CN114946833B

CN114946833B - Application of schisandrin B in preparation of in-vitro biological sample preservation solution

Info

Publication number: CN114946833B
Application number: CN202210537846.3A
Authority: CN
Inventors: 黄小桃; 卫思婷; 林祥华; 张琳; 钟嘉伟
Original assignee: Guangdong Shunde Industrial Design Institute
Current assignee: Guangdong Shunde Industrial Design Institute
Priority date: 2022-05-18
Filing date: 2022-05-18
Publication date: 2023-06-30
Anticipated expiration: 2042-05-18
Also published as: CN114946833A

Abstract

The invention discloses application of schisandrin B in preparation of an in-vitro biological sample preservation solution. The invention innovatively applies the schisandrin B to the cell preservation solution, so that the preservation solution has the protection functions of inhibiting apoptosis, stabilizing cell membranes, scavenging free radicals and the like, can maintain the cell activity of an in-vitro biological sample in a low-temperature state, reduce oxidative stress damage, overcome the defects of low activity and easiness in massive swelling and death of the traditional cell culture solution in a low-temperature environment, ensure that the preservation solution can maintain the cell activity in a low-temperature state, and has the advantages of simple preparation method, convenience in transportation and preservation, and can reduce the preparation cost and transportation cost of the preservation solution.

Description

Application of schisandrin B in preparation of in-vitro biological sample preservation solution

Technical Field

The invention relates to the technical field of biology, in particular to application of schisandrin B in preparation of an in-vitro biological sample preservation solution.

Background

Along with research and application of organ transplantation, the low-temperature preservation solution has the advantages of simple use, simple and feasible preservation operation steps, low environmental requirements, and wider application range, and meets the short-term preservation requirements required in transportation and transplantation. Under the low temperature condition (0-4 ℃), the intracellular enzyme activity is low, the cell metabolism is slow, and cells and tissue organs can be preserved under the low temperature condition to prolong the survival time and improve the cell preservation effect.

The traditional low-temperature preservation method is to directly preserve the separated cells, tissues, organs and the like at a low temperature, such as 4 ℃, but the preservation method can lead to the reduction of the cell survival rate after the low-temperature preservation, shrink cell membranes, seriously damage the cell structures and can not survive after the recovery.

In clinical medicine, physiological saline is often used to maintain cell activity when preserving cells in a short period of time, but the cell activity decreases rapidly in physiological saline and the cell preservation effect is not ideal.

As technology advances, more and more researchers use special cryopreservation solutions to preserve isolated organs/tissues. The internationally common organ preservation solutions are university of wisconsin preservation solution (UW solution) and histidine-tryptophan-ketoglutarate solution (HTK solution) developed in 1987. The traditional cell cryopreservation solution mostly uses organ/tissue cryoprotection solution and lavage solution, such as HTK solution, UW solution, collins solution, etc. Among them, the UW liquid is a "gold standard" for organ preservation due to its excellent performance, but the characteristics of high price, high viscosity, high potassium, insufficient stability, etc. also limit the application range of the UW liquid.

Therefore, reducing the cost of the cryopreservation solution while increasing the viability of cells is a difficulty in improving the conventional cryopreservation solution.

Disclosure of Invention

In order to solve the problems, a first object of the present invention is to provide an application of schisandrin in preparing an in vitro biological sample preservation solution, so as to reduce the preparation cost and preservation cost of the low-temperature preservation solution and improve the survival rate of cells after low-temperature preservation.

The second object of the present invention is to provide an isolated biological sample preservation solution comprising schisandrin b and at least one of an energy substrate, a pH buffer, an osmotic pressure maintaining agent, an osmotic pressure regulator and a salt substance required for preservation of a biological sample.

In one implementation mode of the invention, the concentration of the schisandrin B in the preservation solution is 0.02 g/L-0.04 g/L.

In one implementation mode of the invention, the preservation solution further comprises the following components:

in one implementation of the present invention, the preservation solution includes:

in one implementation mode of the invention, the compound amino acid aqueous solution is a mixed solution of compound amino acid stock solution and water.

