CN116724992A - Whole blood frozen stock solution and preparation method and application thereof - Google Patents
Whole blood frozen stock solution and preparation method and application thereof Download PDFInfo
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Classifications
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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
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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
-
- 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
-
- 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
- A01N1/0278—Physical preservation processes
- A01N1/0284—Temperature processes, i.e. using a designated change in temperature over time
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/38—Diluting, dispersing or mixing samples
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/42—Low-temperature sample treatment, e.g. cryofixation
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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Abstract
The application discloses a whole blood frozen stock solution, which comprises the following components in percentage by volume: 60-80% of RPMI1640 culture medium, 10-30% of Fetal Bovine Serum (FBS) and 10-20% of dimethyl sulfoxide (DMSO); and contains the following components: sucrose 1-20 g/L, disodium ethylenediamine tetraacetate (EDTA-2 Na) final concentration 2-3 mM, pH value 6.0-8.0. The application also discloses a preparation method of the whole blood frozen stock solution, application of the whole blood frozen stock solution in frozen stock of the whole blood, and application of the whole blood frozen stock solution in receptor occupancy detection after resuscitation. The frozen stock solution can be directly used for frozen stock of whole blood, the cell number and the activity rate of the recovered whole blood are obviously superior to those of the traditional frozen stock solution formula, the long-term stable storage of RO whole blood samples can be ensured, the frozen stock and recovery operation of the whole blood are simple and convenient, the cost is lower, the frozen stock solution is used for RO clinical detection, and the samples are easy to transport and store.
Description
Technical Field
The application relates to the field of biological analysis and detection, in particular to a whole blood cryopreservation liquid which can be used for cryopreserving whole blood and can be used for detecting the receptor occupancy rate in the peripheral blood of a cryopreserved human in an antigen-antibody combination mode subsequently by using a flow cytometry.
Background
Human whole blood is an important clinical sample and is widely used in clinical diagnosis and medical research. After blood is collected from human body, it needs to be frozen and preserved to avoid inactivation or death of blood cells in normal temperature environment. At present, the problem of long-term storage of blood is solved mainly by adding a frozen stock solution into the blood to perform low-temperature storage of the blood.
In pharmaceutical research, receptor occupancy (Receptor Occupancy, RO) can detect the degree of occupancy of an antibody drug on a cell surface target, and is the most direct Pharmacodynamic (PD) index for evaluating the level of such drug target effect (Target Engagement). The value of receptor occupancy detection is that the degree of the action of the drug on the target point at a certain moment can be intuitively reflected through a small amount of peripheral blood samples. Although RO does not represent a true pharmacological effect and clinical benefit, it is widely used as a tool in PK/PD model establishment, human first dose selection, drug effect dose and dosing cycle determination, quantitative pharmacological studies, and the like.
Good RO experiment design must fully consider the effect of sample type on detection results. In the RO experiment based on flow, the activity rate and the morphology of cells in the whole blood matrix are superior to those of Peripheral Blood Mononuclear Cells (PBMC) which are isolated and frozen, and the result can be reflected more accurately and intuitively. Preclinical animal experiments are batch operations, and detection and analysis laboratories are generally located in close proximity to animal facilities, with ease of detection, and thus more in the case of whole blood analysis. However, in clinical practice, because clinical research centers are generally located far from analytical laboratories, and even distributed across the country, the difficulty of using whole blood samples is high because: because the stability of a sample may vary from hours to days depending on the nature of the target site itself, this presents a significant challenge for transporting whole blood samples from the clinical center to the testing facility laboratory in new drug clinical trials. First, it is the transport cost. The timeliness of logistics transportation must be ensured, and in addition, in recent years, the influence of new coronavirus epidemic situation is difficult to ensure that all samples can be timely delivered to a detection mechanism. Second, the cost of detection. Once the detection mechanism receives the fresh sample, the detection mechanism generally detects the fresh blood sample at the time of aging, and the fresh blood sample is mostly transported singly and detected singly, thus greatly increasing the transportation cost and the detection cost
And because the frozen PBMC can be processed in batches, the batch errors generated by different batch processing of samples are reduced, and meanwhile, the logistics cost is greatly reduced, so that in clinical experiments, the method development, verification and sample analysis are carried out by adopting the separated frozen PBMC at present.
Therefore, in the clinical test, the detection of RO is less by using fresh whole blood samples, and most of target site occupancy studies adopt isolated frozen PBMCs for detection, but frozen Peripheral Blood Mononuclear Cells (PBMCs) may affect the action balance between the drug and the receptor due to the separation, freezing and other treatment processes, so that the data and the measured result of the fresh blood samples have a certain difference. Secondly, the frozen PBMC sample not only requires the clinical center to extract PBMC in the whole blood in time for freezing after the whole blood of the patient is collected, but also requires the quantity of the extracted PBMC to be enough, and the vitality meets the analysis requirement. However, in many cases, due to improper operations in links such as separation, extraction, freezing or transportation, the number of PBMCs received by the detection center is very small, the activity rate is very low, sometimes even PBMCs cannot be detected, detection of the detection center is affected, and finally effective detection data cannot be obtained, which brings trouble to the whole clinical test.
CN108013025a discloses a whole blood cryopreservation solution, which comprises the following components in volume fraction: dimethyl sulfoxide 20-40% and nutrient solution 60-80%; wherein the nutrient solution comprises any one or a combination of at least two of sodium citrate, glucose, human serum albumin, glycerophospholipids, gamma polyglutamic acid, dexamethasone or polyvinylpyrrolidone. But the patent is mainly aimed at bone marrow whole blood cryopreservation and is not applicable to human whole blood cryopreservation.
