CN110352952A - A kind of composition saved for cell, aqueous compositions and its application - Google Patents
A kind of composition saved for cell, aqueous compositions and its application Download PDFInfo
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- CN110352952A CN110352952A CN201910658888.0A CN201910658888A CN110352952A CN 110352952 A CN110352952 A CN 110352952A CN 201910658888 A CN201910658888 A CN 201910658888A CN 110352952 A CN110352952 A CN 110352952A
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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
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Abstract
The invention discloses a kind of compositions saved for cell, aqueous compositions comprising the composition, and application of the aqueous compositions before flow cytometry cell detection in biological Sample preservation, the composition saved for cell includes following component: potassium dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride, potassium chloride, Dextran 40, tetramisole hydrochloride, poly aldehydes, anti-coagulants, wherein, potassium dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride, potassium chloride, Dextran 40, tetramisole hydrochloride, poly aldehydes, the mass ratio of anti-coagulants is 0.2: 1.4: 8-9: 0.2: 5-25: 25-50: 25: 20-30；Potassium dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride, potassium chloride, Dextran 40, tetramisole hydrochloride, poly aldehydes, anti-coagulants are used in compounding by composition of the invention, biological sample can effectively, steadily be saved up to 60 days, provide extremely important guarantee to detect or diagnosing preceding sample process.
Description
Technical field
The present invention relates to cell preservation technique fields, more particularly to a kind of composition saved for cell, comprising being somebody's turn to do
The application of the aqueous compositions of composition and the aqueous compositions before flow cytometry cell detection in biological Sample preservation.
Background technique
Flow cytometry (Flow Cytometry, FCM) is the present age state-of-the-art cell quantitative technology, is had fast
The advantages that speed, high-precision, multi-parameter.FCM is played an important role in clinical and scientific research field, especially in clinical in vitro diagnosis in vitro
(In Vitro Diagnostic, IVD) existing very extensive and deep application, becomes one of the detection means of most mainstream,
Clinical application includes: immunology, hematology, Clinical Oncology, hematopoietic stem cell transplantation etc.；It is currently leukaemia, lymthoma etc.
The goldstandard of malignant disease clinical diagnosis, and drug susceptibility screening and in terms of play important work
With.
The biological sample of machine testing on FCM, generally peripheral blood, marrow or other tissue samples.Obtaining biology
To before upper machine testing after sample, need properly to save biological sample.Cell is after leaving human body, since environment becomes
Change, the relevant physicochemical characteristics of cell and every biomarker are likely to change therewith, and then influence detection knot
Fruit, or even influence diagnosis and further medical treatment scheme.Therefore, the cell in biological sample appropriately saved, protected
Shield, guarantee cell have with the immediate kilter of original state, it is extremely important in sample process before being detection or diagnosing
Step.
Currently, relatively fewer for cell preservation technique research dedicated before FCM detection, in general, Techniques of preserving is needed
The main problem to be solved includes: the stable state that (1) maintains cell biological marker, especially cell surface marker；(2)
The Sample preservation time can be extended on the basis of guaranteeing detection or diagnosis accuracy；(3) formula for saving reagent is easier to match
System, cost is lower, more convenient operation.
Application publication number CN 107091799A discloses " a kind of blood cell stabilizer ", including A liquid and B liquid, wherein A
Liquid includes following components: anti-coagulants, inhibitors of phosphatases, protease inhibitors；B liquid includes following components: aldehyde polymer, alkali
Property salt, buffer；Wherein, A liquid and B liquid are mixed according to 0.5-2: 0.5-2 volume ratio.Above-mentioned blood cell stabilizer, which has, to be protected
Histiocytic effect is protected, to the chemical substance that protein, enzyme etc. are slowly fixed, has and stablizes blood cell, keeps blood thin
The original form of born of the same parents maintains the original physical property of blood cell, can be well fixed the cell in blood；But it is above-mentioned
Blood cell stabilizer need the separated storage of A agent and B agent, mixed using preceding, made troubles to using again；Separately
Outside, above-mentioned blood cell stabilizer can be reserved for blood only 192 hours, and the holding time is shorter, limits application range.
Summary of the invention
In order to solve the problems, such as that the storable cell stage of current cell-preservation liquid is short and A agent, B agent need separately to store,
The object of the present invention is to provide a kind of compositions saved for cell；In addition, the present invention also provides include the composition
The application of aqueous compositions and the aqueous compositions before flow cytometry cell detection in biological Sample preservation.