In one implementation mode of the invention, the volume ratio of the compound amino acid stock solution to the preservation solution is (1-3): 100; and/or

The compound amino acid stock solution contains 10% of compound amino acid by mass percent; and/or

The compound amino acid stock solution is compound amino acid injection 18aa.

In one implementation of the present invention, the osmotic pressure of the preservation solution is 280 to 310m0sm/kg.

In one implementation of the invention, the pH value of the preservation solution is 7.2-7.4.

A third object of the present invention is to provide a method for preserving an isolated biological sample, wherein the isolated biological sample is preserved at a low temperature in the preservation solution.

In one implementation mode of the invention, the temperature for low-temperature preservation is 0-4 ℃.

In one implementation of the invention, the ex vivo biological sample is at least one of a hepatocyte sample and a liver tissue sample.

The invention innovatively applies the schisandrin B to the cell preservation solution, so that the preservation solution has the protection functions of inhibiting apoptosis, stabilizing cell membranes, scavenging free radicals and the like, can maintain the cell activity of an in-vitro biological sample in a low-temperature state, reduce oxidative stress damage, overcome the defects of low activity and easiness in massive swelling and death of the traditional cell culture solution in a low-temperature environment, ensure that the preservation solution can maintain the cell activity in a low-temperature state, and has the advantages of simple preparation method, convenience in transportation and preservation, and can reduce the preparation cost and transportation cost of the preservation solution.

Drawings

FIG. 1 is a graph showing the result of the trypan blue staining method according to example 5 of the present invention;

fig. 2 is a schematic diagram showing the detection results of AST content and liver SOD content in the preservation solution provided in example 5 of the present invention.

Detailed Description

Reference now will be made in detail to embodiments of the invention, one or more examples of which are described below. Each example is provided by way of explanation, not limitation, of the invention. Indeed, it will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the scope or spirit of the invention. For example, features illustrated or described as part of one embodiment can be used on another embodiment to yield still a further embodiment.

Accordingly, it is intended that the present invention cover such modifications and variations as fall within the scope of the appended claims and their equivalents. Other objects, features and aspects of the present invention will be disclosed in or be apparent from the following detailed description. It is to be understood by one of ordinary skill in the art that the present discussion is a description of exemplary embodiments only, and is not intended as limiting the broader aspects of the present invention.

As above, the damage to cells caused by cryopreservation is mainly several: cell membrane fluidity is reduced, cell expansion is caused by cell membrane damage, oxidative damage is caused by environmental pressure, and cell necrosis and apoptosis are caused by generation of a large amount of lipid peroxide. The traditional preservation method uses common normal saline, has low cell survival rate after preservation, or has expensive preservation solution and unstable components, thus resulting in high low-temperature preservation cost.

In order to at least partially solve at least one of the above technical problems, the first aspect of the present invention provides an application of schisandrin in preparing an isolated biological sample preservation solution, wherein schisandrin enables the preservation solution to have protection functions of inhibiting apoptosis, stabilizing cell membranes, scavenging free radicals and the like, can maintain cell activity of the isolated biological sample in a low temperature state, reduces oxidative stress damage, enables the preservation solution to maintain cell activity in a low temperature, has the advantages of simple preparation, and convenient transportation and preservation, and can reduce preparation cost and transportation cost of the preservation solution.

The second aspect of the present invention provides an isolated biological sample preservation solution, comprising schisandrin b and at least one of an energy substrate, a pH buffer, an osmotic pressure maintaining agent, an osmotic pressure regulator and a salt substance required for a biological sample, so that the isolated biological sample can maintain the survival rate and the activity of cells in long-term preservation and transportation; the schisandrin B can maintain the cell activity of the isolated biological sample in a low-temperature state, reduce oxidative stress damage and keep the form of cell membranes intact, so that the preservation solution can preserve the isolated biological sample in a low-temperature state, reduce the apoptosis phenomenon in the isolated biological sample, improve the survival rate of cells in the isolated biological sample, and has the advantages of simple preparation, convenience in transportation and preservation, and can reduce the preparation cost and transportation cost of the preservation solution.

