissues and organs has attracted much attention, and the breakthrough of cryopreservation technology will provide powerful theoretical and experimental support for the development of regenerative medicine. Active substances such as cells and the like which are separated from the maternal environment consume nutrients in the cells due to life activities such as metabolism and the like in the preservation process, and the activity of the active substances inevitably weakens along with the preservation time until the active substances are completely inactivated. The extremely low temperature, usually 77K (-196 ℃, the boiling point of liquid nitrogen), can effectively inhibit or even stop the vital activities such as metabolism in cells, can prolong the service life of the cells, and realizes the long-term preservation of the cells. Ultra-low temperature liquid nitrogen preservation is widely applied to cryopreservation of biological materials such as cells (oocytes, erythrocytes, stem cells, sperms and the like), tissues and the like.

Uncontrolled freezing during cryopreservation can cause severe mechanical damage, resulting in direct inactivation of cells. The traditional cryopreservation agent usually contains small molecular substances such as DMSO (dimethyl sulfoxide), glycerol and the like, and the substances can enter the interior of cells through cell membranes to enhance the vitrification capability of the cells, so that the cells directly enter a glass state in the rapid cooling process, and the damage of freezing to the cells is avoided. However, glass preservation also presents two important non-negligible problems. Firstly, the realization of the glassy state cryopreservation requires a high-concentration cryopreservation agent and a very fast cooling speed, but the cryopreservation agent such as DMSO has cytotoxicity, and the higher the concentration is, the more harmful the cell is; secondly, cells are also subjected to icing and ice recrystallization in the rewarming process, and the conventional cryopreservation agent commonly used at present does not have the effect of controlling icing and ice recrystallization, so that damage to the cells caused by icing and ice recrystallization in the rewarming process cannot be avoided. Recrystallization as used herein refers to a maturation process in which large ice crystals continue to grow up at the expense of disappearance of small ice crystals during cryopreservation or rewarming, which can mechanically damage biological materials such as cryopreserved cells. Therefore, the ice control molecules are added into the cryopreservation agent, and the scheme of effectively reducing the damage of the ice crystals to the cells in the cryopreservation process is realized by controlling the freezing and inhibiting the recrystallization of the ice crystals.

Eimeria is a common parasitic protozoa, which is spread in a large range in a farm by infecting animals such as chickens and rabbits, and brings huge economic loss to the breeding industry, and the most effective prevention and treatment means at present is through live coccidian vaccines (coccidian oocysts). Therefore, the long-term preservation of coccidia and coccidia live vaccines is important for the study and defense against eimeria. However, due to the existence of the coccidian oocyst wall, the traditional cryopreservation agent is difficult to enter the oocysts through the oocyst wall, so that the damage of the coccidian oocysts caused by the freezing in the ultralow-temperature preservation process cannot be avoided, and the traditional cryopreservation agent is not suitable for the long-term low-temperature preservation of the coccidian oocysts. In addition, the conventional cryopreservation agent contains expensive serum, so that the storage cost of the coccidia is greatly increased, and the conventional cryopreservation agent is not suitable for storing the coccidia vaccine with lower cost. At present, the research and patents on cryopreservation in the field of eimeria are few and have problems.

The invention application named as 'Eimeria coccidium cryopreservation agent and cryopreservation method' under the publication number 'CN 107347873A' discloses an Eimeria coccidium cryopreservation agent and cryopreservation method, wherein the cryopreservation agent comprises a cryopreservation liquid and a cryoprotectant; the frozen stock solution comprises sucrose, monopotassium phosphate, dipotassium phosphate trihydrate, L-glutamic acid and calf serum protein; the cryoprotectant is dimethyl sulfoxide or a composition comprising dimethyl sulfoxide. Although it can achieve a certain preservation effect, it is not suitable for wide popularization and application because it contains toxic DMSO and relatively expensive fetal calf serum components. For this reason, cryopreservation of coccidia is a challenge and urgent problem in this field.

