CN109892320A - A kind of cell-preservation liquid and its preparation method and application - Google Patents
A kind of cell-preservation liquid and its preparation method and application Download PDFInfo
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- CN109892320A CN109892320A CN201910214657.0A CN201910214657A CN109892320A CN 109892320 A CN109892320 A CN 109892320A CN 201910214657 A CN201910214657 A CN 201910214657A CN 109892320 A CN109892320 A CN 109892320A
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Abstract
The present invention relates to a kind of cell-preservation liquids, contain following components: Na2HPO4, glucose, citric acid, adenine, NaCl and water.The present invention also provides the preparation method and application of above-mentioned Cell protective solutions.Cell-preservation liquid provided by the present invention, can be with long-term preservation marrow, peripheral blood, tissue etc., and the stabilization of a variety of leucocyte related test results such as guarantee immunophenotyping, Lymphocyte subtypes test and correct, especially suitable for FCM analysis.And activity is still kept using the cell that liquid saves is saved, can proceed with cell culture and life or death dye marker.
Description
Technical field
The invention belongs to pharmaceutical technology fields, in particular to a kind of cell-preservation liquid and its preparation method and application.
Background technique
With the development of science and technology and further investigation of each medical domain to cell, flow cytometry is as a routine
Detection technique apply in every field, in increasingly consequence in terms of scientific research and clinical diagnosis.Including this
Apply before applicant application No. is including 20161002721.4 and 201610768294.1 Chinese invention patent application
Many prior arts focus on the step of flow cytometric art itself and parameter is studied.However, at present for fluidic cell
The preservation of art sample, but without a kind of generally acknowledged simple and fast store method or product.
In order to guarantee the accuracy of Flow cytometry result, cell sample should guarantee as far as possible it is fresh, even if in 2-8
It saves under the conditions of DEG C, should also use as early as possible, in case sample inactivation leads to result error.
Apply before the applicant application No. is 201610768294.1 Chinese invention patent applications to disclose one
Kind cell-preservation liquid, is exclusively used in the preservation of flow cytometry sample, the stability of sample can be kept for a long time, for peripheral blood sample
This, can stabilized leukocyte divide the expression of group and surface antigen.Product derived from above-mentioned patent application has fixed make to cell
With, through preservation liquid save after cell sample, it is inactive, can not continue culture or life or death cell identify.
Summary of the invention
In view of this, the object of the present invention is to provide a kind of cell-preservation liquids and its preparation method and application.
In order to reach above-mentioned purpose, the present invention provides a kind of cell-preservation liquid, contains following components: Na2HPO4, grape
Sugar, citric acid, adenine, NaCl and water.
Preferably, wherein the water is pure water.
Preferably, wherein the cell-preservation liquid contains the component of following final concentration:
Na2HPO40.1-10g/L；
Glucose 0.1-50g/L；
Citric acid 0.1-10g/L；
Adenine 0.1-10g/L；
NaCl 0.01-500mmol/L；
The solvent of above-mentioned each component is water.
The cell-preservation liquid does not have corrosivity, without oxidisability, can be loaded on the container of various material yet, as glass holds
Device or plastic containers.
The present invention provides the preparation methods of above-mentioned preservation liquid, include the following steps: Na2HPO4, glucose, citric acid,
Adenine, NaCl be uniformly mixed to get.
Preferably, wherein the preparation method is filtered and/or dispenses after further including the steps that mixing.It usually can be with
It does not dispense, is directly loaded in big reagent bottle and stores；Preservation for Hard link Blood collection blood sampling sample is preferably distributed into pipe dress
Preservation liquid, can be directly added in this way into pipe acquisition blood or celliferous sample and saved.Packing can divide
In non-real blank pipe, it can also be divided in vacuum tube.Acquisition volume in view of usual blood sample is 1~2ml, therefore point
The volume of dress corresponds to 100~300 μ l/ pipe, preferably 250 μ l/ pipe.
Invention further provides above-mentioned cell-preservation liquids for the application in flow cytomery.
