CN110352952A - A kind of composition saved for cell, aqueous compositions and its application - Google Patents

Abstract

The invention discloses a composition for cell preservation, an aqueous preparation comprising the composition, and the application of the aqueous preparation in the preservation of biological samples before flow cytometry cell detection. The composition for cell preservation includes the following Components: Potassium dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride, potassium chloride, dextran 40, tetramisole hydrochloride, polyaldehydes, anticoagulant, of which potassium dihydrogen phosphate, disodium hydrogen phosphate , sodium chloride, potassium chloride, dextran 40, tetramisole hydrochloride, polyaldehydes, the mass ratio of anticoagulant is 0.2: 1.4: 8-9: 0.2: 5-25: 25-50: 25: 20‑30; the composition of the present invention will compound potassium dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride, potassium chloride, dextran 40, tetramisole hydrochloride, polyaldehydes, anticoagulant, can Effective and stable preservation of biological samples for up to 60 days, providing an extremely important guarantee for sample processing before testing or diagnosis.

Description

A composition for cell preservation, its aqueous formulation and its application

technical field

The invention relates to the technical field of cell preservation, in particular to a composition for cell preservation, an aqueous preparation comprising the composition, and the application of the aqueous preparation in the preservation of biological samples before flow cytometry cell detection.

Background technique

Flow cytometry (Flow Cytometry, FCM) is the most advanced cell quantitative analysis technology, with the advantages of rapidity, high precision, and multiple parameters. FCM plays an important role in both clinical and scientific research fields, especially in clinical in vitro diagnosis (In Vitro Diagnostic, IVD), which has been widely and deeply applied and has become one of the most mainstream detection methods. Its clinical applications include: immunology, Hematology, clinical oncology, hematopoietic stem cell transplantation, etc.; it is currently the gold standard for clinical diagnosis of malignant diseases such as leukemia and lymphoma, and plays an important role in drug sensitivity screening and pathogenesis research.

Biological samples used for FCM on-board testing are generally peripheral blood, bone marrow or other tissue samples. After the biological sample is obtained and before it is tested on the machine, the biological sample needs to be properly preserved. After the cells leave the human body, due to changes in the environment, the physical and chemical characteristics of the cells and various biomarkers may change accordingly, which will affect the detection results, and even affect the diagnosis and further medical treatment plans. Therefore, it is an extremely important step in sample processing before detection or diagnosis to properly preserve and protect cells in biological samples to ensure that the cells are in a good state closest to the initial state.

At present, there are relatively few researches on the special cell preservation technology before FCM detection. Generally speaking, the main problems that need to be solved in the preservation technology include: (1) maintaining the stable state of cell biomarkers, especially cell surface markers; (2) ) can prolong the storage time of samples on the basis of ensuring the accuracy of detection or diagnosis; (3) the formulation of storage reagents is easier to prepare, the cost is lower, and the operation is more convenient.

Application publication number CN 107091799A discloses "a blood cell stabilizer", including A solution and B solution, wherein A solution includes the following components: anticoagulant, phosphatase inhibitor, protease inhibitor; B solution includes the following components : Aldehyde polymer, alkaline salt, buffer; Wherein, liquid A and liquid B are mixed according to the volume ratio of 0.5-2:0.5-2. The above-mentioned blood cell stabilizer has the function of protecting tissue cells. It can stabilize blood cells, maintain the original shape of blood cells, maintain the original physical properties of blood cells, and can well fix chemical substances such as proteins and enzymes. cells in the blood; however, the above-mentioned blood cell stabilizer needs to store the A agent and the B agent separately, and then mix them before use, which brings inconvenience to the use; in addition, the above-mentioned blood cell stabilizer can preserve the blood for only 192 hours , the storage time is short, which limits the scope of application.

Contents of the invention

In order to solve the problem that the current cell preservation solution can preserve cells for a short time and that agent A and agent B need to be stored separately, the object of the present invention is to provide a composition for cell preservation; in addition, the present invention also provides The aqueous preparation of the substance and the application of the aqueous preparation in the preservation of biological samples before flow cytometry cell detection.