Currently, eimeria and live coccidia vaccines are preserved at 4 degrees C, but the preservation period is usually not more than 6 months, and manual passage intervention for the preservation of the coccidia vaccines is required every two months, which wastes time and labor and increases the cost of the vaccines. Therefore, a novel cryopreservation agent is urgently needed, and the low-temperature long-term preservation of the coccidian oocysts is realized by controlling the freezing in the cryopreservation process, inhibiting the recrystallization of ice crystals and regulating the appearance and growth rate of the ice crystals.

Disclosure of Invention

In view of the above disadvantages, one of the objectives of the present invention is to provide a cryopreservation agent with a reasonable formulation, which has the functions of inhibiting freezing and ice crystal recrystallization, regulating ice crystal morphology, etc., thereby reducing mechanical damage of ice crystals to a sample to be preserved.

The second object of the present invention is to provide a method for preparing the cryopreservation agent.

The invention also aims to provide the application of the cryopreservation agent in coccidian oocysts.

In order to achieve the purpose, the technical scheme provided by the invention is as follows:

a freezing storage agent comprises the following components in percentage by mass: 0.01 to 0.6 percent of lauric acid, 0.01 to 0.4 percent of hydrogenated phosphatidylcholine, 1.7 to 3.6 percent of sodium fluoride, 1.5 to 4.1 percent of ammonium chloride, 0.1 to 0.3 percent of aspartic acid, 3 to 7 percent of proline, 5 to 8 percent of cane sugar, 0.1 to 0.4 percent of sodium dihydrogen phosphate, 0.2 to 0.8 percent of disodium hydrogen phosphate, 2 to 5 percent of PBS buffer solution and the balance of water for injection.

The lauric acid and the hydrogenated phosphatidylcholine form micelles in water, on one hand, hydrophilic and hydrophobic groups of the micelles are gradually enriched on the surface of coccidian oocysts in the stirring process, so that the mechanical damage of ice crystals generated in the freezing storage process to the oocysts is weakened; meanwhile, the free micelle also has the capacity of inhibiting the growth of ice crystals and the recrystallization of the ice, and further reduces the damage caused by the ice crystals.

The sodium fluoride, the ammonium chloride and the aspartic acid can be selectively adsorbed on the surface of the ice crystal, so that the appearance of the ice crystal is modified, the growth of the ice crystal is slowed down, the recrystallization of the ice is inhibited, and the damage of the swollen ice crystal to the oocysts in the freezing storage process is reduced.

The proline and sucrose can interact with water through hydrophilic groups such as hydroxyl and the like, so that the content of ice crystals capable of forming ice water is reduced, and the content of ice crystals generated in the process of cryopreservation is reduced.

The sodium dihydrogen phosphate, the disodium hydrogen phosphate and the PBS buffer solution have the function of adjusting the pH value of the novel cryopreservation agent to be 6.2-8.2 suitable for coccidian oocyst cryopreservation.

The freezing agent reduces the damage of ice crystals to coccidian oocysts by controlling freezing, inhibiting ice crystal recrystallization in the rewarming process, regulating the appearance of the ice crystals and the like; secondly, the ice damage resistance of the oocysts can be enhanced through interaction with the oocyst walls; thirdly, toxic molecules such as DMSO and the like are not contained, so that the damage of DMSO to coccidian is avoided; finally, the composition does not contain expensive components such as serum, reduces the cost of the cryopreservation agent and enhances the applicability of the cryopreservation agent in the field of coccidian vaccines.

