CN113180036B - Cell preservation solution, cell preservation tube, and preservation method - Google Patents

Cell preservation solution, cell preservation tube, and preservation method Download PDF

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CN113180036B
CN113180036B CN202110521016.7A CN202110521016A CN113180036B CN 113180036 B CN113180036 B CN 113180036B CN 202110521016 A CN202110521016 A CN 202110521016A CN 113180036 B CN113180036 B CN 113180036B
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CN113180036A (en
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张磊
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Shenzhen Tianshuo Bio Tech Co ltd
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Shenzhen Tianshuo Bio Tech Co ltd
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts
    • A01N1/0205—Chemical aspects
    • A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
    • A01N1/0221—Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts
    • A01N1/0205—Chemical aspects
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts
    • A01N1/0205—Chemical aspects
    • A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
    • A01N1/0226—Physiologically active agents, i.e. substances affecting physiological processes of cells and tissue to be preserved, e.g. anti-oxidants or nutrients

Abstract

The invention discloses a cell preservation solution, a cell preservation tube and a preservation method, wherein the cell preservation solution comprises 2-2.5 mmol/L KCl, 130-150 mmol/L NaCl and 10-12 mmol/L Na2HPO41-8 mmol/L adenosine, 1-2 mmol/L reduced glutathione, 10-15 g/L anticoagulant and 0.1-1 g/L glucose. The cell preservation solution can stabilize the activity of circulating tumor cells in peripheral blood and the expression of surface antigens thereof for a long time, thereby improving the effects of capturing the circulating tumor cells by immunomagnetic beads and further performing immunofluorescence identification.

Description

Cell preservation solution, cell preservation tube, and preservation method
Technical Field
The invention relates to the technical field of medicines, in particular to a cell preservation solution, a cell preservation tube and a preservation method.
Background
Cancer cells shed from the primary site and enter the peripheral blood of the human body, and are called Circulating Tumor Cells (CTCs). Circulating tumor cells metastasize to other organs of the body through blood transmission and are the leading cause of patient death. The capture of immunomagnetic beads and immunofluorescence assay are common methods for enriching and identifying circulating tumor cells, but the existing reagents have poor preservation effect on circulating tumor cells in peripheral blood, so that the effect of enriching the circulating tumor cells by the immunomagnetic beads and further carrying out the immunofluorescence assay is poor.
The above is only for the purpose of assisting understanding of the technical solutions of the present invention, and does not represent an admission that the above is the prior art.
Disclosure of Invention
The invention mainly aims to provide a cell preservation solution, aiming at improving the preservation effect on circulating tumor cells in peripheral blood, thereby improving the effects of capturing the circulating tumor cells by immunomagnetic beads and carrying out immunofluorescence identification.
In order to achieve the above object, the present invention provides, in a first aspect, a cell preservation solution comprising:
KCl with the concentration of more than or equal to 2mmol/L and less than or equal to 2.5 mmol/L;
NaCl with a concentration of greater than or equal to 130mmol/L and less than or equal to 150 mmol/L;
Na2HPO4the concentration is more than or equal to 10mmol/L and less than or equal to 12 mmol/L;
adenosine at a concentration of 1mmol/L or more and 8mmol/L or less;
reduced glutathione with a concentration of greater than or equal to 1mmol/L and less than or equal to 2 mmol/L;
an anticoagulant comprising at least one of citrate, heparin, or edetate, the concentration of the anticoagulant being greater than or equal to 10g/L and less than or equal to 15 g/L; and
glucose with a concentration of 0.1g/L or more and 1g/L or less.
Optionally, the anticoagulant is edetate.
In a second aspect, the present invention also provides a cell preservation tube comprising a tube body and a cell preservation solution stored in the tube body, the cell preservation solution comprising:
KCl with the concentration of more than or equal to 2mmol/L and less than or equal to 2.5 mmol/L;
NaCl with a concentration of greater than or equal to 130mmol/L and less than or equal to 150 mmol/L;
na2HPO4 with a concentration of 10mmol/L or more and 12mmol/L or less;
adenosine at a concentration of 1mmol/L or more and 8mmol/L or less;
reduced glutathione with a concentration of greater than or equal to 1mmol/L and less than or equal to 2 mmol/L;
an anticoagulant comprising at least one of citrate, heparin, or edetate, the concentration of the anticoagulant being greater than or equal to 10g/L and less than or equal to 15 g/L; and
glucose with a concentration of greater than or equal to 0.1g/L and less than or equal to 1 g/L;
wherein, the volume ratio of the cell preservation solution to the tube body is more than or equal to 1: 10, and less than or equal to 1: 5.
optionally, the volume ratio of the cell preservation solution to the tube body is greater than or equal to 1: 8, and less than or equal to 1: 6.
optionally, the anticoagulant is edetate.