The existing commercial frozen stock solution has poor cell number and activity obtained after frozen whole blood and recovery, and needs to be improved.
Disclosure of Invention
In order to solve the problems of high cost, insufficient quantity of frozen PBMC samples, low activity and the like caused by the detection of fresh whole blood samples, the application develops a whole blood cell frozen solution, and the receptor occupancy can be detected in batches by utilizing a flow cytometry after resuscitation after the fresh peripheral blood is directly frozen.
The technical scheme adopted by the application is as follows:
a whole blood cryopreservation solution comprising the following components in volume fraction:
60-80% of RPMI1640 culture medium;
fetal Bovine Serum (FBS) 10-30%
10-20% of dimethyl sulfoxide (DMSO);
and contains the following components:
sucrose 1-20 g/L;
disodium ethylenediamine tetraacetate (EDTA-2 Na) final concentration 2-3 mM;
the pH value is 6.0-8.0.
In the whole blood frozen stock solution, the pH value is preferably 7.0 to 8.0, more preferably 7.2 to 7.4.
Further, it is preferred that the whole blood cryopreservation solution comprises the following components in volume fraction:
60-70 A% of RPMI1640 culture medium;
20-30% of Fetal Bovine Serum (FBS);
10-15% of dimethyl sulfoxide (DMSO);
and contains the following components:
10-20 g/L of sucrose;
disodium ethylenediamine tetraacetate (EDTA-2 Na) final concentration 2mM;
the pH value is 7.0-8.0.
Further, it is preferred that the whole blood cryopreservation solution comprises the following components in volume fraction:
RPMI1640 medium 70%;
fetal Bovine Serum (FBS) 20%;
dimethyl sulfoxide (DMSO) 10%;
and contains the following components:
sucrose 10g/L;
disodium ethylenediamine tetraacetate (EDTA-2 Na) final concentration 2mM;
the pH value is 7.2-7.4.
The application also provides a preparation method of the whole blood frozen stock solution, which comprises the steps of uniformly mixing the raw materials with the formula amount, and adjusting the pH value to 7.0-8.0 by using sodium hydroxide or potassium hydroxide to prepare the whole blood frozen stock solution.
The application also provides an application of the whole blood cryopreservation liquid in cryopreservation of whole blood, which comprises the following steps of: 1, mixing, cooling to-70 ℃ in a gradient way, and freezing.
Further, the volume ratio of fresh whole blood to whole blood frozen stock solution is preferably 0.2 to 1:1, more preferably 0.5 to 1:1.
The application also provides a method for freezing and storing whole blood, which comprises the following steps: fresh whole blood and the whole blood frozen stock solution according to the application are mixed according to the volume ratio of 0.2-2: 1, mixing, cooling to-70 ℃ in a gradient way, and freezing.
The frozen whole blood can be stored for 3 days at the temperature of minus 20 ℃; storing for 6 months at-70deg.C; can be stored for 6 months under liquid nitrogen.
The application also provides application of the whole blood cryopreservation liquid in cryopreservation of whole blood and receptor occupancy detection after resuscitation, and further the application method comprises the steps of cryopreserving fresh whole blood by using the whole blood cryopreservation liquid, and then carrying out receptor occupancy detection on the resuscitated whole blood after resuscitation.
The method of resuscitation is typically: taking out the frozen whole blood sample, and rapidly shaking for resuscitation in a water bath at 37 ℃.
The application also provides a method for detecting the receptor occupancy after the cryopreserved whole blood and the resuscitation, which comprises the following steps: and freezing fresh whole blood by using the whole blood freezing solution, and then carrying out receptor occupancy detection on the whole blood after recovery.
The application develops a frozen stock solution, and uses the frozen stock solution to freeze whole blood, and compares the difference of the cell number and the cell activity of PBMC obtained after the whole blood is recovered by the frozen stock solution and other 3 frozen stock solutions. Meanwhile, in order to verify the consistency of the detection results of the occupancy of the frozen whole blood, the PBMC and the fresh whole blood receptor by using the frozen stock solution, the precision test and the stability test of the method are respectively carried out. The result shows that the frozen stock solution can be used for detecting the receptor occupancy of the peripheral blood of the frozen stock human, the precision of the frozen stock solution accords with the expected acceptance standard for the whole blood and PBMC of the healthy human, the frozen stock solution detection result is comparable with the fresh whole blood and the PBMC result, and the frozen stock solution can maintain the sample stable for at least 3 days at the temperature of minus 20 ℃ and maintain the sample stable for at least 6 months at the temperature of minus 70 ℃ and the liquid nitrogen.
The frozen PBMC is consistent with the receptor occupancy result in whole blood, and the frozen whole blood is consistent with the receptor occupancy result in fresh whole blood.
The application is based on flow cytometry, utilizes frozen whole blood to carry out RO detection, reduces the extraction process from whole blood to PBMC for clinical centers, provides guarantee for the sample quality of a detection mechanism, reduces the difference between different analysis batches of samples, greatly reduces the sample transportation cost, the detection time and the labor cost, and saves experimental resources.
Compared with the common existing RO detection technology, the application has the following innovation points:
the frozen stock solution can be directly used for frozen stock of whole blood, and the cell number and the activity rate of the resuscitated whole blood are obviously superior to those of the existing frozen stock solution formula.
And the RO whole blood sample can be ensured to be stored stably for a long time. The RO whole blood sample is directly frozen, and RO detection is carried out by using flow cytometry, thus being a novel RO sample treatment mode innovation.
The frozen stock solution has the characteristics of easily obtained formula, simple and convenient whole blood freezing and recovering operation, low cost, application to RO clinical detection, easy transportation and storage of samples and the like.