A preparation method of a cryopreservation agent comprises the following steps:

(1) preparing the following components in percentage by mass: 0.01 to 0.6 percent of lauric acid, 0.01 to 0.4 percent of hydrogenated phosphatidylcholine, 1.7 to 3.6 percent of sodium fluoride, 1.5 to 4.1 percent of ammonium chloride, 0.1 to 0.3 percent of aspartic acid, 3 to 7 percent of proline, 5 to 8 percent of cane sugar, 0.1 to 0.4 percent of sodium dihydrogen phosphate, 0.2 to 0.8 percent of disodium hydrogen phosphate, 2 to 5 percent of PBS buffer solution and the balance of water for injection;

(2) adding lauric acid and hydrogenated phosphatidylcholine to the water for injection to obtain a first mixture, and stirring the first mixture to form micelles;

(3) sequentially adding sodium fluoride, ammonium chloride, aspartic acid, proline and sucrose into the first mixture, and stirring until the sodium fluoride, the ammonium chloride, the aspartic acid, the proline and the sucrose are fully dissolved to obtain a second mixture;

(4) and adding PBS buffer solution, sodium dihydrogen phosphate and disodium hydrogen phosphate into the second mixture, and adjusting the pH of the cryopreservation solution to 6.2-8.2 to prepare the cryopreservation agent.

An application of the cryopreservation agent in coccidian oocyst cryopreservation. The application steps are as follows:

(S1) adding the coccidian oocysts into the cryopreservation agent, stirring or vortex mixing, and standing to obtain a frozen storage solution; before the step (S1), the coccidian oocysts are subjected to a purification treatment in advance. The purification treatment specifically comprises the following steps: taking coccidian oocyst liquid, centrifuging and precipitating; (II) removing the supernatant, adding saturated saline water for resuspension, precipitation and centrifugation; (III) transferring the supernatant liquid containing the oocyst liquid into a centrifuge tube filled with injection water, and centrifuging again to obtain oocyst precipitation liquid; (IV) transferring the oocyst sediment solution into a centrifuge tube filled with injection water, repeating the previous step for three times, and finally obtaining the coccidia oocyst solution without disinfectant components.

(S2) subpackaging the frozen preservation solution into a freezing tube, and then placing the tube into a freezing box;

(S3) putting the freezing storage box into a program temperature controller, cooling for a period of time, and transferring into liquid nitrogen for long-term storage.

After the coccidian oocysts are frozen in liquid nitrogen for a period of time, resuscitation needs to be carried out and the state of the oocysts is observed, and the method comprises the following steps:

1) and (3) recovering oocysts: preheating the water bath in advance, taking the freezing tube out of the liquid nitrogen, quickly putting the freezing tube into the water bath, and recovering the freezing tube;

2) and (3) oocyst cleaning: diluting the melted frozen stock solution in 10 times of volume of injection water, carrying out vortex mixing and centrifugation, taking the pellet solution containing coccidian oocysts, diluting and centrifuging in 10 times of volume of injection water again, repeating the process for five times, and taking the coccidian oocysts pellet solution to be suspended in 2.5% potassium dichromate solution.

3) Observing the state of oocysts: taking the oocyst suspension, placing under a microscope, and observing the structural integrity and the sporangium form inside the oocysts.

The invention has the beneficial effects that: the cryopreservation agent disclosed by the invention is reasonable in formula, can effectively inhibit an ice crystal recrystallization process in a cryopreservation process, regulate and control the appearance of ice crystals, reduce the growth rate of the ice crystals, and reduce the damage of the ice crystals to coccidian oocysts, and meanwhile, phospholipid molecules can be adsorbed on the walls of the coccidian oocysts to enhance the ice damage resistance of the coccidian oocysts, so that the coccidian oocysts can be preserved in liquid nitrogen for a long time. Compared with the traditional cryopreservation agent which does not contain toxic molecules such as DMSO and the like and components such as expensive serum and the like, the cryopreservation agent not only avoids the damage of DMSO to coccidian oocysts, but also reduces the production cost, effectively enhances the applicability of the cryopreservation agent in the coccidian vaccine field, and is beneficial to wide popularization and application.

The preparation method of the cryopreservation agent provided by the invention is simple in steps and easy to realize, and the cryopreservation agent which has the functions of inhibiting icing and ice crystal recrystallization, regulating and controlling the appearance of the ice crystal and the like can be quickly prepared, so that the mechanical damage of the ice crystal to a sample to be preserved is reduced.