Optionally, the size of the tube body is greater than or equal to 2ml and less than or equal to 10 ml.
In a third aspect, the present invention further provides a method for preserving blood circulation tumor cells, comprising the following steps:
(1) preparing a cell preservation solution;
(2) adding the collected peripheral blood into the cell preservation solution, and uniformly mixing, wherein the volume ratio of the cell preservation solution to the peripheral blood is more than or equal to 1: 9, and less than or equal to 1: 4;
wherein the cell preservation solution comprises 2-2.5 mmol/L KCl, 130-150 mmol/L NaCl and 10-12 mmol/L Na2HPO41-8 mmol/L adenosine, 1-2 mmol/L reduced glutathione, 10-15 g/L anticoagulant and 0.1-1 g/L glucose.
Optionally, the volume ratio of the cell preservation fluid to the peripheral blood is greater than or equal to 1: 6, and less than or equal to 1: 5.
the cell preservation solution comprises 2-2.5 mmol/L KCl, 130-150 mmol/L NaCl and 10-12 mmol/L Na2HPO41-8 mmol/L adenosine, 1-2 mmol/L reduced glutathione, 10-15 g/L anticoagulant and 0.1-1 g/L glucose; thus, a peripheral blood sample is added into the vessel and mixed with a cell preservation solution, KCl, NaCl and Na2HPO4The pH value of the solution is maintained between 7.2 and 7.5, so that the condition that the cell is influenced by peracid in the solution is avoided; adenosine and reduced glutathione provide energy substrates for circulating tumor cells, and contribute to prolonging the survival time of the cells; the anticoagulant prevents red blood cells from hemolysis, platelet aggregation and the like, and reduces the influence on the survival of circulating tumor cells; glucose is an energy source of living cells and an intermediate product of metabolism, and can maintain normal metabolism of circulating tumor cells, thereby further prolonging the survival time of the circulating tumor cells. Therefore, the cell preservation solution can effectively stabilize the activity of the circulating tumor cells in peripheral blood and the expression of the surface antigens thereof for a long time, thereby improving the effects of capturing the circulating tumor cells by the immunomagnetic beads and further performing immunofluorescence identification. The cell preservation tube of the present invention contains the cell preservation solution, and the method for preserving cells of the present invention uses the cell preservation solution, and has at least all of the advantageous effects of the cell preservation solution.
Detailed Description
It should be noted that if the description of "first", "second", etc. is provided in the embodiment of the present invention, the description of "first", "second", etc. is only for descriptive purposes and is not to be construed as indicating or implying relative importance or implicitly indicating the number of indicated technical features. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one such feature. In addition, the meaning of "and/or" appearing throughout is to include three juxtapositions, exemplified by "A and/or B" including either scheme A, or scheme B, or a scheme in which both A and B are satisfied.
The invention provides a cell preservation solution.
The cell preservation solution provided by the invention contains KCl, NaCl and Na2HPO4Adenosine, reduced glutathione, anticoagulant and glucose; the concentration of KCl is more than or equal to 2mmol/L and less than or equal to 2.5 mmol/L; the concentration of the NaCl is more than or equal to 130mmol/L and less than or equal to 150 mmol/L; the Na is2HPO4The concentration of (A) is more than or equal to 10mmol/L and less than or equal to 12 mmol/L; the concentration of adenosine is greater than or equal to 1mmol/L and less than or equal to 8 mmol/L; the concentration of the reduced glutathione is more than or equal to 1mmol/L and less than or equal to 2 mmol/L; the anticoagulant comprises at least one of citrate, heparin or edetate, and the concentration of the anticoagulant is greater than or equal to 10g/L and less than or equal to 15 g/L; the concentration of the glucose is more than or equal to 0.1g/L and less than or equal to 1 g/L.