To achieve the goals above, the present invention adopts the following technical scheme:
The first aspect of the present invention provides a kind of composition saved for cell, including following component: biphosphate
Potassium, disodium hydrogen phosphate, sodium chloride, potassium chloride, Dextran 40, tetramisole hydrochloride, poly aldehydes, anti-coagulants, wherein phosphoric acid
The quality of potassium dihydrogen, disodium hydrogen phosphate, sodium chloride, potassium chloride, Dextran 40, tetramisole hydrochloride, poly aldehydes, anti-coagulants
Than being 0.2: 1.4: 8-9: 0.2: 5-25: 25-50: 25: 20-30.
Preferably, the potassium dihydrogen phosphate, disodium hydrogen phosphate, 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.
Preferably, the potassium dihydrogen phosphate, disodium hydrogen phosphate, 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.
Above-mentioned tetramisole hydrochloride is inhibitors of phosphatases.
Wherein, the poly aldehydes is the mixing of one of Metaldehyde or metapropionaldehyde or both.
Wherein, the anti-coagulants is the mixing of one of EDTA, citrate or both.
The second aspect of the present invention provides a kind of aqueous compositions saved for cell, includes combinations of the above object.
The third aspect of the present invention provides above-mentioned aqueous compositions biological Sample preservation before flow cytometry cell detection
In application.
Wherein, the biological sample is the blood or tissue samples of people.
Wherein, the biological sample is people's marrow or human peripheral.
The fourth aspect of the present invention provides a kind of method that cell saves, includes the following steps:
S1 mixes combinations of the above object 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.
Compared with prior art, the present invention realize the utility model has the advantages that composition of the invention by potassium dihydrogen phosphate, phosphoric acid hydrogen
Disodium, sodium chloride, potassium chloride, Dextran 40, tetramisole hydrochloride, poly aldehydes, anti-coagulants are used in compounding, by fixed thin
Born of the same parents keep the original form of cell, maintain the original physical property of cell, inhibit effect of the enzyme to cell membrane and albumen, Neng Gouyou
Effect steadily saved biological sample up to 60 days, provided extremely important guarantee to detect or diagnosing preceding sample process.
Specific embodiment
Embodiments of the present invention are illustrated by particular specific embodiment below, those skilled in the art can be by this explanation
Content disclosed by book is understood other advantages and efficacy of the present invention easily.
The present invention is the addition Dextran 40, four on the basis of potassium dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride, potassium chloride
Imidazole hydrochloride, poly aldehydes and anti-coagulants, Dextran 40, tetramisole hydrochloride, poly aldehydes and anti-coagulants compatibility can
Fixed cell keeps the original cell of cell, can stablize and save biological sample up to 60 days, before being convenient for detection or diagnosing at sample
Reason.
The present inventor is by chronically research and experiment it was unexpectedly observed that aqueous compositions are to cell infiltration appropriate
Pressure energy enough increases the preservation effect of cell, by adjusting the dosage of sodium chloride, reduces a possibility that being damaged when cell saves, protects
Hold the original form of cell.
One, the preparation of aqueous compositions
1, it under conditions of temperature is 20-25 DEG C, humidity is less than 60%, weighs each raw material and is placed in 50mL sterile centrifugation tube
In to get to for cell save composition；
2, the above-mentioned composition for being used for cell preservation is dissolved in a certain amount of water for injection, obtains colorless and transparent liquid
Body；
3, in the gnotobasis of local laminar flow clean by laminar flow condition, above-mentioned colourless transparent liquid is straight by 0.22 μm
The filter membrane of diameter, filtrate go in the sterile centrifugation tube of another 50mL to get to the aqueous compositions saved for cell, at 4 DEG C
It is kept in dark place spare.
The aqueous compositions of different compositions, the composition of each aqueous compositions such as table 1, table 1 are obtained by the dosage of feed change
By weight percentage, surplus is water for injection.
Table 1
Two, the method that aqueous compositions carry out cell preservation
Randomly choose 20 healthy adult volunteers, volunteer's age between 20-40, male or female.Take volunteer's
Anticoagulation cirumferential blood sample (n=20) is mixed with the embodiment 1-12 and comparative example 1-5 aqueous compositions prepared respectively, anticoagulant outer
All blood samples and the volume ratio of aqueous compositions are 10: 1, and mixed sample is placed in 2-8 DEG C of preservation, the taking-up when needing to detect
Sample.