The invention is further described with reference to the following figures and examples.

Drawings

FIG. 1 shows the results of an ice crystal recrystallization experiment for a PBS solution control and a frozen stock solution of the present invention.

FIG. 2 shows the results of experiments on the growth of single crystal ice in PBS control and frozen stocks of the invention.

FIG. 3 is a graph showing the results of cryopreservation of E.maxima oocysts using the cryopreservation solution of the present invention.

Detailed Description

Example 1: this example illustrates cryopreservation and recovery of E.maxima oocysts, but is not intended to limit the scope of the invention.

Preparing a freezing storage agent: putting 90ml of injection water into a beaker placed in a water bath at 4 ℃, adding a stirrer, and adjusting the rotating speed to 500 rpm; adding 0.02g of lauric acid and 0.2g of hydrogenated phosphatidylcholine, and stirring for 10 minutes; 3g of sodium fluoride, 3g of ammonium chloride, 0.3g of aspartic acid, 7g of proline, 8g of sucrose, 5ml of standard PBS buffer solution, 0.3g of sodium dihydrogen phosphate and 0.7g of disodium hydrogen phosphate are added in sequence, and the mixture is stirred for 30 minutes.

Secondly, pretreating oocysts, and carrying out the following purification pretreatment before adding the E.maxima oocysts:

1. centrifuging 2ml of Eimeria maxima oocyst solution (3000rpm, 5min) for precipitation;

2. removing supernatant, adding 2ml saturated sodium chloride aqueous solution, and centrifuging (3000rpm, 5 min);

3. taking out supernatant containing Eimeria maxima oocyst liquid, transferring into a centrifuge tube filled with 2ml of injection water, and centrifuging again (3000rpm for 5min) to obtain oocyst precipitation liquid;

4. transferring the oocyst sediment solution into a centrifuge tube filled with 2ml of injection water, centrifuging (3000rpm for 5min), and repeating the process for three times to finally obtain the coccidia oocyst solution without disinfectant.

Adding the treated coccidian oocyst solution into a freezing tube containing 3ml of freezing preservation solution, stirring for 60 minutes in a vortex mode, and standing for 10 minutes;

respectively filling the cryopreservation liquid containing the coccidian oocysts into 1ml cryopreservation tubes, and putting the cryopreservation tubes into a cryopreservation box;

(III) cryopreservation of coccidian oocysts: putting the freezing storage box into a program temperature controller to cool to-100 ℃, and setting the cooling program as follows: the cooling rate in the temperature range of 4 ℃ to-25 ℃ is 2 ℃/min; the cooling rate in the temperature range of minus 25 ℃ to minus 100 ℃ is 15 ℃/min;

and (5) preserving the frozen box at the temperature of 100 ℃ below zero for 5 hours, and transferring the frozen box into liquid nitrogen for preservation.

(IV) recovery of coccidian oocysts: after coccidian oocysts are frozen in liquid nitrogen for 10 days, the coccidian oocysts are revived and the state of the oocysts is observed, and the method comprises the following steps:

(1) and (3) recovering oocysts: preheating a water bath kettle in advance to stabilize the liquid in the bathtub at 37 ℃, taking out the freezing tube from the liquid nitrogen, quickly putting the freezing tube into the water bath kettle at 37 ℃, and melting the freezing tube within 1-2 minutes;

(2) and (3) oocyst cleaning: diluting the melted frozen stock solution in 10ml of injection water, centrifuging (3000rpm, 5min) after vortex mixing, taking the pellet solution containing the coccidian oocysts, diluting and centrifuging again in 10ml of injection water, repeating the process for five times, taking the pellet solution of the coccidian oocysts, and then suspending the pellet solution in 2.5% potassium dichromate solution.

(3) Observing the state of oocysts: taking the oocyst suspension, placing under a microscope, and observing the structural integrity and the sporangium form inside the oocysts.