KCl, NaCl, Na in the cell preservation tube of the invention2HPO4The buffer solution system is a common buffer solution system, can keep the pH value of the solution stable, and can offset and lighten the influence of external strong acid or strong base on the pH value of the solution to a certain extent. Adenosine (Adenosine) is an important intermediate for synthesizing Adenosine Triphosphate (ATP), adenine, adenylic acid and vidarabine as an energy substrate, can be rapidly taken up by cells, and plays an important role in the life activities of the cells. The adenosine can also transfer energy in the form of Adenosine Triphosphate (ATP) or Adenosine Diphosphate (ADP), or signal with cyclic adenosine monophosphate (cAMP)And so on. The reduced glutathione is an active component of glutathione in vivo, comprises glutamic acid, cystine and glycine, is a prosthetic group of glyceraldehyde dehydrogenase, is a coenzyme of glyoxalase and triose phosphate dehydrogenase, and participates in tricarboxylic acid cycle and sugar metabolism in vivo. Glutathione can activate various enzymes such as sulfhydryl (-SH) enzyme, coenzyme and the like in vivo, thereby promoting the metabolism of saccharides, fats and proteins, and also influencing the metabolic process of cells, and is an important substance for regulating metabolism in cells.
The anticoagulant comprises at least one of citrate, heparin or edetate. The citrate can be sodium citrate, and the sodium citrate can be combined with calcium ions in blood to form a chelate so as to prevent blood coagulation. The heparin is a physiological anticoagulant, is mucopolysaccharide containing sulfate groups, has more negative charges, and can strengthen antithrombin III (AT-III) to inactivate serine protease, thereby preventing thrombin from forming, avoiding platelet aggregation and the like. The ethylene diamine tetraacetic acid salt (EDTA) can be EDTA sodium salt and EDTA potassium salt, and the EDTA can be combined with calcium ions in blood to form a chelate so that the calcium ions lose the coagulation effect, thereby achieving the anticoagulation purpose. The glucose can be directly utilized by the cells to provide energy, which helps to prolong the survival time of the circulating tumor cells.
The cell preservation solution comprises 2-2.5 mmol/L KCl, 130-150 mmol/L NaCl and 10-12 mmol/L Na2HPO41-8 mmol/L adenosine, 1-2 mmol/L reduced glutathione, 10-15 g/L anticoagulant and 0.1-1 g/L glucose. Thus, a peripheral blood sample is added into the vessel and mixed with a cell preservation solution, KCl, NaCl and Na2HPO4The pH value of the solution is maintained between 7.2 and 7.5, so that the condition that the cell is influenced by peracid in the solution is avoided; adenosine and reduced glutathione provide energy substrates for circulating tumor cells, and contribute to prolonging the survival time of the cells; the anticoagulant prevents red blood cells from hemolysis, platelet aggregation and the like, and reduces the influence on the survival of circulating tumor cells; glucose is an energy source of living cells and an intermediate product of metabolism, can maintain the normal metabolism of circulating tumor cells,thereby further prolonging the survival time of the circulating tumor cells. Therefore, the cell preservation solution can effectively stabilize the activity of the circulating tumor cells in peripheral blood and the expression of the surface antigens thereof for a long time, thereby improving the effects of capturing the circulating tumor cells by the immunomagnetic beads and further performing immunofluorescence identification.
Optionally, the anticoagulant is edetate. Through multiple times of experimental verification, the invention discovers that when the anticoagulant is ethylenediamine tetraacetate, the influence on the capture of subsequent circulating tumor cells by immunomagnetic beads and the identification by immunofluorescence is the minimum.
The invention also provides a cell preservation tube.
The cell preservation tube provided by the invention comprises a tube body and cell preservation liquid stored in the tube body, wherein the cell preservation liquid contains KCl, NaCl and Na2HPO4Adenosine, reduced glutathione, anticoagulant and glucose; the concentration of KCl is more than or equal to 2mmol/L and less than or equal to 2.5 mmol/L; the concentration of the NaCl is more than or equal to 130mmol/L and less than or equal to 150 mmol/L; the Na is2HPO4The concentration of (A) is more than or equal to 10mmol/L and less than or equal to 12 mmol/L; the concentration of adenosine is greater than or equal to 1mmol/L and less than or equal to 8 mmol/L; the concentration of the reduced glutathione is more than or equal to 1mmol/L and less than or equal to 2 mmol/L; the anticoagulant comprises at least one of citrate, heparin or edetate, and the concentration of the anticoagulant is greater than or equal to 10g/L and less than or equal to 15 g/L; the concentration of the glucose is more than or equal to 0.1g/L and less than or equal to 1 g/L; wherein, the volume ratio of the cell preservation solution to the tube body is more than or equal to 1: 10, and less than or equal to 1: 5.
