CN108517311A - For detaching and the separating liquid of enrichment of cell, kit and its application - Google Patents
For detaching and the separating liquid of enrichment of cell, kit and its application Download PDFInfo
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- CN108517311A CN108517311A CN201810359903.7A CN201810359903A CN108517311A CN 108517311 A CN108517311 A CN 108517311A CN 201810359903 A CN201810359903 A CN 201810359903A CN 108517311 A CN108517311 A CN 108517311A
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- separating liquid
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
- C12N2500/34—Sugars
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
Abstract
It is a kind of for detach with the separating liquid of enrichment of cell, kit and its application, separating liquid includes separating medium, viscosity modifier, preservative and ultra-pure water；Kit includes separating liquid；Separation method using separating liquid includes following process：S1:Centrifuge tube is added in a certain amount of separating liquid；S2:Sample to be separated is slowly added to the separating liquid upper layer in S1 steps；Two steps of S3 centrifuge, and upper layer sample is abandoned in centrifugation for the first time, and separating liquid is abandoned in second of centrifugation, and the cell precipitation of last tube bottom is the relatively single cell being enriched with.Operability of the present invention is strong, stability is good, reliability is high, is suitable for separation, the purification of the large-scale pathological cells sample of hospital, has higher market popularization value.
Description
Technical field
The present invention relates to a kind of for detaching and the separating liquid of enrichment of cell, kit and its application.
Background technology
Since late 1990s, liquid-based thin layer tabletting technology gradually instead of traditional manual smear and scraping blade,
Liquid-based method of drawing material is more scientific, easy, and production effect is also significantly better than Conventional slide under mirror, integrally improves sectioning cells effect
Fruit, and then so that cytodiagnosis positive rate and accuracy is had and greatly improve.However because of inflammatory cell, mucus, blood cell fragment
Influence and reasons for its use it is dirty and messy become its blemish in an otherwise perfect thing；It seriously affects slice-making quality, interfering with pathological doctor diagnosed.We
Trial removes the ingredient of no diagnostic significance with cell separating liquid and gradient separations method, is not only advantageous under the microscope, and
It is more advantageous to diagnosis.
Meanwhile there is complicated for operation, feed rate is slow, is unfavorable for scale, sample-adding for existing cell separating liquid
The shortcomings of technology difficulty is grasped, operator easily contradicts greatly affected follow-up popularization and application work.When detaching cell, pole is needed
Cell sample is slowly added in it, to prevent the destruction of cell separating liquid liquid level, if cell suspension is mixed with separating liquid,
Cell separating effect will have a greatly reduced quality.This process has seriously constrained the progress of mass cell separation, becomes processing sample
Bottleneck.
Invention content
The technical problem to be solved in the present invention is to provide it is a kind of for detach with the separating liquid of enrichment of cell, kit and its
Using preventing the diagnosis of the impurity interfering with pathological doctor such as excessive inflammatory cell, cell fragment, overcome in real work and often meet
It is excessive to inflammatory cell, background do not lead to not totally diagnosis the shortcomings that.
In order to solve the above-mentioned technical problem, the technical scheme is that：It is a kind of to be used to detach and the separation of enrichment of cell
Liquid, which is characterized in that including the separating liquid includes separating medium, viscosity modifier, preservative and ultra-pure water.
Further, each component quality accounting in the separating liquid：Separating medium 1%-10%, viscosity modifier 1%-
10%, preservative 0.1%-5%, ultra-pure water supplies required grammes per square metre.
Further, which is characterized in that the separating medium uses sucrose, ficoll or Percoll (silica colloidal state
Suspension).
Further, which is characterized in that the viscosity modifier uses cardiografin, Sodium Amidotrizoate and carboxymethyl cellulose
Two kinds of combinations in plain sodium.
Further, in the viscosity modifier two kinds combination proportionings 1：5-7.
Further, which is characterized in that the preservative uses phenol, benzoic acid, potassium sorbate or Metagin
Ester.
It is a kind of to be used to detach and the kit of enrichment of cell, which is characterized in that the kit contains above-mentioned separating liquid.
A method of separation and enrichment of cell, which is characterized in that used in the method above-mentioned separating liquid or
Using above-mentioned kit.
Further, a method of separation and enrichment of cell, which is characterized in that include the following steps：
S1：Centrifuge tube is added in separating liquid；
S2：Sample to be separated is slowly added to the separating liquid upper layer in S1；
S3：It centrifuges for the first time, is inhaled after centrifugation and abandon upper layer sample；It is centrifuged into second, remaining separating liquid is abandoned after centrifugation,
The cell precipitation of tube bottom is the single cell being enriched with；
S4：Cell precipitation in S3 passes through liquid based cytology film-making.
Further, the liquid based cytology film-making in the S4 is using sedimentation method of tableting or drop piece method.
Further, the sedimentation method of tableting includes the following steps：
S11:Cell precipitation in S3 is diluted with buffer solution, with oscillator mixing cell mass；
S12:Cell diluent is transferred on sedimentation pelleter, automatic film-making, dyeing.