Drawings
FIG. 1 shows a flow chart of the staining of a dead dye after storage at-70℃for 7 days after whole blood was frozen using the frozen stock solution 1 in example 6.
FIG. 2 shows a flow chart of the dye stained with the live dye after storage at-70℃for 1 month after whole blood was frozen using the frozen solution 1 in example 6.
FIG. 3 shows a flow chart of the dye stained with the live dye after storage at-70℃for 3 months after whole blood was frozen using the frozen stock solution 1 in example 6.
FIG. 4 shows a flow chart of the staining of the dead dye after 6 months of storage at-70℃after whole blood was frozen using the frozen stock solution 1 in example 6.
Detailed Description
The following describes the technical scheme of the present application with specific examples, but the scope of the present application is not limited thereto.
Example 1
The volume ratio of the frozen stock solution 1 is as follows: 70% RPMI1640 medium (Gibco, 1835055)
+20% FBS+10% dimethyl sulfoxide (DMSO) +10g/L sucrose+EDTA-Na 2 (final concentration 2 mM), and small amounts of sodium hydroxide and potassium hydroxide to adjust the pH to 7.2-7.4.
The freezing solution 1 is adopted to freeze and store human peripheral blood (K2-EDTA anticoagulation) whole blood, and the specific steps are as follows:
(1) Freezing: taking appropriate amount of peripheral blood (K) 2 EDTA anticoagulation) sample, adding the frozen stock solution 1 according to the volume ratio of the whole blood to the frozen stock solution 1:1, uniformly mixing, adding 1.8mL of frozen stock sample into each frozen stock tube, placing the frozen stock sample into a program cooling box, placing the program cooling box into a refrigerator at the temperature of-70 ℃, and storing for at least 4 hours for detection.
(2) Resuscitating: the frozen samples were removed and rapidly shaken in a 37℃water bath for resuscitation, transferred to 10mL of 1 XPBS after resuscitation, centrifuged at 500 Xg for 5min at room temperature, the supernatant discarded, resuspended in 1mL of 1 XPBS and counted.
Example 2
The volume ratio of the frozen stock solution 2 is as follows: 60% RPMI1640 medium+30% FBS+10% dimethyl sulfoxide (DMSO) +10g/L sucrose+EDTA-Na 2 (final concentration 2 mM), and small amounts of sodium hydroxide and potassium hydroxide to adjust the pH to 7.2-7.4.
Human peripheral blood (K) was obtained using frozen stock solution 2 2 EDTA anticoagulation) whole blood cryopreservation and resuscitation, the specific steps are as follows:
(1) Freezing: taking appropriate amount of peripheral blood (K) 2 EDTA anticoagulation) sample, adding the frozen stock solution 2 according to the volume ratio of the whole blood to the frozen stock solution of 1:1, uniformly mixing, adding 1.8mL of frozen stock sample into each frozen stock tube, placing the frozen stock sample into a program, placing the program cooling box into a refrigerator at the temperature of-70 ℃, and storing for at least 4 hours for detection.
(2) Resuscitating: the frozen samples were removed and rapidly shaken in a 37℃water bath for resuscitation, transferred to an appropriate volume of 1 XPBS after resuscitation, centrifuged at 500 Xg for 5min at room temperature, the supernatant discarded, resuspended in 1mL of 1 XPBS and counted.
Example 3
The volume ratio of the frozen stock solution 3 is as follows: 80% RPMI1640 medium+10% FBS+10% dimethyl sulfoxide (DMSO) +1% sucrose (m/v) +EDTA-Na2 (final concentration 2 mM), and small amounts of sodium hydroxide and potassium hydroxide to adjust pH to 7.2-7.4.
Human peripheral blood (K) was obtained using the frozen stock solution 3 2 EDTA anticoagulation) whole blood cryopreservation and resuscitation, the specific steps are as follows:
(1) Freezing: taking appropriate amount of peripheral blood (K) 2 EDTA anticoagulation) sample, adding the frozen stock solution 3 according to the volume ratio of the whole blood to the frozen stock solution of 1:1, uniformly mixing, adding 1.8mL of frozen stock sample into each frozen stock tube, placing the frozen stock sample into a program, placing the program cooling box into a refrigerator at the temperature of-70 ℃, and storing for at least 4 hours for detection.
(2) Resuscitating: the frozen samples were removed and rapidly shaken in a 37℃water bath for resuscitation, transferred to an appropriate volume of 1 XPBS after resuscitation, centrifuged at 500 Xg for 5min at room temperature, the supernatant discarded, resuspended in 1mL of 1 XPBS and counted.
Comparative example 1
Compared with example 1, the frozen stock solution 4 is a commercial cell frozen stock solution.
Human peripheral blood (K) was obtained using the frozen stock solution 4 2 EDTA anticoagulation) whole blood cryopreservation and resuscitation, the specific steps are as follows:
(1) Freezing: taking appropriate amount of peripheral blood (K) 2 EDTA anticoagulation) sample, adding a proper amount of frozen stock solution 4 according to the specification, uniformly mixing, adding 1.8mL of frozen stock sample into each frozen stock tube, placing the frozen stock tube in a program, placing the program cooling box in a refrigerator at-70 ℃, and storing for at least 4 hours for detection.
(2) Resuscitating: the frozen samples were removed and rapidly shaken in a 37℃water bath for resuscitation, transferred to an appropriate volume of 1 XPBS after resuscitation, centrifuged at 500 Xg for 5min at room temperature, the supernatant discarded, resuspended in 1mL of 1 XPBS and counted.
Comparative example 2
Compared with example 1, the frozen stock solution 5 is a commercial serum-free frozen stock solution.