Referring to fig. 1, it can be seen that the ice crystals formed using the frozen stock solutions of the present invention had a much smaller recrystallized size than the aqueous PBS solution. It should be noted that the recrystallization process of ice in the actual cryopreservation and rewarming process is very fast, and direct in-situ observation is difficult to realize, and fig. 1 is an ice crystal recrystallization experiment in the simulated rewarming process, which is a method generally accepted in the field of cryopreservation.

Referring to fig. 2, it can be known that the single crystal ice in the frozen stock solution of the invention grows slowly and has the effect of modifying the appearance. The single crystal ice growth experiment is a main means for researching the interaction between the ice control material and ice, the single crystal ice in a normal solution grows isotropically to show sphericity, and the single crystal ice adsorbed on the ice surface by the ice control material grows slowly and shows a special shape.

The recovery results show that: after the eimeria maxima is subjected to liquid nitrogen cryopreservation for 10 days by using the novel cryopreservation agent, the shape is not changed, and the eimeria maxima is not obviously different from a fresh oocyst. Referring to fig. 3 in particular, as a result of cryopreservation of eimeria maxima oocysts by using the cryopreservation liquid of the invention, the coccidium oocysts are intact in form, the sporangium inside the oocysts are intact in form, the preservation effect is good, and the eimeria oocysts are preserved in a frozen manner.

Example 2: the embodiment is basically the same as the embodiment 1, and the difference is that the cryopreservation agent comprises the following components in percentage by mass: 0.03 percent of lauric acid, 0.2 percent of hydrogenated phosphatidylcholine, 1.7 percent of sodium fluoride, 1.5 percent of ammonium chloride, 0.2 percent of aspartic acid, 5 percent of proline, 5 percent of sucrose, 0.2 percent of sodium dihydrogen phosphate, 0.5 percent of disodium hydrogen phosphate, 3 percent of PBS buffer solution and the balance of water for injection.

Example 3: the embodiment is basically the same as the embodiment 1, and the difference is that the cryopreservation agent comprises the following components in percentage by mass: 0.02% of lauric acid, 0.05% of hydrogenated phosphatidylcholine, 3.6% of sodium fluoride, 4.1% of ammonium chloride, 0.1% of aspartic acid, 3% of proline, 7% of sucrose, 0.4% of sodium dihydrogen phosphate, 0.2% of disodium hydrogen phosphate, 2% of PBS buffer solution and the balance of water for injection.

Example 4: the embodiment is basically the same as the embodiment 1, and the difference is that the cryopreservation agent comprises the following components in percentage by mass: 0.01% of lauric acid, 0.4% of hydrogenated phosphatidylcholine, 2% of sodium fluoride, 3.5% of ammonium chloride, 0.2% of aspartic acid, 4% of proline, 6% of sucrose, 0.1% of sodium dihydrogen phosphate, 0.8% of disodium hydrogen phosphate, 4% of PBS buffer solution and the balance of water for injection.

Example 5: the embodiment is basically the same as the embodiment 1, and the difference is that the cryopreservation agent comprises the following components in percentage by mass: 0.6% of lauric acid, 0.01% of hydrogenated phosphatidylcholine, 2.8% of sodium fluoride, 2.5% of ammonium chloride, 0.25% of aspartic acid, 4% of proline, 6% of sucrose, 0.3% of sodium dihydrogen phosphate, 0.7% of disodium hydrogen phosphate, 2% of PBS buffer solution and the balance of water for injection.

The above examples are only preferred embodiments of the present invention, and the present invention is not limited to all embodiments, and any technical solution using one of the above examples or equivalent changes made according to the above examples is within the scope of the present invention.