Preferably, wherein it is described application the following steps are included:
1) sample liquid containing cell is provided；
2) above-mentioned cell-preservation liquid is mixed with the sample liquid, wherein the body of the cell-preservation liquid and the sample liquid
Product is than being 1:5~1:10；
3) mixture for obtaining step 2) saves；
4) mixture that step 3) obtains 50-100 μ l is taken out to mix with the 20 μ l of streaming monoclonal antibody of fluorescent marker
Streaming pipe is added, incubation 15 minutes is protected from light after mixing；
5) hemolysin 2ml is added in the streaming pipe being incubated for step 4), is incubated at room temperature 10 minutes, 200-300g after mixing,
Centrifugation 5 minutes is toppled over and discards supernatant and sample is resuspended with 200 μ lPBS；
6) the streaming pipe that flow cytomery step 5) is resuspended, and calculated result；
7) it takes out after a week and carries out retest.
Preferably, wherein sample liquid containing cell inherently can be liquid, e.g., blood, marrow, providing in this way should
Sample liquid itself.Sample liquid itself containing cell can be tissue or cell deposition, and needing in this way will be thin in tissue
Born of the same parents separate or disperse cell deposition, cell suspending liquid are formed, to be provided.
Preferably, wherein the mixture can be saved in room temperature (such as 25 DEG C), 2~8 DEG C of preservations are preferable over, in this way may be used
To there is longer storage life.It is preferred that storage life is 3 days or more, more preferably 5 days or more, further preferably 7 days.
Invention further provides the preservative agents for containing above-mentioned cell-preservation liquid.
Preferably, wherein the preservative agent also includes sample liquid, the volume of the cell-preservation liquid and the sample liquid
Than for 1:5~1:10.
Preferably, wherein sample liquid described in step 1) is blood, marrow or cell suspending liquid；It is flowed described in step 4)
Formula monoclonal antibody includes CD45-FITC, CD13-PE, CD33-PerCP-cy5.5 and CD7-APC of each 5 μ l.
Invention further provides above-mentioned preservative agents to be used for fluidic cell for flow cytomery or in preparation
Application in the kit of instrument detection.
Preferably, wherein the flow cytomery is the detection of Swine lymphocyte antigen and antigen intracellular.
The beneficial effects of the present invention are:
1, cell-preservation liquid provided by the present invention, can be with long-term preservation marrow, peripheral blood, tissue etc., and guarantees immune
The stabilization of a variety of leucocyte related test results such as parting, Lymphocyte subtypes test and correct, especially suitable for fluidic cell
Detection.And the cell by saving still keeps activity, can proceed with cell culture and life or death dye marker.
2, cell-preservation liquid provided by the present invention, containing special dietary preparation needed for cell survival, (glucose, gland are fast
Purine), the various physical indexs of cell and antigen presentation are not influenced, the stabilization of cell activity and surface antigen can be effectively kept.
Added the cell sample of the cell-preservation liquid, it is subsequent to continue to cultivate, life or death dye marker can be carried out, will not haemolysis,
And sample can stablize preservation seven days.
Specific embodiment
It will describe to invent by specific embodiment herein below.It, can be according to this field skill such as not specified place
" cell experiment guide " known to art personnel (Science Press, Beijing, China, 2001), " immunoassay technology " (science
Publishing house, Beijing, China, 1991) etc. in laboratory manuals and bibliography cited herein listed method implement.Its
In, reagent raw material used is commercially available product, can be obtained by open channel purchase.
The preparation of the bottled cell-preservation liquid 1 of embodiment 1
The formula of the bottled cell-preservation liquid 1 is as follows: Na2HPO40.4g/L；Glucose 0.5g/L；Citric acid 2.68g/
L；Adenine 2g/L；NaCl 5.8g/L.
Weigh Na2HPO40.4g, glucose 0.5g, citric acid 2.68g, adenine 2g, NaCl 5.8g, it is pure to be dissolved in 900ml
After changing water, it is settled to 1L, 0.22 μm of film filters, and in filling to sterile reagent bottle, thus cell-preservation liquid is made in closed reagent bottle
1L, as needed in packing to the bottle of suitable capacity.After tested, it at least stable can save 2 years, be suitble under 2~8 DEG C of environment
Long term storage.