In order to achieve the above object, the present invention adopts the following technical solutions:

The first aspect of the present invention provides a composition for cell preservation, including the following components: potassium dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride, potassium chloride, dextran 40, tetramisole hydrochloride, Polyaldehydes, anticoagulant, wherein, the mass ratio of potassium dihydrogen phosphate, disodium hydrogenphosphate, sodium chloride, potassium chloride, dextran 40, tetramisole hydrochloride, polyaldehydes, anticoagulant is 0.2:1.4:8-9:0.2:5-25:25-50:25:20-30.

Preferably, the mass ratio of potassium dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride, potassium chloride, dextran 40, tetraimidazole hydrochloride, polyaldehydes, and anticoagulant is 0.2:1.4: 9:0.2:5:25:25:30.

Preferably, the mass ratio of potassium dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride, potassium chloride, dextran 40, tetraimidazole hydrochloride, polyaldehydes, and anticoagulant is 0.2:1.4: 9:0.2:15:25:25:30.

The above-mentioned tetramisole hydrochloride is a phosphatase inhibitor.

Wherein, the paraaldehydes are one or a mixture of paraacetaldehyde or parapropionaldehyde.

Wherein, the anticoagulant is one of EDTA, citrate or a mixture of both.

The second aspect of the present invention provides an aqueous preparation for cell preservation, comprising the above-mentioned composition.

The third aspect of the present invention provides the application of the above-mentioned aqueous preparation in the preservation of biological samples before flow cytometry cell detection.

Wherein, the biological sample is human blood or tissue sample.

Wherein, the biological sample is human bone marrow or human peripheral blood.

A fourth aspect of the present invention provides a method for cell preservation, comprising the following steps:

S1, mixing the above composition with water to form an aqueous formulation;

S2, the biological sample is mixed with the aqueous preparation in S1, and stored at 2-8°C.

Compared with the prior art, the beneficial effects realized by the present invention: the composition of the present invention combines potassium dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride, potassium chloride, dextran 40, tetramisole hydrochloride, polyaldehyde It is used in combination with class and anticoagulant. By fixing the cells to maintain the original shape of the cells, maintain the original physical properties of the cells, and inhibit the action of enzymes on the cell membrane and proteins, it can effectively and stably preserve biological samples for up to 60 days. Or pre-diagnosis sample processing provides extremely important safeguards.

Detailed ways

The implementation of the present invention will be illustrated by specific specific examples below, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

The present invention adds dextran 40, tetraimidazole hydrochloride, polyaldehydes and anticoagulant on the basis of potassium dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride and potassium chloride, dextran 40, tetramisole hydrochloride The compatibility of polyaldehydes and anticoagulants can fix cells, maintain the original cells of cells, and can stably store biological samples for up to 60 days, which is convenient for sample processing before detection or diagnosis.

The inventors of the present invention unexpectedly found through long-term research and experiments that the proper osmotic pressure of the aqueous preparation on the cells can increase the preservation effect of the cells, and by adjusting the amount of sodium chloride, the possibility of cell damage during preservation is reduced, and the cells original form.

1. Preparation of aqueous formulations

1. Under the condition that the temperature is 20-25°C and the humidity is less than 60%, weigh each raw material and place it in a 50mL sterile centrifuge tube to obtain the composition for cell preservation;

2. Dissolving the above-mentioned composition for cell preservation in a certain amount of water for injection to obtain a colorless transparent liquid;

3. In a sterile environment with local 100-level laminar flow purification conditions, pass the above-mentioned colorless transparent liquid through a filter membrane with a diameter of 0.22 μm, and transfer the filtrate to another 50 mL sterile centrifuge tube to obtain the The preserved aqueous preparation was stored at 4°C in the dark for future use.