Lauric acid and hydrogenated phosphatidylcholine in the cryopreservation agent can form micelles in water, so that hydrophilic and hydrophobic groups of the micelles can be gradually enriched on the surface of coccidian oocysts in the stirring process, and mechanical damage of ice crystals generated in the cryopreservation process to the oocysts is weakened; meanwhile, the free micelle also has the capacity of inhibiting the growth of ice crystals and the recrystallization of the ice, and further reduces the damage caused by the ice crystals. The sodium fluoride, the ammonium chloride and the aspartic acid can be selectively adsorbed on the surface of the ice crystal, so that the appearance of the ice crystal is modified, the growth of the ice crystal is slowed down, the recrystallization of the ice is inhibited, and the damage of the swollen ice crystal to the oocysts in the freezing storage process is reduced. The proline and sucrose can interact with water through hydrophilic groups such as hydroxyl and the like, so that the content of ice crystals capable of forming ice water is reduced, and the content of ice crystals generated in the process of cryopreservation is reduced. The sodium dihydrogen phosphate, the disodium hydrogen phosphate and the PBS buffer solution have the function of adjusting the pH value of the novel cryopreservation agent to enable the pH value to be in a range value suitable for cryopreservation of coccidian oocysts.

Variations and modifications to the above-described embodiments may occur to those skilled in the art, which fall within the scope and spirit of the above description. Therefore, the present invention is not limited to the specific embodiments disclosed and described above, and some modifications and variations of the present invention should fall within the scope of the claims of the present invention. In addition, although specific terms are used herein, they are used for convenience of description only and do not limit the present invention in any way, and other refrigerants that can be obtained by the same or similar method as the present invention, and their preparation methods and uses are within the scope of the present invention.

Claims

1. The cryopreservation agent is characterized by comprising the following components in percentage by mass: 0.01 to 0.6 percent of lauric acid, 0.01 to 0.4 percent of hydrogenated phosphatidylcholine, 1.7 to 3.6 percent of sodium fluoride, 1.5 to 4.1 percent of ammonium chloride, 0.1 to 0.3 percent of aspartic acid, 3 to 7 percent of proline, 5 to 8 percent of cane sugar, 0.1 to 0.4 percent of sodium dihydrogen phosphate, 0.2 to 0.8 percent of disodium hydrogen phosphate, 2 to 5 percent of PBS buffer solution and the balance of water for injection.

2. A preparation method of a freezing storage agent is characterized by comprising the following steps: which comprises the following steps:

(3) adding sodium fluoride, ammonium chloride, aspartic acid, proline and sucrose to the first mixture, and stirring until the sodium fluoride, ammonium chloride, aspartic acid, proline and sucrose are fully dissolved to obtain a second mixture;

3. The method for preparing a cryopreservation agent according to claim 2, wherein: the sodium fluoride, the ammonium chloride, the aspartic acid, the proline and the sucrose are sequentially added into the first mixture from front to back.

4. The use of the cryopreservation agent of claim 1 or the cryopreservation agent prepared by the preparation method of any one of 2-3 in cryopreservation of coccidian oocysts.

5. The use of the cryopreservation agent in coccidian oocyst cryopreservation as claimed in claim 4, wherein the application steps are as follows:

(S1) adding coccidian oocysts into the cryopreservation agent of any one of claims 1 or 2 to 3, stirring or vortex mixing, and standing to obtain a cryopreservation solution;

6. The use of the cryopreservation agent in cryopreservation of coccidial oocysts as claimed in claim 5, wherein the coccidial oocysts are purified in advance before the step (S1).

7. The use of the cryopreservation agent in coccidian oocyst cryopreservation as claimed in claim 6, wherein the purification treatment specifically comprises the following steps:

taking coccidian oocyst liquid, centrifuging and precipitating;

(II) removing the supernatant, adding saturated saline water for resuspension, precipitation and centrifugation;

(III) transferring the supernatant liquid containing the oocyst liquid into a centrifuge tube filled with injection water, and centrifuging again to obtain oocyst precipitation liquid;

(IV) transferring the oocyst sediment solution into a centrifuge tube filled with injection water, repeating the previous step for three times, and finally obtaining the coccidia oocyst solution without disinfectant components.