Wherein, the concentration of the schisandrin B is 0.02 g/L-0.04 g/L, and further 0.03 g/L-0.04 g/L, so as to realize better low-temperature preservation effect.

In some embodiments, the preservation solution further comprises the following components: 5g/L to 6g/L sodium chloride, 0.3g/L to 0.5g/L potassium chloride, 0.01g/L to 0.03g/L calcium chloride, 0.8g/L to 1g/L disodium hydrogen phosphate, 0.18g/L to 0.23g/L magnesium sulfate, 0.4g/L to 1.2g/L sodium bicarbonate, 45g/L to 55g/L dextran 40,3.1g/L to 4.0g/L mannitol, 1g/L to 1.6g/L glucose, 4g/L to 5.5g/LHEPES,1g/L to 1.5g/L adenosine, 0.7g/L to 1g/L LD-trehalose, 0.04g/L to 0.07g/L reduced peptide, 0.1g/L to 0.3g/L vitamin C and compound amino acid aqueous solution.

The invention utilizes the components in the formula to form basic salts, pH buffering agent, hibernation protective agent, energy substrate, scavenger for removing metabolic products, colloid osmotic pressure maintaining agent and the like required by cells, thereby forming the hepatocyte preservation solution with stable components, multiple nutrient substances and strong buffering capacity of a solution osmotic system. When the preservation solution is used for preserving cells or organs, the preservation solution not only can provide nutrition for the cells and provide proper osmotic pressure to prevent cell edema caused by low temperature, but also can well maintain the activity of the cells, reduce the oxidative damage of the liver cells and improve the survival rate of the cells after low-temperature preservation by correcting acidosis in the cells through a buffer system.

The invention is preservedThe reduced glutathione and the trehalose in the liquid are good protective agents for the low-temperature preservation of the isolated cells, and the isolated cells in the low-temperature preservation can be protected through the channels of scavenging oxygen free radicals, inhibiting calcium channels, increasing the stability of cell membranes, inhibiting apoptosis caused by low temperature and the like; glucose, adenosine, provides energy to cells; non-CO ₂ The dependency buffer pair can reduce the CO content of the preservation solution during transportation and air exposure ₂ Reducing the fluctuation of the pH value of the preservation solution; schisandrin B has antiinflammatory, free radical scavenging and antioxidant effects. The invention optimizes the formula of the low-temperature preservation solution, and maintains the biological activity and the specific function of the cells by a low-temperature preservation method; meanwhile, under the condition of low-temperature preservation at 4 ℃, no nutrition and oxygen supply are needed in the transportation and preservation process of the cells, the operation is simple and feasible, the convenience is brought to the preservation and transportation of the isolated cells, and the method can also be used for the in-vitro static cold preservation of the liver transplantation donor.

In some embodiments, the compound amino acid aqueous solution comprises a mixture of a compound amino acid stock solution and water for providing multiple nutrients for cell preservation.

In some specific embodiments, the compound amino acid stock solution contains 10% compound amino acid by mass percent, the compound amino acid stock solution is compound amino acid injection 18aa, and the volume ratio of the compound amino acid stock solution to the preservation solution is (1-3): 100, further 20:100, which is compounded with the other components in the preservation solution to provide multiple nutrients for cell preservation.