Human peripheral blood (K) was obtained by using the frozen stock solution 5 2 EDTA anticoagulation) whole blood cryopreservation and resuscitation, the specific steps are as follows:
(1) Freezing: taking appropriate amount of peripheral blood (K) 2 EDTA anticoagulation) sample, adding proper amount of frozen stock solution 5 according to the instruction, mixing, adding 1.8mL of frozen stock sample into each frozen stock tube, placing in a program, placing in a refrigerator at-70 ℃, storing for at least 4 hours, and detecting.
(2) Resuscitating: the frozen samples were removed and rapidly shaken in a 37℃water bath for resuscitation, transferred to an appropriate volume of 1 XPBS after resuscitation, centrifuged at 500 Xg for 5min at room temperature, the supernatant discarded, resuspended in 1mL of 1 XPBS and counted.
Comparative example 3
Compared with example 1, the frozen stock solution 6 is a commercial non-animal-derived frozen stock solution.
Human peripheral blood (K) was obtained by using the frozen stock solution 6 2 EDTA anticoagulation) whole blood cryopreservation and resuscitation, the specific steps are as follows:
(1) Freezing: taking appropriate amount of peripheral blood (K) 2 EDTA anticoagulation) sample, adding proper amount of frozen stock solution 6 according to the instruction, mixing, adding 1.8mL of frozen stock sample into each frozen stock tube, placing in a program, placing in a refrigerator at-70 ℃, storing for at least 4 hours, and detecting.
(2) Resuscitating: the frozen samples were removed and rapidly shaken in a 37℃water bath for resuscitation, transferred to an appropriate volume of 1 XPBS after resuscitation, centrifuged at 500 Xg for 5min at room temperature, the supernatant discarded, resuspended in 1mL of 1 XPBS and counted.
Comparative example 4
Compared with example 1, the volume percentage of DMSO is 5%, the volume percentage of RPMI1640 medium is 75%, and other components and volume or mass percentages are the same as in example 1. Peripheral blood of human (K) 2 EDTA anticoagulation) whole blood was frozen and resuscitated and counted.
Example 4
Compared with example 1, the volume percentage of DMSO is 20%, the volume percentage of RPMI1640 medium is 60%, and other components and volume or mass percentages are the same as in example 1. Peripheral blood of human (K) 2 EDTA anticoagulation) whole blood was frozen and resuscitated and counted.
Comparative example 5
In comparison with example 1, the components do not contain sucrose, and the other components and the volume percentages are the same as in example 1. Peripheral blood of human (K) 2 EDTA anticoagulation) whole blood was frozen and resuscitated and counted.
Comparative example 6
In comparison with example 1, EDTA-Na2 was not contained in the components, and the other components and the volume or mass percentages were the same as in example 1. Peripheral blood of human (K) 2 EDTA anticoagulation) whole blood was frozen and resuscitated and counted.
Comparative example 7
Compared with example 1, the frozen stock solution was added in a volume ratio of whole blood to frozen stock solution of 2:1. Peripheral blood of human (K) 2 EDTA anticoagulation) whole blood was frozen and resuscitated and counted.
Comparative example 8
Compared with example 1, the frozen stock solution was added in a volume ratio of whole blood to frozen stock solution of 5:1. Peripheral blood of human (K) 2 EDTA anticoagulation) whole blood was frozen and resuscitated and counted.
Comparative example 9
Compared with example 1, the frozen stock solution was added in a volume ratio of whole blood to frozen stock solution of 100:1. Peripheral blood of human (K) 2 EDTA anticoagulation) whole blood was frozen and resuscitated and counted.
The whole blood was frozen by testing the frozen stock solution 1 and the frozen stock solutions 2 and 3, respectively, and resuscitated, and the measured PBMC viability and concentration result are shown in table 1 below.
The frozen stock solution 1 and the frozen stock solutions 4, 5 and 6 were respectively tested for the recovery after different batches of whole blood were frozen, and the results of the measured PBMC viability and concentration are shown in table 2 below.
Table 1: cryopreservation liquid 1 and cryopreservation liquid 2 and 3 cryopreservation resuscitates results
| Frozen stock solution | Concentration (cells/mL) | Activity (%) |
| Example 1 | 3.31E+06 | 55.37 |
| Example 2 | 1.62E+06 | 57.03 |
| Example 3 | 5.69E+05 | 44.37 |
Table 2: cryopreservation solution 1 and cryopreservation solutions 4, 5, 6 for resuscitating results
Remarks: whole blood 1: split red, wash once, use normal AO/PI pattern, cell type PBMC; whole blood 2 and 3: split red, washed twice, using normal AO/PI mode, cell type PBMC.