Aqueous formulations with different compositions are obtained by changing the amount of raw materials. The composition of each aqueous formulation is shown in Table 1. Table 1 is in weight percentage, and the balance is water for injection.

Table 1

2. Aqueous preparations for cell preservation

Randomly select 20 healthy adult volunteers, aged between 20-40, regardless of gender. The anticoagulated peripheral blood samples (n=20) of the volunteers were mixed with the aqueous preparations prepared in Examples 1-12 and Comparative Examples 1-5 respectively, and the volume ratio of the anticoagulated peripheral blood samples to the aqueous preparation was 10:1. Store the mixed sample at 2-8°C, and take out the sample when testing is required.

Three, the water-containing preparation of the present invention and control example carry out the effect evaluation of cell preservation

The mixture of anticoagulated peripheral blood samples and aqueous preparations was mixed upside down 10 times, stored in a refrigerator at 4°C, and tested by FCM on 0d, 10d, 20d, 30d, 40d, 50d, and 60d, respectively. The results are shown in Table 2 and Table 2. 3. Table 2 and Table 3 only show the changes in the cells of one of the anticoagulated peripheral blood samples after storage in Examples 1-12 and Control Examples 1-5, and Table 2 shows the changes in granulocytes, monocytes, and lymphocytes Ratio changes, Table 3 shows the ratio changes of T cells, B cells, NK cells, and DC cell surface markers. A total of 20 cases of the above-mentioned FCM tests were carried out in parallel, and the test results were all highly similar to those in Table 2 and Table 3, proving that the aqueous preparation of the present invention can effectively and stably preserve biological samples for up to 60 days.

The general experimental steps of FCM detection are as follows: (1) Antibodies are added to the bottom of the flow tube respectively according to the dosage in the instructions. (2) Take 100ul biological samples and add them along the tube wall, mix well, and incubate at room temperature in the dark for 20 minutes. (3) Add 2ml of erythrocyte lysate, mix thoroughly, and keep away from light for 10 minutes at room temperature. (4) Centrifuge at 1500 rpm for 5 minutes, discard the supernatant. (5) Add 2ml of washing solution, mix well, centrifuge at 1500rpm for 5 minutes, and discard the supernatant. (6) Add 0.4ml of PBS wash solution, mix well, and perform flow cytometry detection. (7) Analyze the results.

For the Lin-FITC antibody, due to the relatively high viscosity, the operation method was improved on the basis of the general experimental procedures. The specific experimental procedures are as follows: (1) Take 100ul EDTA biological sample and add it to the bottom of the tube along the tube wall. (2) Add 2ml of erythrocyte lysate, mix well, and keep away from light for 10 minutes at room temperature. (3) Centrifuge at 1500 rpm for 5 minutes, discard the supernatant. (4) Add 2ml of washing solution, mix well, centrifuge at 1500rpm for 5 minutes, and discard the supernatant. (5) Antibodies were added to the bottom of the flow tube respectively according to the dosage specified in the instructions. Incubate at room temperature for 20 minutes in the dark. (6) Add 2ml of washing solution, mix well, centrifuge at 1500rpm for 5 minutes, and discard the supernatant. (7) Add 0.4ml of PBS washing solution, mix well, and perform flow cytometry detection.