Three, aqueous compositions of the invention and reference examples carry out the effect assessment of cell preservation
The mixture of anticoagulation cirumferential blood sample and aqueous compositions is mixed by inversion 10 times, 4 DEG C of refrigerators is put into and saves, exist respectively
0d, 10d, 20d, 30d, 40d, 50d, 60d carry out FCM detection, and as a result such as table 2 and table 3, table 2 and table 3 only show wherein 1
Cellular change situation of the anticoagulation cirumferential blood sample after embodiment 1-12 and reference examples 1-5 is saved, wherein table 2 is granulocyte, list
The ratio variation of nucleus, lymphocyte, table 3 are the ratio variation of T cell, B cell, NK cell, DC cell surface marker.
Above-mentioned FCM detection has carried out 20 altogether in parallel, and testing result is similar to the height in table 2 and table 3, it was demonstrated that of the invention is aqueous
Preparation can effectively, steadily save biological sample up to 60 days.
FCM detects general experimental procedure are as follows: (1) antibody by specification dosage is separately added into streaming tube bottom.(2) 100ul is taken
Biological sample is added along tube wall, mixes, and room temperature is protected from light incubation 20 minutes.(3) 2ml erythrocyte cracked liquid is added, mixes well, room
Temperature is protected from light 10 minutes.(4) 1500rpm is centrifuged 5 minutes, abandons supernatant.(5) plus 2ml washing lotion, mixing, 1500rpm are centrifuged 5 minutes, are abandoned
Supernatant.(6) 0.4ml PBS washing lotion is added to mix, flow cytometer detection.(7) result is analyzed.
Operation side is carried out on the basis of general experimental procedure since viscosity is bigger for Lin-FITC antibody
Method improves, and steps are as follows for specific experiment: (1) taking 100ul EDTA biological sample that tube bottom is added along tube wall.(2) it is red thin that 2ml is added
Cellular lysate liquid, mixes well, and room temperature is protected from light 10 minutes.(3) 1500rpm is centrifuged 5 minutes, abandons supernatant.(4) add 2ml washing lotion, mix
Even, 1500rpm is centrifuged 5 minutes, abandons supernatant.(5) antibody by specification dosage is separately added into streaming tube bottom.Room temperature is protected from light incubation
20 minutes.(6) plus 2ml washing lotion, mixing, 1500rpm are centrifuged 5 minutes, abandon supernatant.(7) 0.4ml PBS washing lotion is added to mix, on
Flow cytomery.
Table 2
Table 3
As shown in table 1, reference examples 1 are compared with embodiment 12, K2HPO4、NaH2PO4, the additive amount of NaCl, KCl it is identical, and
Dextran 40, tetramisole hydrochloride, poly aldehydes, anti-coagulants is not added in reference examples 1；Reference examples 2 are compared with embodiment 12,
K2HPO4、NaH2PO4, NaCl, KCl, tetramisole hydrochloride, poly aldehydes, the additive amount of anti-coagulants it is all the same, and in reference examples 1
Dextran 40 is not added；Reference examples 3 are compared with embodiment 6, K2HPO4、NaH2PO4, it is NaCl, KCl, tetramisole hydrochloride, more
Polyacetals class, the additive amount of anti-coagulants are all the same, and Dextran 40 is not added in reference examples 3；12 phase of reference examples 4 and embodiment
Than K2HPO4、NaH2PO4, NaCl, KCl, tetramisole hydrochloride, poly aldehydes, the additive amount of anti-coagulants it is all the same, and reference examples
The additive amount of Dextran 40 is down to 0.5% by 2.5% in 4；Reference examples 5 are compared with embodiment 12, K2HPO4、NaH2PO4、
NaCl, KCl, tetramisole hydrochloride, poly aldehydes, the additive amount of anti-coagulants be all the same, and Dextran 40 adds in reference examples 5
Dosage is down to 1.5% by 2.5%；Reference examples 4 are compared with embodiment 10, K2HPO4、NaH2PO4, KCl, Dextran 40, four miaows
Triazole hydrochloride, poly aldehydes, the additive amount of anti-coagulants are all the same, and the additive amount of NaCl is near by 0.9% in reference examples 4
0.8%；Reference examples 5 are compared with embodiment 11, K2HPO4、NaH2PO4, KCl, Dextran 40, tetramisole hydrochloride, poly aldehyde
Class, the additive amount of anti-coagulants are all the same, and the additive amount of NaCl is by 0.9% near 0.8% in reference examples 4.