Table 3: cryopreservation resuscitatory results of cryopreservation solution 1 and the cryopreservation solutions of comparative examples 4, 5, 6, 7, 8, 9, example 4
| Frozen stock solution | Concentration (cells/mL) | Activity (%) |
| Example 1 | 3.31E+06 | 55.37 |
| Comparative example 4 | 1.12E+06 | 37.88 |
| Example 4 | 2.58E+06 | 52.53 |
| Comparative example 5 | 1.97E+06 | 54.56 |
| Comparative example 6 | 1.91E+06 | 59.61 |
| Comparative example 7 | 3.42E+05 | 31.04 |
| Comparative example 8 | 4.64E+04 | 18.53 |
| Comparative example 9 | 2.95E+04 | 3.14 |
From the above data, it can be seen that the frozen stock solution 1 and the frozen stock solution of example 4 have better cell viability and more cell numbers than the PBMC obtained after whole blood is recovered by other frozen stock solutions, and have obvious advantages. Meanwhile, in order to demonstrate that whole blood frozen using the frozen stock solution 1 can be used for detection of the occupancy of clinical receptors, the following examples 5 and 6 were examined:
EXAMPLE 5 Whole blood and PBMC precision test
Reagent: anti-PD-L1 antibody drugs, biotinylatedanti-PD-L1 antibody (Shanghai-John), biotinylated IgG from rabbit serum (Shanghai-John), PE Anti-human CD3 (BioLegend, 300441), brilliant Violet 421 stretavidin (BioLegend, 405225), zombie NIR TM Fixable Viabillity Kit (BioLegend, 423106), cytofix Fixation Buffer (BD, 554655), mouse serum powder (Jackson Immuno Research, 015-000-120), rabbit serum (Sbjbio, SBJ-SE-RAB001-100 mL), starting buffer (BioLegend, 420201), 1 XPBS (Corning, 21-040-CVR), DPBS (Corning, 21-031-CVC), 0.4% trypan blue dye (Invitrogen, T10282), AO/PI dye (Count star, RE 010212), 10X RBC Lysing Buffer (BD, 555899), and redissolving a portion of the reagents according to instructions to give a complex solution thereof: mouse serum powder and 10× RBC Lysing Buffer, and adding 5mL pure water per bottle of mouse serum powder to obtain mouse bloodClearing stock solution; 10X RBC Lysing Buffer with pure water 1:9 dilution to obtain 1X erythrocyte lysate. In this method, the dead dye is formulated: mu.L stock solution was taken into 995. Mu.LPBS (or DPBS) to prepare a working solution of dead dye for use on the day. Preparing a sealing liquid: contains 10% mouse serum and 10% rabbit serum.
Experimental group detection antibody preparation: a) Taking 180 mu LPE anti-human CD3 to 3420 mu L staining buffer, fully and uniformly mixing, preparing PE anti-human CD3 working solution, and preparing the working solution for use in the day; b) And (3) mixing the materials with 3 mu LBiotinylatedanti-PM8001 to 297 mu LPE anti-human CD3 working solution thoroughly, and preparing the mixture for use in the day.
Isotype control group detection antibody preparation: and (3) taking 3 mu L Biotinylated IgG from rabbit serum to 297 mu L PE anti-human CD3 working solution, fully and uniformly mixing, and preparing the PE anti-human CD3 working solution for use in the day.
The Brilliant Violet 421 strepitavidin working solution is prepared: mixing 3 μ L Brilliant Violet to 4997 μl of stainingbuffer, and making into dosage form for use in the day.
The experimental process comprises the following steps:
preparing a whole blood sample: in the experiment, anticoagulated whole blood and Anti-PD-L1 antibody medicines with different concentrations are adopted to prepare quality control samples (HQC, MQC, LQC), the quality control samples are uniformly mixed, and the samples are incubated for 25min plus or minus 5min at room temperature after preparation is completed, so that clinical samples are simulated. And (3) distributing proper volume according to the requirement, adding the frozen stock solution with the same volume, sub-packaging, adding about 1.5mL of sample to be frozen into each frozen stock pipe, placing the sample into a program cooling box, placing the sample into a refrigerator with the temperature of-70 ℃, preserving the sample for at least 4 hours, transferring the sample into a refrigerator with the temperature of-20 ℃ and the temperature of-70 ℃ or liquid nitrogen, preserving the sample, and waiting for subsequent detection.
PBMC sample treatment procedure: 3 different batches of human peripheral blood (anticoagulant sodium citrate) were added with Anti-PD-L1 antibody drugs (HQC, MQC, LQC) at different concentrations to prepare quality control samples, and after preparation, incubated at room temperature for 25 min.+ -. 5min, and PBMC were extracted using CPT tubes.
Precision:
to evaluate the in-batch precision of the method, the experiment validated at least one acceptable precision analysis batch, and the target occupancy of PD-L1 antibodies to PD-L1 in 3 different batches of human whole blood and the target occupancy of PD-L1 antibodies to PD-L1 in cryopreserved PBMC were tested separately. The method can be carried out synchronously with a stability baseline test, and the average value is used for subsequent stability evaluation, and can also be carried out by using frozen samples:
a. including 3 different batches of human peripheral blood and corresponding extracted PBMCs, 3 levels (HQC, MQC, LQC) of quality control samples each were repeated 2 times (i.e., 2-well assays), including experimental and isotype control groups.
The PD-L1 target occupancy of the experimental group was used to calculate the precision.
Unless there is evidence of well-documented evidence or technical operation errors, all data generated for precision analysis need to be taken into account in statistical calculations.
d, calculating the precision in batch of the quality control samples of the concentration levels, and independently calculating the precision in batch of the human peripheral blood and the PBMC of each batch.
Within-batch precision acceptance criteria:
a for human peripheral blood and PBMC: at least 2/3 of the quality control sample at each level needs to meet the requirement that the composite pore% CV is less than or equal to 25.0 percent, and the precision in the batch is calculated independently.
b for detection items whose precision in the batch cannot meet the above criteria, an additional 2 analytical batches of detection may be performed. Quality control
Part 1: whole blood split red
1.1 Whole blood split red: the prepared whole blood quality control sample was mixed with 1×red blood cell lysate 1:9, i.e., 1.5mL whole blood quality control sample was added with 13.5mL of 1×red blood cell lysate. And standing at RT for 15min.
Centrifuge at room temperature for 5min at 1.2500 Xg, discard supernatant, resuspend with appropriate volume of PBS, count.
Centrifuging at 1.3500 Xg at room temperature for 5min, discarding supernatant, and adjusting cell density to 5×10 with a stainingbuffer 6 cells/mL, 100. Mu.L/well (5X 10) 5 cells/well), 4 wells (containing experiments and isotype groups, 2 wells each) were allocated for each concentration, and if the number of cells was insufficient, all cells were resuspended according to the required number of wells and 100 μl/well was dispensed into each well.