In some embodiments, the preservation solution comprises the following components: 5.5g/L to 6g/L sodium chloride, 0.4g/L to 0.5g/L potassium chloride, 0.02g/L to 0.03g/L calcium chloride, 0.9g/L to 1g/L disodium hydrogen phosphate, 0.2g/L to 0.23g/L magnesium sulfate, 0.8g/L to 1.2g/L sodium bicarbonate, 50g/L to 55g/L dextran 40,3.6g/L to 4.0g/L mannitol, 1.3g to 1.6g/L glucose, 4.8g to 5.5g/L LHEPES,1.2g to 1.5g/L adenosine, 0.8g to 1g/L LD-trehalose, 0.06g to 0.07g/L reduced glutathione, 0.2g to 0.3g/L vitamin C,0.03g to 0.04g/L compound schisandra fruit of an amino acid solution. In some embodiments, the preservation solution comprises: 6g/L sodium chloride, 0.5g/L potassium chloride, 0.02g/L calcium chloride, 0.8g/L disodium hydrogen phosphate, 0.2g/L magnesium sulfate, 0.5g/L sodium bicarbonate, 52g/L dextran 40,3.7g/L mannitol, 1.5g/L glucose, 4.8g/LHEPES,1g/L adenosine, 1 g/LD-trehalose, 0.05g/L reduced glutathione, 0.1g/L vitamin C,0.03g/L schisandrin and compound amino acid aqueous solution, so that the preservation solution has better preservation effect.

The osmotic pressure of the preservation solution is 280m0 sm/kg-310 m0sm/kg, more can be 290m0 sm/kg-310 m0sm/kg, still more can be 300m0 sm/kg-310 m0sm/kg, and can avoid the phenomenon of massive death caused by swelling of cells due to osmotic pressure; the pH value of the protective solution is 7.2-7.4, and further 7.3-7.4, so that the buffer force of the preservation solution can be maintained, and a reasonable pH space is provided for the survival of cells at low temperature.

The third aspect of the present invention provides a method for preserving an isolated biological sample, comprising placing the isolated biological sample in the preservation solution for low-temperature preservation, wherein the preservation solution of the present invention can alleviate the conditions of cell membrane fluidity decrease, cell membrane damage causing cell expansion, oxidative damage caused by environmental pressure, cell necrosis and apoptosis caused by the generation of a large amount of lipid peroxides, and the like, maintain cell membrane morphology, reduce cell oxidative damage, and thereby improve cell activity of cells in the low-temperature environment.

In some specific embodiments, the temperature for low-temperature preservation is 0-4 ℃, further can be 2-4 ℃, and can be 3 ℃, and the preservation solution can achieve a better activity preservation effect on the damage of cells caused by a low-temperature environment of 0-4 ℃.

In some embodiments, the ex vivo biological sample is at least one of a hepatocyte sample and a liver tissue sample, and it is understood that the preservation solution can be used for low temperature transport preservation of the ex vivo hepatocyte sample, and also for static low temperature preservation of liver tissue during liver transplantation.

Therefore, the fourth aspect of the present invention also provides a method for preparing a preservation solution of an isolated biological sample, comprising: and (3) dissolving the components in water according to the concentration of the components in the preservation solution, stirring and uniformly mixing to obtain a mixed solution, regulating the pH value of the mixed solution to be 7.2-7.4, and regulating the osmotic pressure to be 280m0 sm/kg-310 m0sm/kg to obtain the in-vitro biological sample preservation solution.

In some embodiments, the in vitro biological sample preservation solution is further subjected to a sterilization operation. Specifically, the preparation is completed, a sterile PES filter (0.22 mu m) is used for filtration and sterilization to obtain a hepatocyte preservation solution, the hepatocyte preservation solution is placed in a 4-DEG refrigerator for preservation in a dark place, and after the sterile PES filter (0.22 mu m) is used for filtration and sterilization, a sample is extracted for bacterial culture inspection. The preservation solution provided by the invention has the advantages of simple preparation method and convenience in transportation and preservation while improving the cell survival rate, and can reduce the preparation cost and transportation cost of the preservation solution.

Embodiments of the present invention will be described in detail below with reference to examples.

The reagents according to the following examples of the invention are all obtained by conventional commercial means.