As shown in table 2, the cell that embodiment 12 saves is detected through FCM, and granulocyte is down to 60d's by the 76.122% of 0d
65.450%, monocyte is down to the 5.266% of 60d, lymphocyte by the 5.69% of 0d and rises to 60d's by the 16.336% of 0d
16.468%；The cell that reference examples 1 save is detected through FCM, granulocyte by the 76.232% of 0d be down to 60d 54.466%, it is single
Nucleus is down to the 3.701% of 60d, lymphocyte by the 5.78% of 0d and is down to the 10.87% of 60d by the 16.466% of 0d.It is right
As usual 1 and the corresponding testing result of embodiment 12 show reference examples 1 save after cell occur biggish damage in 60d, and
Embodiment 12 save after cell in 60d damage it is smaller, similarly, identical result is also embodied in table 3, it is seen then that only
K2HPO4、NaH2PO4, NaCl, KCl solution can not play the protective effect to cell, and in K2HPO4、NaH2PO4、NaCl、
Dextran 40, tetramisole hydrochloride, poly aldehydes, anti-coagulants are added on the basis of KCl and is remarkably improved the effect of the preservation to cell
Fruit.
Keep other components constant in reference examples 2, reference examples 4, reference examples 5 and embodiment 12, only Dextran 40 adds
Dosage changes, the additive amount of Dextran 40 such as table 4.
Table 4
| The additive amount (%) of Dextran 40 | |
| Reference examples 2 | 0 |
| Reference examples 4 | 0.5 |
| Reference examples 5 | 1.5 |
| Embodiment 12 | 2.5 |
Reference examples 2, reference examples 4, reference examples 5 and the corresponding granulocyte of embodiment 12, monocyte, lymphocyte test knot
Fruit is as shown in table 5.
Table 5
As shown in table 5, reference examples 2, reference examples 4, reference examples 5 testing result corresponding with embodiment 12 show in 60d
When, granulocyte, the content of monocyte are suitable, and there are larger differences for the content of lymphocyte, and reference examples 2 are when saving 60d
The content of corresponding lymphocyte is 12.154%, and the content of corresponding lymphocyte when saving 60d of reference examples 4 is
13.063%, the content of the corresponding lymphocyte when saving 60d of reference examples 5 is 13.605%, and embodiment 12 is when saving 60d
The content of corresponding lymphocyte is 16.468%, it is seen then that Dextran 40 and tetramisole hydrochloride, poly aldehydes compatible use
Cell preservation effect can be greatly improved, and only the combination of both tetramisole hydrochloride, poly aldehydes can not play identical effect, dextrorotation
The additive amount of sugared acid anhydride 40 influences the preservation effect of cell obvious.
Such as table 2, compare reference examples 4 and the corresponding testing result of embodiment 10, in 60d, granulocyte, monocyte contain
Quite, and the content of lymphocyte is there are larger difference for amount, and the content of corresponding lymphocyte when saving 60d of reference examples 4 is
13.063%, the content of the corresponding lymphocyte when saving 60d of embodiment 10 is 16.843%,.And reference examples 4 and embodiment
10 there is only the difference of NaCl additive amount, and the additive amount of NaCl is 0.8% in reference examples 4, the additive amount of NaCl in embodiment 10
It is 0.9%, it is seen then that the smaller difference of NaCl additive amount brings larger impact to cell preservation effect.
Inventor, by repeatedly studying and testing, anticipates on the basis of 10 corresponding testing result of reference examples 4 and embodiment
Other places discovery, NaCl additive amount are attributed to the variation of osmotic pressure, embodiment 10 and embodiment 11 to the influence of cell preservation effect
Osmolarity data when preservation cell is compared to reference examples 4 and 5, high about 30mOsm/kg.In addition, embodiment 10,11 is corresponding
Testing result testing result corresponding with embodiment 1-9 and embodiment 12 be compared discovery, embodiment 10,11 save it is thin
Born of the same parents 60d differs smaller with granulocyte, monocyte, the lymphocyte content when 0d, further verifies osmotic pressure and protects to cell
The influence of effect is deposited, but as known to those skilled in the art, not the higher the better for osmotic pressure, and osmotic pressure appropriate saves cell and sends out
Key effect is waved, the additive amount of NaCl is particularly important.
The above-described embodiments merely illustrate the principles and effects of the present invention, and is not intended to limit the present invention.It is any ripe
The personage for knowing this technology all without departing from the spirit and scope of the present invention, carries out modifications and changes to above-described embodiment.Cause
This, institute is complete without departing from the spirit and technical ideas disclosed in the present invention by those of ordinary skill in the art such as
At all equivalent modifications or change, should 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.
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Cited By (1)
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| CN111504887A (en) * | 2020-05-09 | 2020-08-07 | 苏州四正柏生物科技有限公司 | Hemolysin and preparation method thereof |
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Application publication date: 20191022 |