Part 2: cell staining vital dye
2.1 centrifugation at 500 Xg for 5min at RT, discarding the supernatant, re-suspending the cells at 1300rpm for 2min at RT, adding dead and live dye working solution, 100. Mu.L/well, mixing well, and incubating at RT for 25 min.+ -. 5min in the absence of light.
2.2 washing with 100. Mu.L/well of stainingbuffer, and centrifugation at 500 Xg for 5min at room temperature.
2.3 washing solution was discarded, and cells were resuspended in a plate shaker at room temperature of 1300rpm for 2min, washed by adding 200. Mu.L/well stainingbuffer, and centrifuged at 500 Xg at room temperature for 5min.
Part 3: sealing liquid seal
3.1 discarding the washing solution, re-suspending the cells at room temperature of 1300rpm for 2min by using a plate shaking machine, adding 50 mu L/hole of sealing solution (containing 10% of mouse serum and 10% of rabbit serum), uniformly mixing, and incubating at 2-8 ℃ in the absence of light for 25 min+/-5 min;
part 4: antibody incubation
4.1, respectively adding an experimental group detection antibody and a isotype control group detection antibody, mixing uniformly at 50 mu L/hole, and incubating for 60min plus or minus 10min at 2-8 ℃ in a dark place;
4.2 washing with 100. Mu.L/well of stainingbuffer, and centrifuging at 500 Xg for 5min at room temperature;
4.3 washing solution is discarded, the cells are resuspended by using a plate shaking machine at room temperature of 1300rpm for 2min, 200 mu L/well of stainingbuffer is added for washing, and the cells are centrifuged at 500 Xg for 5min at room temperature;
part 5: brilliant Violet 421 strepitavidin working solution staining
5.1 discarding the washing solution, re-suspending the cells by using a plate shaking machine at the room temperature of 1300rpm for 2min, adding 100 mu L/hole Brilliant Violet 421 strepitavidin working solution, uniformly mixing, and incubating at 2-8 ℃ in a dark place for 25 min+/-5 min;
5.2 washing with 100. Mu.L/well of stainingbuffer, and centrifuging at 500 Xg for 5min at room temperature;
5.3 washing solution is discarded, the cells are resuspended by using a plate shaking machine at room temperature of 1300rpm for 2min, 200 mu L/well of stainingbuffer is added for washing, and the cells are centrifuged at 500 Xg for 5min at room temperature;
part 6: fixing
6.1, discarding the washing solution, re-suspending the cells by using a plate shaking machine at the room temperature of 1300rpm for 2min, adding 100 mu L/hole Cytofix Fixation Buffer, uniformly mixing, and incubating at the temperature of 2-8 ℃ in a dark place for 25 min+/-5 min;
6.2 washing with 100. Mu.L/well of stainingbuffer, and centrifuging at 500 Xg for 5min at room temperature;
6.3 washing solution is discarded, the cells are resuspended by using a plate shaking machine at room temperature of 1300rpm for 2min, 200 mu L/well of stainingbuffer is added for washing, and the cells are centrifuged at 500 Xg for 5min at room temperature;
part 7: a rotary pipe;
7.1 washing was discarded, the samples were resuspended in stainingbuffer, 200. Mu.L/well, and the samples were transferred to a 5mL flow tube. The sample can be immediately collected, or can be stored at 2-8 ℃ in a dark place, and the sample collection is completed within 48 hours;
part 8: collecting a sample;
part 9: and (5) data storage.
The results of the experiments are shown in tables 4 and 5, in which Table 4 shows the results of the precision test of whole blood and PBMC, and Table 5 shows the precision and difference between whole blood and PBMC.
Table 4 results of whole blood and PBMC precision tests.
Remarks: bold italics indicates that the acceptance criteria are not met.
TABLE 5 precision and differences between whole blood and PBMC lots
The results show that:
through data analysis, the precision in whole blood batch is the% CV range in HQC batch: 0.7% -4.0%, all quality control samples meet the acceptance standard; MQC intra-lot% CV range: 0.3% -4.2%, all quality control samples meet the acceptance standard; LQC in-batch% CV range: 1.2 to 6.4 percent, and all quality control samples meet the acceptance standard. Precision in PBMC batch%cv range in HQC batch: 0.3% -1.8%, all quality control samples meet the acceptance standard; MQC intra-lot% CV range: 2.5% -19.5%, all quality control samples meet the acceptance standard; LQC in-batch% CV range: 2.4% -37.7%, and 2/3 quality control samples meet the acceptance standard. Inter-batch precision% CV range of PBMC and whole blood: 0.0 to 17.5 percent. For whole blood and PBMC of healthy people, the precision meets the expected acceptance standard, and the cryopreserved PBMC is consistent with the PD-L1 target occupancy result in the whole blood.
EXAMPLE 6 Whole blood-20 ℃, -70 ℃ and liquid nitrogen cryopreservation stability test
The sample stability test needs to include 3 different batches of human peripheral blood and PBMCs extracted correspondingly, and examine 3 levels (HQC, MQC, LQC) of stability samples without duplicate wells (if baseline detection is performed in synchronization with precision, with reference to precision, treatment) including experimental and isotype control groups.
a. Sample preparation
3 different batches of human peripheral blood are added with Anti-PD-L1 antibody medicines (HQC, MQC, LQC) with different concentrations to prepare quality control samples, and the quality control samples are separated into one part for whole blood baseline detection, and when the whole blood baseline detection is carried out synchronously with precision, two-hole detection is needed, no hole is arranged when only baseline detection is carried out, and the measurement is needed within 2 hours of sample preparation. PBMC were extracted with CPT tubes from 4mL whole blood. The rest whole blood sample is distributed into proper volumes according to the requirement, the cell freezing solution with the same volume is added, and the whole blood sample is split into sub-packages, and 1.5mL of sample to be frozen is added into each freezing tube.