Example 1

The embodiment provides a cell preservation solution, 1000mL of which comprises the following components: 6g/L of sodium chloride, 0.5g/L of potassium chloride, 0.02g/L of calcium chloride, 0.8g/L of disodium hydrogen phosphate, 0.2g/L of magnesium sulfate, 48g/L of dextran 40, 3.9g/L of mannitol, 1.5g/L of glucose, 4g/L of HEPES,1g/L of adenosine, 0.05g/L of reduced glutathione, 0.1g/L of vitamin C and 20mL of supplementary 10% compound amino acid, and the balance of sterile water for injection or double distilled water. The medicines are stirred and mixed uniformly to obtain a mixed solution, and the pH value of the cell buffer solution is regulated to 7.4.

Example 2

The embodiment provides a cell preservation solution, 1000mL of which comprises the following components: :6g/L of sodium chloride, 0.5g/L of potassium chloride, 0.02g/L of calcium chloride, 0.8g/L of disodium hydrogen phosphate, 0.2g/L of magnesium sulfate, 0.5g/L of sodium bicarbonate, 52g/L of dextran 40,3.7g/L of mannitol, 1.5g/L of glucose, 4.8g/L of HEPES,1g/L of adenosine, 1g/L of trehalose, 0.05g/L of reduced glutathione, 0.1g/L of vitamin C, 0.02g/L of schisandrin and 15mL of supplementary 10% compound amino acid, and the balance of sterile water for injection or double distilled water. The mixture is stirred and mixed evenly to obtain a mixed solution, and the pH value of the cell buffer solution is regulated to 7.2.

Example 3

The embodiment provides a cell preservation solution, 1000mL of which comprises the following components: :6g/L of sodium chloride, 0.5g/L of potassium chloride, 0.02g/L of calcium chloride, 0.8g/L of disodium hydrogen phosphate, 0.2g/L of magnesium sulfate, 0.5g/L of sodium bicarbonate, 50g/L of dextran 40,3.7g/L of mannitol, 1.5g/L of glucose, 4.7g/L of HEPES,1g/L of adenosine, 1g/L of trehalose, 0.05g/L of reduced glutathione, 0.1g/L of vitamin C, 0.02g/L of ligustrazine hydrochloride and 15mL of supplementary 10% compound amino acid, and the balance of sterile water for injection or double distilled water. The medicines are stirred and mixed uniformly to obtain a mixed solution, and the pH value of the cell buffer solution is regulated to 7.3.

Example 4

The embodiment provides a cell preservation solution, 1000mL of which comprises the following components: :6g/L of sodium chloride, 0.5g/L of potassium chloride, 0.02g/L of calcium chloride, 0.8g/L of disodium hydrogen phosphate, 0.2g/L of magnesium sulfate, 1g/L of sodium bicarbonate, 52g/L of dextran 40,3.7g/L of mannitol, 1.5g/L of glucose, 4.7g/L of HEPES,1g/L of adenosine, 0.05g/L of reduced glutathione, 0.15g/L of vitamin C, 0.02g/L of catechin and 15mL of 10% compound amino acid supplement, and the balance of sterile water for injection or double distilled water. The medicines are stirred and mixed uniformly to obtain a mixed solution, and the pH value of the cell buffer solution is regulated to 7.3.

The preservation solutions of examples 1 to 4 were subjected to a 48-hour cell activity test. When the cell growth reaches the logarithmic growth phase, the collected cells are digested, the cell density is adjusted to be 2 multiplied by 105/ml, the cells are inoculated into a six-hole culture plate, the liquid adding amount of each hole reaches 2ml, and after the cells are subjected to the wall-attached culture for 24 hours in a 37 ℃ carbon dioxide incubator, the cell growth reaches 80% confluence. The original culture medium from each well was aspirated, and washed with PBS (2 ml). 2ml of the protective solution in examples 1 to 4 was added, and the mixture was kept at 2℃to 8℃for 48 hours. Cell viability was measured by Trypan Blue staining (Trypan Blue) of each sample, and the results are shown in table 1.