the cell preservation tube contains the cell preservation solution, so that the activity of circulating tumor cells in peripheral blood and the expression of surface antigens of the circulating tumor cells can be effectively stabilized for a long time, and the effects of capturing the circulating tumor cells by immunomagnetic beads and identifying immunofluorescence are improved.
Optionally, the volume ratio of the cell preservation solution to the tube body is greater than or equal to 1: 8, and less than or equal to 1: 6.
optionally, the anticoagulant is edetate.
Optionally, the size of the tube body is greater than or equal to 2ml and less than or equal to 10 ml. The tube body comprises a plurality of specifications, for example, 2ml, 5ml and 10ml, and is suitable for collecting peripheral blood samples with different volumes under different detection requirements.
Optionally, a label is adhered to the circumferential side surface of the tube body. By the arrangement, a detector can conveniently and directly name and record the sample added into the tube body. In the invention, the cell preservation tube further comprises a tube cover, one end of the tube body is open, and the tube cover is arranged at the opening. The tube cover can be set to be different colors or different shapes, so that different samples can be distinguished by a detector conveniently.
The invention also provides a preservation method of the blood circulation tumor cells.
The blood circulation tumor cell provided by the invention comprises the following steps: (1) preparing a cell preservation solution; (2) adding the collected peripheral blood into the cell preservation solution, and uniformly mixing, wherein the volume ratio of the cell preservation solution to the peripheral blood is more than or equal to 1: 9, and less than or equal to 1: 4; wherein the cell preservation solution comprises 2-2.5 mmol/L KCl, 130-150 mmol/L NaCl and 10-12 mmol/L Na2HPO41-8 mmol/L adenosine, 1-2 mmol/L reduced glutathione, 10-15 g/L anticoagulant and 0.1-1 g/L glucose.
In the preservation method of the present invention, when the volume ratio of the cell preservation solution to the peripheral blood is less than 1: and 9, the content of the cell preservation solution is not enough to effectively stabilize the activity of the circulating tumor cells in the tube body and the expression of the surface antigen thereof for a long time, so that the effects of capturing the circulating tumor cells by immunomagnetic beads and further identifying by immunofluorescence are reduced. When the volume ratio of the cell preservation solution to the peripheral blood is more than 1: 4, the content of the cell preservation solution is too high, the ion concentration is too high, the subsequent specific binding of the antibody to the circulating tumor cell surface antigen is influenced, and the effects of capturing the circulating tumor cell by the immunomagnetic beads and further identifying by immunofluorescence are reduced.
The cell preservation tube contains the cell preservation solution, and can effectively stabilize the activity of circulating tumor cells in peripheral blood and the expression of surface antigens thereof for a long time, thereby improving the effects of capturing the circulating tumor cells by immunomagnetic beads and further performing immunofluorescence identification.
Optionally, the volume ratio of the cell preservation fluid to the peripheral blood is greater than or equal to 1: 6, and less than or equal to 1: 5. when the volume ratio of the cell preservation solution to the peripheral blood is in the above range, the preservation effect of the cell preservation solution on the circulating tumor cells is good.
Example one
(one) preparing cell preserving fluid with different formulas
1. Respectively collecting KCl, NaCl and Na2HPO4Preparing mother liquor from adenosine and reduced glutathione, wherein the concentration of the mother liquor of KCl is 100mmol/L, the concentration of the mother liquor of NaCl is 1mol/L, and Na2HPO4The concentration of the mother liquor of (2) is 1mol/L, the concentration of the mother liquor of adenosine is 100mmol/L, and the concentration of the mother liquor of reduced glutathione is 100 mmol/L;
2. mixing said KCl, said NaCl, said adenosine, said reduced glutathione and said Na2HPO4Adding EDTA-2Na and glucose into the mother solution, diluting with distilled water, uniformly stirring, and adjusting the pH to 7.2;
3. carrying out constant volume on the solution;
4. filtering and sterilizing the solution obtained in the previous step by adopting a filtering membrane with the diameter of a filtering hole of 0.2 mu m to obtain cell preservation solution, and pouring the cell preservation solution into a tube body;
5. vacuumizing by using vacuumizing equipment at 0.29 standard atmospheric pressure, sealing a tube plug and a tube body of the cell preservation tube, and stably preserving the cell preservation tube at the temperature of 2-8 ℃ (the cell preservation tube can be preserved for 2 years), wherein the volume ratio of the cell preservation solution to the tube body is 1: 10.