Table 2

table 3

As shown in Table 1, compared with Example 12, the addition amount of K ₂ HPO ₄ , NaH ₂ PO ₄ , NaCl, KCl is the same in Comparative Example 1, while in Comparative Example 1 no dextran 40, tetramisole hydrochloride, Polyaldehydes, anticoagulant; comparison example 2 and embodiment 12, K ₂ HPO ₄ , NaH ₂ PO ₄ , NaCl, KCl, tetraimidazole hydrochloride, polyaldehydes, anticoagulant addition All are the same, but no dextran 40 was added in Comparative Example 1; compared with Example 6 in Comparative Example 3, K ₂ HPO ₄ , NaH ₂ PO ₄ , NaCl, KCl, tetramisole hydrochloride, polyaldehydes, anticoagulant The addition amount of the agent is the same, but no dextran 40 is added in the control example 3; compared with the example 12 in the control example 4, K ₂ HPO ₄ , NaH ₂ PO ₄ , NaCl, KCl, tetramisole hydrochloride, polyaldehyde The added amount of anticoagulant and anticoagulant were the same, but the added amount of dextran 40 in Comparative Example 4 was reduced from 2.5% to 0.5%; compared with Example 12, K ₂ HPO ₄ , NaH ₂ PO ₄ , NaCl , KCl, tetramisole hydrochloride, polyaldehydes, and anticoagulant additions are all the same, while the addition of dextran 40 in comparative example 5 is reduced from 2.5% to 1.5%; comparative example 4 is compared with embodiment 10 , K ₂ HPO ₄ , NaH ₂ PO ₄ , KCl, dextran 40, tetraimidazole hydrochloride, polyaldehydes, and anticoagulants were all added in the same amount, while the amount of NaCl added in Comparative Example 4 was reduced from 0.9% to 0.8%; compared with Example 11, the additions of K ₂ HPO ₄ , NaH ₂ PO ₄ , KCl, dextran 40, tetraimidazole hydrochloride, polyaldehydes, and anticoagulant were the same, while the control The addition of NaCl is reduced to 0.8% by 0.9% among the example 4.

As shown in Table 2, the cells preserved in Example 12 are detected by FCM, and granulocytes are reduced from 76.122% of 0d to 65.450% of 60d, monocytes are reduced from 5.69% of 0d to 5.266% of 60d, and lymphocytes are reduced from 0d to 5.266%. 16.336% rose to 16.468% of 60d; the cells preserved in control example 1 were detected by FCM, granulocytes were reduced from 76.232% of 0d to 54.466% of 60d, monocytes were reduced from 5.78% of 0d to 3.701% of 60d, lymphocytes From 16.466% at 0d to 10.87% at 60d. The test results corresponding to Comparative Example 1 and Example 12 show that the cells preserved in Comparative Example 1 have greater damage at 60 days, while the cells preserved in Example 12 have less damage at 60 days. 3 also showed the same result, it can be seen that only K ₂ HPO ₄ , NaH ₂ PO ₄ , NaCl, and KCl could not protect the cells, while K ₂ HPO ₄ , NaH ₂ PO ₄ , NaCl , KCl, adding dextran 40, tetramisole hydrochloride, polyaldehydes, and anticoagulants can significantly improve the preservation effect on cells.

In Comparative Example 2, Comparative Example 4, Comparative Example 5 and Example 12, the other components remained unchanged, only the amount of dextran 40 added changed, and the amount of added dextran 40 was shown in Table 4.

Table 4

The amount of dextran 40 added (%) Comparative example 2 0 Comparative example 4 0.5 Comparative example 5 1.5 Example 12 2.5

The test results of granulocytes, monocytes and lymphocytes corresponding to Comparative Example 2, Comparative Example 4, Comparative Example 5 and Example 12 are shown in Table 5.

table 5

As shown in Table 5, the test results corresponding to Comparative Example 2, Comparative Example 4, Comparative Example 5 and Example 12 showed that at 60 days, the contents of granulocytes and monocytes were equivalent, while the contents of lymphocytes were quite different. , the content of lymphocytes corresponding to control example 2 was 12.154% when it was preserved for 60 days, the content of lymphocytes corresponding to control example 4 was 13.063% when it was preserved for 60 days, and the content of lymphocytes corresponding to control example 5 was 13.605% when it was preserved for 60 days %, the content of lymphocytes corresponding to Example 12 was 16.468% when preserved for 60 days. It can be seen that the compatible use of dextran 40, tetramisole hydrochloride and polyaldehydes can greatly improve the cell preservation effect, while only tetramisole hydrochloride The combination of polyaldehydes and polyaldehydes cannot exert the same effect, and the addition of dextran 40 has a significant impact on the preservation effect of cells.