TABLE 1

Grouping	Survival rate of 48h
		Example 1	73.8％
Example 2	89.1％
		Example 3	81.4％
Example 4	75.9％

As can be seen from Table 1, the preservation solution added with Schisandra chinensis extract has better effect of preserving cell-protecting activity than the preservation solution without Schisandra chinensis extract, the preservation solution added with ligustrazine hydrochloride or the preservation solution of catechin, and the preservation solution added with Schisandra chinensis extract has higher cell survival rate.

Example 5

The preservation solution, HTK solution, UW solution and ringer's solution of example 2 were used for comparison, and L-02 hepatocytes of the same origin were subjected to preservation test study by the same method, and the preservation results were recorded and compared. The cell culture plate is produced by Corning company, 1640 culture medium is purchased to Shanghai Biotechnology (Shanghai) stock company, and medicines such as schizandrin B are purchased to Shanghai Michelin Biochemical technology Co.

1. LDH cell activity assay:

the damaged cell membrane of the cell is ruptured and LDH is released, so that the survival of the cell can be assessed using Lactate Dehydrogenase (LDH) levels. The amount of Lactate Dehydrogenase (LDH) released from different groups of cells was determined using a kit.

When the fusion degree of the liver cells in the culture flask reaches more than 80%, carrying out passage operation and paving 6-hole plates according to the density of 3X 106 pieces/ml, 2 ml/hole, 37 ℃ and 5% CO ₂ After the carbon dioxide incubator is subjected to wall-attached culture for 24 hours, the carbon dioxide incubators are grouped: washing with PBS after cold preservation for 48 hours, adding complete culture medium into each group, and placing the groups in an incubator for re-culturing for 6 hours; taking cell supernatants of each group, and centrifuging for later use. LDH leakage was measured in each supernatant set using LDH assay kit (supplied by nanjing built biosystems). The test steps are as follows:

(1) Sample addition was performed as shown in table 1, with three duplicate wells per set;

TABLE 2

	Blank hole	Standard hole	Measuring hole	Control wells
					Ultrapure water (mu l)	25	5		5
0.2mmol/L standard solution (μl)		20
					Sample to be measured (mul)			20	20
Substrate buffer (μl)	25	25	25	25
					Coenzyme (mul)			5

(2) After the addition is completed, slightly and uniformly mixing, and incubating in a constant temperature box at 37 ℃ for 15min;

(3) (3) adding 25 μl of 2, 4-dinitrotoluene into each well, mixing well, and incubating in a incubator at 37deg.C for 15min;

(4) 250 μl of 0.4mmol/LNaOH solution is added into each hole, the mixture is placed at room temperature for 5min, and the OD value of each hole with the wavelength of 450nm is measured by using a multifunctional enzyme-labeled instrument;

(5) LDH activity was calculated according to the calculation formula: LDH activity (U/L) = (determination OD-control OD)/(Standard OD-blank OD). Times.standard concentration. Times.1000 (note: standard concentration is 0.2 mmol/L).

TABLE 3 Table 3

Grouping	LDH content (U/L)
		HTK group	193.25
UW group	165.02
		Ringer's solution	233.45
Example 1	147.37
		Example 2	136.36
Normal group	87.12

The LDH content test results of each group of supernatants detected by LDH kit are shown in table 3. As can be seen from Table 3, the cells were recovered after 48 hours of low temperature storage and then cultured, and the state of example 2 was better, and the cells were recovered after recovery, which was significantly different from those of ringer's solution and HTK solution, and the effect of cell preservation was significantly better than those of ringer's solution and HTK solution. Has no obvious difference with the LDH release amount of the UW liquid group.

The results prove that the best maintenance of the bioactivity of the liver cells in the example 2 group in the experimental group is realized, and the addition of the schisandrin is beneficial to enhancing the preservation effect and plays a positive role in reducing the apoptosis of the liver cells; can maintain the survival rate and bioactivity of liver cells during long-term preservation and transportation.