The preparation of the bottled cell-preservation liquid 2 of embodiment 2
The formula of the bottled cell-preservation liquid 2 is as follows: Na2HPO45g/L；Glucose 2.5g/L；Citric acid 1.25g/L；
Adenine 0.5g/L；NaCl 11.6g/L.
Weigh Na2HPO45g, glucose 2.5g, citric acid 1.25g, adenine 0.5g, NaCl 11.6g, are dissolved in 900ml
After purified water, it is settled to 1L, 0.22 μm of film filters, and in filling to sterile reagent bottle, thus closed reagent bottle is made cell and saves
Liquid 1L, as needed in packing to the bottle of suitable capacity.After tested, it at least stable can save 2 years, fit under 2~8 DEG C of environment
Close long term storage.
The preparation of the non-real blank pipe of embodiment 3 dress cell-preservation liquid 3
Example 1 prepare cell-preservation liquid, it is filling into plastic tube, every pipe filling amount be 250 μ l, closed pipe close,
Thus cell-preservation liquid 3 is made, after tested, at least stable can be saved 2 years under 2~8 DEG C of environment.It can directly be used.
The preparation of 4 vacuum tube of embodiment dress cell-preservation liquid 4
Cell-preservation liquid prepared by Example 1, filling into plastic tube, every pipe filling amount is 250 μ l, is set with vacuumizing
For with 0.29 standard atmospheric pressure vacuumize process and closed pipe close, cell-preservation liquid 4 is thus made, after tested, in 2~8 DEG C
It at least stable can be saved 2 years under environment.It can directly be used.
Application of the cell-preservation liquid of the invention of embodiment 5 in streaming Sample preservation
The plastic tube for the cell-preservation liquid being prepared according to embodiment 4 is taken, every pipe is directly added into an example Normal human peripheral
Blood 2mL, 2~8 DEG C of placements save, and carry out cell on the 7th and save test, it is thin to carry out streaming for the 3rd, 5, the 7 day period sample deposited of going bail for
The detection of born of the same parents' instrument, including being detected according to the method for the Chinese patent application application No. is 20161002721.4, as a result well
(can effectively keep cytotostatic one month).
The plastic tube for the cell-preservation liquid being prepared according to embodiment 4 is taken, every pipe is directly added into an example Normal human peripheral
Blood 2mL, 2~8 DEG C of placements save, and carry out Cell viability test on the 7th as follows.50 μ l blood samples are added into streaming pipe
This, is added 2ml hemolysin and mixes, be incubated at room temperature 10 minutes.With 1300rpm centrifugation 5 minutes, liquid is discarded supernatant, 500 μ l are added
PBS is resuspended, and repeated washing is primary, finally with 500 μ l PBS resuspension.It takes 90 μ l that cell liquid is resuspended, 10 μ l, 0.4wt% platforms is added
Expect blue dyeing liquor, mix, dye 3 minutes, that is, be carved into microscopically observation, calculates cell (living cells) the Zhan Suoyou cell that is unstained
The percentage of sum, as a result as shown in table 1 below.
Table 1
From table 1 it follows that being not added with compared to the Cell viability of fresh sample and saving the Cell viability of liquid after a week
Up to 43.8%, addition saves the Cell viability of liquid after a week and does not find to be decreased obviously for decline, after this illustrates that addition saves liquid
Cell death can be slowed down, to keep stable.
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, fall within the scope of the claimed invention without departing from theon the basis of the spirit of the present invention.
Claims (14)
1. a kind of cell-preservation liquid, which is characterized in that contain following components: Na2HPO4, glucose, citric acid, adenine, NaCl
And water.
2. cell-preservation liquid as described in claim 1, which is characterized in that the water is pure water.
3. cell-preservation liquid as claimed in claim 2, which is characterized in that the cell-preservation liquid contains the group of following final concentration
Point:
Na2HPO40.1-10g/L；
Glucose 0.1-50g/L；
Citric acid 0.1-10g/L；
Adenine 0.1-10g/L；
NaCl 0.01-500mmol/L；
The solvent of above-mentioned each component is water.