As shown in Table 2, comparing the test results corresponding to Control Example 4 and Example 10, at 60 days, the contents of granulocytes and monocytes were equivalent, but there was a large difference in the contents of lymphocytes. The content of lymphocytes is 13.063%, and the corresponding content of lymphocytes in Example 10 is 16.843% when stored for 60 days. And comparative example 4 and embodiment 10 only have the difference of NaCl addition, the addition of NaCl is 0.8% among the comparison example 4, and the addition of NaCl is 0.9% among the embodiment 10, as seen, the small difference of NaCl addition, have a greater impact on cell preservation.

The inventor has repeatedly studied and experimented on the basis of the detection results corresponding to Comparative Example 4 and Example 10, and unexpectedly found that the effect of NaCl addition on the effect of cell preservation is due to the change of osmotic pressure. Example 10 and Example 11 Compared with the control examples 4 and 5, the osmotic pressure data when the cells were preserved were about 30 mOsm/kg higher. In addition, comparing the detection results corresponding to Examples 10 and 11 with the detection results corresponding to Examples 1-9 and Example 12, it was found that the granulocytes, monocytes, The difference in the content of lymphocytes was small, further verifying the influence of osmotic pressure on the effect of cell preservation, but those skilled in the art know that the higher the osmotic pressure, the better, and the appropriate osmotic pressure plays a key role in cell preservation. especially important.

The above-mentioned embodiments only illustrate the principles and effects of the present invention, but are not intended to limit the present invention. Anyone skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or changes made by those skilled in the art without departing from the spirit and technical ideas disclosed in the present invention should still be covered by the claims of the present invention.

Claims (10)

1. it is a kind of for cell save composition, which is characterized in that including following component: potassium dihydrogen phosphate, disodium hydrogen phosphate, Sodium chloride, potassium chloride, Dextran 40, tetramisole hydrochloride, poly aldehydes, anti-coagulants, wherein potassium dihydrogen phosphate, phosphoric acid hydrogen Disodium, sodium chloride, potassium chloride, Dextran 40, tetramisole hydrochloride, poly aldehydes, anti-coagulants mass ratio be 0.2: 1.4: 8-9∶0.2∶5-25∶25-50∶25∶20-30。

2. the composition saved as described in claim 1 for cell, which is characterized in that the potassium dihydrogen phosphate, phosphoric acid Disodium hydrogen, sodium chloride, potassium chloride, Dextran 40, tetramisole hydrochloride, poly aldehydes, anti-coagulants mass ratio be 0.2: 1.4 ∶9∶0.2∶5∶25∶25∶30。

3. the composition saved as described in claim 1 for cell, which is characterized in that the potassium dihydrogen phosphate, phosphoric acid Disodium hydrogen, sodium chloride, potassium chloride, Dextran 40, tetramisole hydrochloride, poly aldehydes, anti-coagulants mass ratio be 0.2: 1.4 ∶9∶0.2∶15∶25∶25∶30。

4. the composition as described in any one of claims 1-3 saved for cell, which is characterized in that the poly aldehydes For the mixing of one of Metaldehyde or metapropionaldehyde or both.

5. the composition as described in any one of claims 1-3 saved for cell, which is characterized in that the anti-coagulants is The mixing of one of EDTA, citrate or both.

6. a kind of aqueous compositions saved for cell, which is characterized in that include the described in any item combinations of claim 1-5 Object.

7. a kind of aqueous compositions as claimed in claim 6 answering in biological Sample preservation before flow cytometry cell detection With.

8. the use as claimed in claim 7, which is characterized in that the biological sample is the blood or tissue samples of people.

9. the use as claimed in claim 7, which is characterized in that the biological sample is people's marrow or human peripheral.

10. a kind of method that cell saves, which comprises the steps of:

S1 mixes the described in any item compositions of claim 1-5 with water, forms aqueous compositions；

Biological sample is mixed with the aqueous compositions in S1, is placed at 2-8 DEG C and saves by S2.