2. Cell viability assay for long-term cold storage

Cell culture: conventional cell culture was carried out in a cell culture chamber, once every 3 days, and after the confluence of cell growth reached 80%, the hepatocytes were collected by digestion, resuspended in a cell culture medium, and then cultured in a medium of 2X 10 ⁵ Inoculating the cell density/ml into a six-hole cell culture plate, adding 2ml of liquid into each hole, carrying out wall-attached culture for 24 hours at 37 ℃ by a carbon dioxide incubator, and preserving at a low temperature of 4 ℃ until the cell growth reaches 80% confluency.

2. Preserving at low temperature: after the original culture medium of each hole is sucked and washed twice by PBS buffer solution, 2ml of preservation solution prepared according to the example 2 is added into each hole of the six-hole culture plate, the six-hole culture plate is placed in a refrigerator with the temperature of 4 ℃ for cold preservation, the culture plate is sealed by a sealing film, and the cold preservation time is 1 to 6 days.

3. Survival rate was measured by trypan blue staining: after the cell culture plates were taken out of the refrigerator at 4℃on time from 1 to 6 days of cold preservation, after each set of preservation solutions was sucked out, the cells were collected and the cell viability was measured for a cold preservation time of 1 to 6 days using the trypan blue dye exclusion test. Samples were stained with trypan blue (TrypanBlue), the number of living and dead cells counted, and the cell viability was calculated as cell viability = living cell number/(living cell number + dead cell number). The trypan blue staining results are shown in figure 1. Cell viability was measured using a cell counter for about 3min with trypan blue exclusion, and data were collected to build a comparison table of cell preservation test results, the results of which are shown in table 4.

TABLE 4 cell viability Table for 1-6 days of Cold preservation

Grouping	Cell viability
		Cold preserving for 1 day	91％
Cold preserving for 2 days	85.6％
		Cold preserving for 3 days	77.8％
Cold preserving for 4 days	70.1％
		Cold preserving for 5 days	50.5％
Cold preserving for 6 days	44.6％

4. Cell re-proliferation assay: collecting cells after cold preservation for 1-6 days, centrifuging, re-suspending with complete medium, counting total number of cells by using a blood cell counting plate, and counting at 2.5X10 per group ⁵ Cell density of wells was re-cultured in six well plates and placed at 37℃in 5% CO ₂ The cells were normally cultured in the incubator for 48 hours, all cells were collected by digesting the cells with pancreatin, the total number of cells was counted, the fold-increase of the cells after 48 hours of re-proliferation was calculated from the initial density, and the results are shown in table 5, and the statistical data were analyzed.

TABLE 5

Grouping	Cell count/well	Multiplication factor
			Cold preserving for 1 day	1.42×106	5.68
Cold preserving for 2 days	1.33×106	5.32
			Cold preserving for 3 days	1.09×106	4.36
Cold preserving for 4 days	8.1×105	3.24
			Cold preserving for 5 days	6.4×105	2.56
Cold preserving for 6 days	4.5×105	1.80

As is clear from Table 4, when the liver cells were stored at low temperature using the storage solution of example 2, the 24-hour cell survival rate was 90% or more, the 48-hour cell survival rate was 85% or more, and the 72-hour cell survival rate was 75% or more. The preservation solution of example 2 was considered to have a good effect of protecting the viability of hepatocytes because the viability of hepatocytes was maintained at 50% or more, and the preservation solution of the present invention was used to preserve hepatocytes at low temperature for about 5 days when the viability of hepatocytes was 50% or more. As is clear from Table 5, the preservation solution of the present invention has a good protective effect on the physiological state of liver cells, and the cells are observed to be normal in morphology after re-proliferation by an optical microscope. Therefore, the cell preservation solution in the embodiment 2 of the invention has excellent cell protection effect and good application and development prospects.