For a sample with the stability of-20 ℃, directly placing the program cooling box in a refrigerator with the temperature of-20 ℃ for overnight; for-70 ℃ and liquid nitrogen stable samples, the temperature programming cassette was placed in a-70 ℃ refrigerator overnight. The next day, the samples were transferred to corresponding storage conditions (-20 ℃, -70 ℃ and liquid nitrogen) for storage, and the transfer time was adjusted according to the actual situation. All stability samples were formulated and stored under each test condition until stability testing was performed.
And (3) re-suspending the extracted PBMC by using cell freezing solution, placing a program cooling box, placing the box in a refrigerator at the temperature of-70 ℃, transferring the box out the next day, and adjusting the transfer time according to actual conditions. After being stored for a period of time at the temperature of-70 ℃ in a refrigerator (generally 1-3 days, the actual storage condition of clinical samples is simulated), the PBMC are recovered and used for freezing PBMC baseline detection, and the detection is carried out synchronously with precision, namely, two-hole detection is carried out. Comparing the result with a whole blood baseline detection result, and evaluating the consistency of the frozen PBMC and the PD-L1 target point occupation ratio result in the whole blood by adopting the average value of the target point occupation ratio generated by precision detection.
b. Acceptance criteria:
% TO is less than or equal TO 25.0% CV compared with baseline detection results in whole blood.
% TO is within + -20.0% of baseline measurements in whole blood.
Stability samples at least 2/3 of each concentration level meet the criteria described above.
c. Test content
Whole blood cryopreservation stability: stability was assessed for at least 1 day, 3 days at-20 ℃ storage conditions; the stability was evaluated at-70 ℃ storage for at least 7 days, 1 month, 3 months, 6 months; stability was assessed for at least 3 days, 3 months and 6 months under liquid nitrogen storage conditions.
d. Acceptance criteria
PD-L1 target occupancy of experimental group is used for statistical calculation, and the base line detection acceptance standard is required to meet 'precision acceptance standard in batch'
Remaining stability test acceptance criteria:
% TO is less than or equal TO 25.0% as compared with baseline detection results.
% TO is within + -20.0% of baseline detection.
For each stability condition, at least 2/3 of the stability sample per concentration level meets the criteria described above.
For stability tests, one assay does not meet the acceptance criteria, two assays can be added, and 2/3 pass is the condition where the sample is deemed stable.
The experimental steps are as follows:
the samples were taken out and tested after being stored at-20 c for 1 day, 3 days, and-70 c for 3 days, 1 month, 3 months, and 6 months, respectively, and the stability test procedure was substantially the same as in example 5, except that the samples were recovered first, then the drug incubation was performed, and the red burst step was not required, and the test results were as shown in table 6 below.
And (3) resuscitating the quality control sample:
(1) Taking out the frozen sample, rapidly shaking and resuscitating in a water bath at 37 ℃, and transferring to a proper volume of PBS after resuscitating.
(2) Centrifuge at room temperature for 5min at 400 to 500 Xg, discard supernatant.
(3) Appropriate volumes of PBS were resuspended, counted and the same sample (containing different drug treatment levels) was counted only 1 time.
(4) Centrifuging at room temperature of 400-500 Xg for 5min, discarding supernatant, and adjusting cell density to 1×10 with a stainingbuffer 7 cells/mL, 50. Mu.L/well (5X 10) 5 cells/well), and dispensed into 96-well V-well plates. If the cell number is insufficient, all cells are resuspended according to the required hole number, and 50 mu L/hole is split into each hole;
flow charts of the stained live dye after the whole blood is frozen by using the frozen stock solution 1 and stored at the temperature of-70 ℃ for 7 days, 1 month, 3 months and 6 months are shown in figures 1-4 respectively. The cell viability was seen to be maintained substantially between 65% and 90%.
And (3) a drug incubation step:
(5) Adding a stable buffer into the quality control sample, mixing uniformly at 50 mu L/hole each, and incubating at 2-8 ℃ for 25min plus or minus 5min;
note that: the cell density and the packed volume can be adjusted according to the requirement, and the number of the packed cells and the final cell suspension volume are only required to be consistent (5 multiplied by 10) 5 cells/well, 100 μl/well). For analysis batches such as qualification confirmation, the test wells are incubated without supersaturated drugs in the well plate, and the step (9) can be directly carried out after cell plating.
(6) Adding 100 mu L/well of stainingbuffer for washing, and centrifuging at 500 Xg for 5min at room temperature;
(7) The wash was discarded, the cells were resuspended using a plate shaker at room temperature of 1300rpm for 2min and washed with 200. Mu.L/well stainingbuffer.
TABLE 6 results of Whole blood storage stability at-20℃and-70 ℃
Remarks: bold and italic represent that the acceptance criteria are not met.
Samples were taken out for detection after 3 days, 3 months and 6 months of storage under liquid nitrogen, respectively, and the detection procedure was the same as above, and the detection results are shown in Table 7 below.
TABLE 7 Nitrogen storage stability results of whole blood
Remarks: bold and italic represent that the acceptance criteria are not met.
The results of example 6 show that whole blood stability samples are stable for 3 days at-20 ℃; the whole blood stable sample is stable for 6 months at-70 ℃; whole blood stable samples were stable for 6 months under liquid nitrogen conditions. The result shows that the frozen stock solution developed by the application can preserve whole blood for a long time and can achieve the technical effect of stable receptor occupancy.