according to the preparation method, seven groups of cell preservation solutions with different concentrations and components are prepared, and see table 1 specifically. This example also used a commercially available cell preservation solution (group number: eight) as a comparative reagent.
TABLE 1 Components and concentrations of eight groups of cell-preserving fluids
Figure BDA0003062656800000071
Preparation of (di) NCI-H1993 cells
1. Culturing NCI-H1993 until the volume coverage rate reaches 80-90%;
2. sucking off the original culture medium, adding 3ml of PBS for rinsing, and washing away the PBS;
3. adding 1ml of trypsin, and digesting at 37 ℃ for 3 min;
4. after the cells were rounded, 1ml of serum-containing medium was added to stop digestion;
5. blowing and beating the suspended cells by using a pipette;
6. sucking the cells into a centrifuge tube, gently blowing and beating the cells until the cells are dispersed, and centrifuging the cells for 5min at 300 Xg;
7. pouring out the supernatant, adding 2ml of culture medium, and blowing off the cells for suspension;
8. NCI-H1993 cells were collected and divided into several portions, 10 each4And (4) respectively.
Incubation of (tri) NCI-H1993 cells with peripheral blood samples
1. Collecting 4ml of peripheral blood of a healthy person in a conventional arm vein blood collection mode;
2. adding 10 into peripheral blood respectively1、102、103、104、105An NCI-H1993 cell;
3. 1ml of the eight cell-preserving solution of example one was added thereto, the mixture was turned upside down and mixed, and the mixture was left at room temperature for 3 days, 5 days and 7 days, respectively.
(IV) separation of circulating tumor cells in blood by immunomagnetic beads
1. Taking 15mL of separation solution, adding 50mL of sterile LeucosepTMCentrifuging 1000g in a separation tube for 1 min;
2. adding 3mL of PBS, 7.5mL of anticoagulated peripheral whole blood and 4.5mL of PBS in sequence, and centrifuging for 15min at 800 g;
3. removing liquid on the tunica albuginea layer by a vacuum pump filtration pump, carefully pouring the residual liquid into a new 50mL centrifuge tube, adding 5mL PBS along the wall to wash the separation tube, pouring the washing liquid into the 50mL centrifuge tube, and repeatedly washing for 3 times;
4. centrifuging at 300g for 10min, and discarding the supernatant;
5. adding 20 mu L of confining liquid into a 50mL centrifuge tube, flicking the bottom of the centrifuge tube, completely dispersing cells, and standing in a refrigerator at the temperature of 2-8 ℃ for 10 min;
6. uniformly oscillating immunomagnetic beads, taking 50 mu L of each sample into a 1.5mL centrifuge tube, subpackaging according to the sample amount, absorbing a magnetic bead protection solution, and carrying out incubation rinsing for 3 times for later use;
7. mixing the cells with the magnetic beads, and incubating for 90min at 10rpm/min and 2-8 ℃ on a rotary shaking table;
8. taking down from the rotary table, placing into a magnetic frame, standing for 3min, and removing liquid after the magnetic beads are completely adsorbed;
9. taking the centrifuge tube off the magnetic frame, washing the centrifuge tube with 1mL PBS, placing the centrifuge tube into the magnetic frame, standing for 3min, removing liquid after the magnetic beads are completely adsorbed, and repeating the steps twice;
10. taking down the centrifuge tube from the magnetic frame, and adding 100 mu L of incubation liquid;
11. and (4) preserving the separated cells at the temperature of 2-8 ℃ for subsequent analysis.
In the above experiment, the immunomagnetic beads may be CD45 immunomagnetic beads or CA72-4 immunomagnetic beads for enriching leukocytes, or various immunomagnetic beads such as CEA immunomagnetic beads, EpCAM immunomagnetic beads or EGFR immunomagnetic beads for enriching NCI-H1993 cells.