3. Liver low-temperature preservation test verification

1. Extracting isolated livers: SPF-grade healthy male SD rats were selected to be fasted for 12 hours prior to surgery without water withdrawal. The hind limb is injected with anesthesia and hypnotic agent at a speed of 0.6ml/kg. After successful anesthesia, the rats were fixed on an operating table, the abdomen was dehaired, and sterilized with 75% alcohol. After shaving, the abdomen of the rat is crossed and cut from the upper edge to the xiphoid process, and the lower edge is pubic symphysis. The stomach and intestine in the abdominal cavity is lightly pushed to the left iliac region by a cotton swab, and the saline gauze is covered for moisturizing, so that the perihepatic ligament and the abdominal aorta are passively separated, and the liver is exposed. The hepatic inferior vena cava was free and the portal vein was free for in vivo lavage. After the abdominal aorta is subjected to intubation treatment, the liver is slowly flushed to be earthy yellow by using 40ml of heparinized normal saline for flushing and shearing the inferior hepatic vena cava as an outlet. Portal catheterization was then performed, and the liver was rinsed thoroughly and isolated by portal intravenous injection of approximately 20ml of each set of preservative fluid at 4 ℃.

2. Cold preservation of liver: taking liver, placing in an organ preservation bag filled with 100ml preservation solution at 4deg.C, fully soaking the liver, sealing the organ preservation bag, soaking the organ in the preservation solution, cold preserving at 4deg.C for 24 hr, and sampling for analysis. And (3) centrifuging the preservation solution sample at 3000rpm for 5min, taking supernatant, preserving in an ultralow temperature refrigerator at-80 ℃, and detecting AST content by using a full-automatic biochemical analyzer. The SOD is measured by xanthine oxidase method, and the generation amount of SOD is measured by corresponding commercial kit according to the instruction of manufacturer (Nanjing built biological engineering institute). The organ preservation solution is the preservation solution of the embodiment 2, the UW solution and the HTK solution in sequence, and the sample size of each group is 4. The experimental results are expressed as mean ± standard deviation, as shown in fig. 2.

As can be seen from fig. 2, the AST content and the liver tissue SOD content in the preservation solution detected after the preservation was completed, respectively, indicate that the preservation solution of example 2 can effectively resist oxidative stress injury and protect liver tissue. After the liver is preserved for 24 hours at low temperature, the AST content of the group is not obviously different from that of the UW liquid preservation group, but is obviously better than that of the HTK liquid group, and the preservation effect is better. The SOD content of the preservation solution of example 2 is the highest, and the SOD content of the preservation solution is remarkably different from the other two types, and the antioxidant stress effect of the preservation solution of example 2 is better. The preservation solution provided by the invention provides a good preservation effect for preserving liver organs at low temperature, and has low production cost and practical application value.

The technical features of the above embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.

The foregoing examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.

Claims

1. An isolated biological sample preservation solution, which is characterized by comprising the following components in 1000 mL: 6g/L of sodium chloride, 0.5g/L of potassium chloride, 0.02g/L of calcium chloride, 0.8g/L of disodium hydrogen phosphate, 0.2g/L of magnesium sulfate, 0.5g/L of sodium bicarbonate, 52g/L of dextran 40,3.7g/L of mannitol, 1.5g/L of glucose, 4.8g/L of HEPES,1g/L of adenosine, 1g/L of trehalose, 0.05g/L of reduced glutathione, 0.1g/L of vitamin C, 0.02g/L of schisandrin and a compound amino acid aqueous solution;

the isolated biological sample is an L-02 hepatocyte sample.

2. The preservation solution according to claim 1, wherein the compound amino acid aqueous solution is a mixed solution of a compound amino acid stock solution and water.

3. The preservation solution according to claim 2, wherein the compound amino acid stock solution contains 10% of compound amino acid by mass percent or the compound amino acid stock solution is compound amino acid injection 18aa;

the volume ratio of the compound amino acid stock solution to the preservation solution is 1-3: 100.

4. a preservation solution according to claim 3, characterized in that the osmotic pressure of the preservation solution is 280-310 m0sm/kg; and/or

The pH value of the preservation solution is 7.2-7.4.

5. An isolated biological sample storage method, characterized in that an isolated biological sample is stored at a low temperature in the storage solution according to any one of claims 1 to 4.

6. The method of claim 5, wherein the low temperature storage temperature is 0-4 ℃.