In conclusion, the whole blood frozen stock solution developed by the application can better preserve whole blood, the number and the living rate of PBMC living cells obtained after the whole blood frozen by the frozen stock solution is recovered are higher than those of the frozen stock solution which is generally commercialized, and the frozen stock solution can be used for freezing human peripheral blood for a long time to detect the receptor occupancy rate.
Claims (10)
1. The whole blood cryopreservation liquid is characterized by comprising the following components in percentage by volume:
60-80% of RPMI1640 culture medium;
10-30% of fetal bovine serum;
10-20% of dimethyl sulfoxide;
and contains the following components:
sucrose 1-20 g/L;
disodium ethylenediamine tetraacetate final concentration is 2-3 mM;
the pH value is 6.0-8.0.
2. The whole blood cryopreservation solution of claim 1, wherein the whole blood cryopreservation solution comprises the following components in volume fraction:
60-70% of RPMI1640 culture medium;
20-30% of fetal bovine serum;
10-15% of dimethyl sulfoxide;
and contains the following components:
10-20 g/L of sucrose;
disodium edetate final concentration 2mM;
the pH value is 7.0-8.0.
3. The method for preparing the whole blood cryopreservation solution according to claim 1 or 2, which is characterized in that the method comprises the following steps: uniformly mixing the raw materials in the formula amount, and regulating the pH value to 6.0-8.0 by using sodium hydroxide or potassium hydroxide to prepare the whole blood frozen stock solution.
4. The use of the whole blood cryopreservation solution according to claim 1 or 2 in the cryopreservation of whole blood, characterized in that the method of the use comprises the steps of mixing fresh whole blood with the whole blood cryopreservation solution according to a volume ratio of 0.2-2: 1, mixing, cooling to-70 ℃ in a gradient way, and freezing.
5. The use according to claim 4, wherein the volume ratio of fresh whole blood to whole blood frozen stock is 0.2-1:1.
6. A method for cryopreserving whole blood, characterized in that the method comprises the steps of: mixing fresh whole blood and the whole blood frozen stock solution according to the volume ratio of 0.2-2: 1, mixing, cooling to-70 ℃ in a gradient way, and freezing.
7. The method of claim 6, wherein the whole blood after cryopreservation is stored at-20 ℃ for 3 days; storing for 6 months at-70deg.C; can be stored for 6 months under liquid nitrogen.
8. Use of whole blood cryopreservation solution according to claim 1 or 2 for receptor occupancy detection after cryopreservation of whole blood and resuscitation, wherein the method of use is to use the whole blood cryopreservation solution for cryopreservation of fresh whole blood and then to perform receptor occupancy detection of the resuscitated whole blood after resuscitation.
9. The method for detecting the receptor occupancy after cryopreservation of whole blood and resuscitation is characterized by comprising the following steps of: the method for detecting receptor occupancy of whole blood comprises the steps of cryopreserving fresh whole blood using the whole blood cryopreservation solution according to claim 1 or 2, and then resuscitating the whole blood.
10. The method according to claim 9, wherein the method of cryopreserving is: fresh whole blood and whole blood frozen stock solution are mixed according to the volume ratio of 0.2-2: 1, mixing, cooling to-70 ℃ in a gradient way, and freezing; the resuscitation method comprises the following steps: shaking and recovering the frozen whole blood at 37 ℃.
Priority Applications (1)
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| CN102669087A (en) * | 2012-05-15 | 2012-09-19 | 深圳市博泰生物医疗机构管理有限公司 | Freeze-storage liquid of peripheral blood mononuclear cells and freeze-storage method |
| CN108094411A (en) * | 2018-01-29 | 2018-06-01 | 山东省齐鲁细胞治疗工程技术有限公司 | A kind of cryopreservation methods and method for resuscitation of PBMC cells |
| CN108651439A (en) * | 2018-04-13 | 2018-10-16 | 上海韵飞生物科技有限公司 | A kind of complete serum-free frozen stock solution of peripheral blood mononuclear cells and its method |
| CN110140716A (en) * | 2019-07-08 | 2019-08-20 | 方艳秋 | A kind of peripheral blood mononuclear cells improvement frozen stock solution |
| CN110447636A (en) * | 2019-08-07 | 2019-11-15 | 广东万海细胞生物科技有限公司 | A kind of frozen stock solution of peripheral blood mononuclear cells, cryopreservation methods and method for resuscitation |
| CN114964966A (en) * | 2022-05-25 | 2022-08-30 | 上海市东方医院（同济大学附属东方医院） | Method for separating and freezing blood sample |
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| CN102669087A (en) * | 2012-05-15 | 2012-09-19 | 深圳市博泰生物医疗机构管理有限公司 | Freeze-storage liquid of peripheral blood mononuclear cells and freeze-storage method |
| CN108094411A (en) * | 2018-01-29 | 2018-06-01 | 山东省齐鲁细胞治疗工程技术有限公司 | A kind of cryopreservation methods and method for resuscitation of PBMC cells |
| CN108651439A (en) * | 2018-04-13 | 2018-10-16 | 上海韵飞生物科技有限公司 | A kind of complete serum-free frozen stock solution of peripheral blood mononuclear cells and its method |
| CN110140716A (en) * | 2019-07-08 | 2019-08-20 | 方艳秋 | A kind of peripheral blood mononuclear cells improvement frozen stock solution |
| CN110447636A (en) * | 2019-08-07 | 2019-11-15 | 广东万海细胞生物科技有限公司 | A kind of frozen stock solution of peripheral blood mononuclear cells, cryopreservation methods and method for resuscitation |
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