4. a kind of preparation method of cell-preservation liquid as claimed in claim 3, which comprises the steps of: will be formulated
The Na of amount2HPO4, glucose, citric acid, adenine, NaCl be uniformly mixed with water to get.
5. preparation method as claimed in claim 4, which is characterized in that the preparation method further includes being filtered after mixing
And/or the step of packing.
6. preparation method as claimed in claim 5, which is characterized in that the volume of the packing is 100~300 μ l/ pipe.
7. a kind of application of cell-preservation liquid as claimed in claim 3 in flow cytomery.
8. the use as claimed in claim 7, which is characterized in that it is described application the following steps are included:
1) sample liquid containing cell is provided；
2) above-mentioned cell-preservation liquid is mixed with the sample liquid, wherein the volume ratio of the cell-preservation liquid and the sample liquid
For 1:5~1:10；
3) mixture for obtaining step 2) saves；
4) mixture that step 3) obtains is taken out into 50-100 μ l and mixes addition with the 20 μ l of streaming monoclonal antibody of fluorescent marker
Streaming pipe is protected from light incubation 15 minutes after mixing；
5) hemolysin 2ml is added in the streaming pipe being incubated for step 4), is incubated at room temperature 10 minutes, 200-300g after mixing, centrifugation 5
Minute, topple over and discards supernatant and sample is resuspended with 200 μ lPBS；
6) the streaming pipe that flow cytomery step 5) is resuspended, and calculated result；
7) it takes out after a week and carries out retest.
9. application as claimed in claim 8, which is characterized in that sample liquid described in step 1) is that blood, marrow or cell are outstanding
Supernatant liquid；Streaming monoclonal antibody described in step 4) includes CD45-FITC, CD13-PE, CD33-PerCP-cy5.5 of each 5 μ l
And CD7-APC.
10. a kind of preservative agent containing cell-preservation liquid as claimed in claim 3.
11. preservative agent as claimed in claim 10, which is characterized in that the preservative agent also includes sample liquid, and the cell is protected
The volume ratio of liquid storage and the sample liquid is 1:5~1:10.
12. preservative agent as claimed in claim 10, which is characterized in that the sample liquid is blood, marrow or cell suspending liquid.
13. a kind of described in any item preservative agents of claim 10-12 are being used for flow cytomery or in preparation for flowing
Application in the kit of formula cell instrument detection.
14. application as claimed in claim 13, which is characterized in that the flow cytomery be Swine lymphocyte antigen and
The detection of antigen intracellular.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
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| CN201910214657.0A CN109892320A (en) | 2019-03-20 | 2019-03-20 | A kind of cell-preservation liquid and its preparation method and application |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201910214657.0A CN109892320A (en) | 2019-03-20 | 2019-03-20 | A kind of cell-preservation liquid and its preparation method and application |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112056309A (en) * | 2020-11-16 | 2020-12-11 | 广州杜德生物科技有限公司 | Cell preservation solution and application thereof in preservation of cord blood NK cells |
| CN113180036A (en) * | 2021-05-12 | 2021-07-30 | 深圳天烁生物科技有限公司 | Cell preservation solution, cell preservation tube, and preservation method |
| CN114762497A (en) * | 2021-01-11 | 2022-07-19 | 京东方再生医学科技有限公司 | Cell cryopreservation solution and cell cryopreservation method |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112056309A (en) * | 2020-11-16 | 2020-12-11 | 广州杜德生物科技有限公司 | Cell preservation solution and application thereof in preservation of cord blood NK cells |
| CN114762497A (en) * | 2021-01-11 | 2022-07-19 | 京东方再生医学科技有限公司 | Cell cryopreservation solution and cell cryopreservation method |
| CN114762497B (en) * | 2021-01-11 | 2023-08-11 | 京东方再生医学科技有限公司 | Cell cryopreservation liquid and cell cryopreservation method |
| CN113180036A (en) * | 2021-05-12 | 2021-07-30 | 深圳天烁生物科技有限公司 | Cell preservation solution, cell preservation tube, and preservation method |
| CN113180036B (en) * | 2021-05-12 | 2022-02-25 | 深圳天烁生物科技有限公司 | Cell preservation solution, cell preservation tube, and preservation method |
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