(V) CK, CD45 and DAPI stain isolated cells
The steps for CD45 staining isolated cells were as follows:
1. diluting 100 multiplied CD45 and 200 multiplied developing solution into 1 multiplied working solution by using an incubation solution, preserving at 2-8 ℃, and pre-cooling a stationary solution at-20 ℃;
2. taking a sample incubated by the magnetic beads, adsorbing the beads by using a magnetic frame, removing the incubation liquid, adding 100 mu L of a fixing liquid precooled at the temperature of minus 20 ℃, fixing for 5min, adsorbing the beads by using the magnetic frame, and removing the fixing liquid;
3. adding 100 mu L of incubation liquid for resuspension, adsorbing the beads by a magnetic frame, and removing the incubation liquid;
4. adding 40 μ L of permeation solution, mixing, standing for 5min, and removing the permeation solution with magnetic frame adsorption beads;
5. adding 40 μ L of blocking solution, resuspending for 20min, adsorbing beads with magnetic frame, and removing blocking solution;
6. adding 40 μ L of 1 × CD45, resuspending, incubating for 20min (resuspending every 10 min), adsorbing the magnetic beads with a magnetic frame, and removing 1 × CD 45;
7. adding 100 mu L of incubation solution for heavy suspension, and removing the suspension by using a magnetic frame to adsorb beads;
8. adding 40 μ L of 1 × color developing solution, re-suspending, dyeing for 20min (re-suspending every 10 min), adsorbing magnetic beads with magnetic frame, and removing 1 × color developing solution;
9. adding 100 μ L of the double-dyeing liquid for heavy suspension, adsorbing the beads by a magnetic frame for 1min, and removing the double-dyeing liquid;
10. adding 100 mu L of incubation liquid for resuspension, quickly dropping the incubation liquid into a prepared slide glass immunohistochemical ring, and keeping the immunohistochemical ring covered with a magnet for adsorption flaking;
11. adding 3 mu L of the blocking tablet into the immunohistochemical ring, and covering a glass slide;
12. the staining results were observed under a fluorescent microscope.
The steps for CK and DAPI staining of isolated cells are similar to CD45 and will not be described further.
(VI) observing the staining result under a fluorescent microscope
And scanning and counting the dyed products. The amounts of leukocytes and NCI-H1993 obtained by comparing the amounts of the eight groups of cell preservation solutions after 3 days, 5 days and 7 days of preservation of the NCI-H1993 cells and the mixed cells in peripheral blood, respectively. TABLE 2 shows 101、102、103、104、105Results of staining counts after mixing individual NCI-H1993 cells with 4ml of peripheral blood.
Table 2 example a cell staining count result
Figure BDA0003062656800000091
Figure BDA0003062656800000101
Figure BDA0003062656800000111
As is clear from Table 2, the cell preservation solutions (group one and group two) of the present invention stably preserved NCI-H1993 and peripheral blood for 7 days while maintaining the expression of leukocyte surface antigens in NCI-H1993 cells and peripheral blood. Changes in the concentration of the reagents in the cell preservation solution (group three and group four) affected the NCI-H1993 and the preservation effect of leukocytes, and the results of capture of cells by immunomagnetic beads and further immunofluorescence identification were poor. Cell preservation solutions lacking adenosine, reduced glutathione or glucose (groups four to five) also showed poor preservation of NCI-H1993 and leukocytes. The effect of the conventional cell preservation solution (group VIII) on preservation of NCI-H1993 and peripheral blood for 7 days was inferior to that of the cell preservation solution of the present invention.
Example two
4ml of peripheral blood was collected from 27 lung cancer patients, and 1ml of the eight cell preservation solutions of example one was added thereto, and the mixture was mixed by turning upside down and left at room temperature for 3 days, 5 days and 7 days, respectively. The preservation effect of the cells is detected by using the immunomagnetic bead capture and immunofluorescence identification method in the first embodiment. The results of cell staining counts are shown in Table 3, and the values are mean values.
Table 3 example two cell stain count results
Figure BDA0003062656800000121
As can be seen from table 3, the cell preservation solution of the present invention can stably preserve peripheral blood of a lung cancer patient for 7 days, and can maintain the expression of surface antigens of leukocytes and Circulating Tumor Cells (CTCs) during preservation, so that the cells can be captured by immunomagnetic beads and further immunofluorescence-labeled. The cell preservation solution has important clinical application value, and can be used for diagnosis of cancer, real-time monitoring of disease conditions, prevention and control of prognosis and the like.
EXAMPLE III
4ml of peripheral blood was collected from 31 patients with gastric cancer, 20 patients with breast cancer, and 25 patients with liver cancer, 1ml of the cell preservation solution of the eighth group of examples was added thereto, the mixture was turned upside down and mixed, and the mixture was left at room temperature for 7 days. The preservation effect of the cells is detected by using the immunomagnetic bead capture and immunofluorescence identification method in the first embodiment. See table 4 for cell staining counts.
TABLE 4 example three cell staining count results
Figure BDA0003062656800000122
Figure BDA0003062656800000131
As can be seen from table 4, the cell preservation solution of the present invention can stably preserve peripheral blood of patients with gastric cancer, breast cancer and liver cancer for 7 days, and can maintain the expression of surface antigens of circulating tumor cells of gastric cancer, breast cancer and liver cancer during the preservation period.

Claims (6)

1. A cell preservation solution for preserving circulating tumor cells, comprising:
KCl with the concentration of more than or equal to 2mmol/L and less than or equal to 2.5 mmol/L;
NaCl with a concentration of greater than or equal to 130mmol/L and less than or equal to 150 mmol/L;
Na2HPO4the concentration is more than or equal to 10mmol/L and less than or equal to 12 mmol/L;
adenosine at a concentration of 1mmol/L or more and 8mmol/L or less;
reduced glutathione with a concentration of greater than or equal to 1mmol/L and less than or equal to 2 mmol/L;
the anticoagulant is ethylenediamine tetraacetate, and the concentration of the anticoagulant is more than or equal to 10g/L and less than or equal to 15 g/L; and
glucose with a concentration of 0.1g/L or more and 1g/L or less.
2. A cell preservation tube, characterized in that, the cell preservation tube includes the body and deposits in the cell preservation liquid in the body, the cell preservation liquid is used for circulating tumor cell's preservation, the cell preservation liquid contains:
KCl with the concentration of more than or equal to 2mmol/L and less than or equal to 2.5 mmol/L;
NaCl with a concentration of greater than or equal to 130mmol/L and less than or equal to 150 mmol/L;
na2HPO4 with a concentration of 10mmol/L or more and 12mmol/L or less;
adenosine at a concentration of 1mmol/L or more and 8mmol/L or less;
reduced glutathione with a concentration of greater than or equal to 1mmol/L and less than or equal to 2 mmol/L;
the anticoagulant is ethylenediamine tetraacetate, and the concentration of the anticoagulant is more than or equal to 10g/L and less than or equal to 15 g/L; and
glucose with a concentration of greater than or equal to 0.1g/L and less than or equal to 1 g/L;
wherein, the volume ratio of the cell preservation solution to the tube body is more than or equal to 1: 10, and less than or equal to 1: 5.
3. the cell preservation tube according to claim 2, wherein the volume ratio of the cell preservation fluid to the tubular body is greater than or equal to 1: 8, and less than or equal to 1: 6.
4. the cell storage tube of claim 2, wherein the tube body has a gauge of greater than or equal to 2ml and less than or equal to 10 ml.
5. A method for preserving blood circulation tumor cells, which is characterized by comprising the following steps:
(1) preparing a cell preservation solution;
(2) adding the collected peripheral blood into the cell preservation solution, and uniformly mixing, wherein the volume ratio of the cell preservation solution to the peripheral blood is more than or equal to 1: 9, and less than or equal to 1: 4;
wherein the cell preservation solution comprises 2-2.5 mmol/L KCl, 130-150 mmol/L NaCl and 10-12 mmol/L Na2HPO41-8 mmol/L adenosine, 1-2 mmol/L reduced glutathione, 10-15 g/L anticoagulant and 0.1-1 g/L glucose; the anticoagulant is ethylenediamine tetraacetate.
6. The preservation method according to claim 5, wherein the volume ratio of the cell preservation solution to the peripheral blood is 1: 6, and less than or equal to 1: 5.
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