Further, the drop piece method includes the following steps：
S21：Cell precipitation in S3 is diluted with buffer solution, with oscillator mixing cell mass；
S22：S21 treated cell diluents places 1-2mm right over slide are inhaled with dropper, two drop cell liquid of drop are made
At a uniform cell thin slice；
S23：After drying, 15min is fixed with 95% ethyl alcohol, is dyed.
Further, the condition centrifuged twice in the S3 is：It centrifuges for the first time：100-600g, 1-10min, the second
The condition of secondary centrifugation is：200-2000g, 1-20min.
The present invention has the following advantages：
1, have feed rate fast, the advantages of separating liquid liquid level is not easy to be destroyed greatly shortens sample-adding and cell separation
Time, easy operating process detaches especially suitable for beginner and large-scale cell；
2, removal inflammatory cell, mucus, cell fragment effect are good, and film-making clean background is conducive to cell pathology doctor's
Diagnosis；
3, phenomena such as a variety of liquid-based tabletting technologies can be compatible with, be not in cell detachment；
4, the problems such as cell can be protected, cell is made not deformed in separating liquid, distort, be swollen；
5, separating effect is stable, reliable, can be enriched with the aim cell for having diagnostic significance of high concentration.
Description of the drawings
Specific embodiments of the present invention will be described in further detail below in conjunction with the accompanying drawings.
Fig. 1 is the schematic diagram that cell separating liquid of the present invention detaches cervical epithelial cells, is enriched with；
Fig. 2 to Fig. 7 is to carry out liquid-based film-making using the cell of the separating liquid separation after different component proportioning in the present invention
Effect diagram.
Specific implementation mode
With reference to the accompanying drawings and detailed description, the present invention will be further described.
It is a kind of for detach with the separating liquid of enrichment of cell, including, the separating liquid includes separating medium, viscosity-adjusting agent
Agent, preservative and ultra-pure water.Wherein, each component quality accounting in separating liquid：Separating medium 1%-10%, viscosity modifier 1%-
10%, preservative 0.1%-5%, ultra-pure water supplies required grammes per square metre.In separating liquid, separating medium using sucrose, ficoll or
Percoll (silica colloidal suspension liquid), viscosity modifier use two in cardiografin, Sodium Amidotrizoate and sodium carboxymethylcellulose
Kind combination, and in viscosity modifier two kinds combination proportionings 1：5-7；Preservative using phenol, benzoic acid, potassium sorbate or
Person's methyl hydroxybenzoate.
It is a kind of to contain above-mentioned separating liquid with the kit of enrichment of cell, the kit for detaching, for cell point
From.
A method of separation and enrichment of cell using above-mentioned separating liquid or use above-mentioned kit.
Specifically, a kind of method of separation and enrichment of cell, includes the following steps：
S1：Centrifuge tube is added in separating liquid；
S2：Sample to be separated is slowly added to the separating liquid upper layer in S1；
S3：It centrifuges for the first time, is inhaled after centrifugation and abandon upper layer sample；It is centrifuged into second, remaining separating liquid is abandoned after centrifugation,
The cell precipitation of tube bottom is the single cell being enriched with；
S4：Cell precipitation in S3 passes through liquid based cytology film-making.
As shown in Figure 1, being divided into three layers after detaching twice, in centrifuge tube, top layer is sample raffinate, and centre is inflammatory
The ingredients such as cell, mucus, cell fragment, bottom are that separating liquid includes the aim cell containing enrichment.
In the above method：The condition centrifuged twice in S3 is：It centrifuges for the first time：100-600g, 1-10min, second from
The condition of the heart is：200-2000g, 1-20min.Liquid based cytology film-making in S4 is using sedimentation method of tableting or drop piece method.
The sedimentation method of tableting includes the following steps：
S11:Cell precipitation in S3 is diluted with buffer solution, with oscillator mixing cell mass；
S12:Cell diluent is transferred on sedimentation pelleter, automatic film-making, dyeing.
The drop piece method includes the following steps：
S21：Cell precipitation in S3 is diluted with buffer solution, with oscillator mixing cell mass；
S22：S21 treated cell diluents places 1-2mm right over slide are inhaled with dropper, two drop cell liquid of drop are made
At a uniform cell thin slice；
S23：After drying, 15min is fixed with 95% ethyl alcohol, is dyed.
According to above-mentioned separating liquid and separation method, multiple embodiment situations are carried out according to different proportionings, to aid in illustrating.
Separating liquid embodiment one：
| Component | Weight |
| Ficoll | 5.18g |
| Cardiografin | 6.00g |
| Sodium Amidotrizoate | 1.00g |
| Phenol | 0.40g |
| Ultra-pure water | Supply 100g |
100g separating liquids are configured to according to said components, upper separating medium is dissolved in ultra-pure water, viscosity-adjusting agent is added
Agent, preservative stir and evenly mix, and ultra-pure water supplies 100g.
Specific separation process：
1) centrifuge tube is added in the separating liquid of 4ml；
2) sample 8ml to be separated is slowly added to the separating liquid upper layer in 1) step；
3) two steps centrifuge, and are carefully inhaled after first time 200g, 2min centrifugation and abandon upper layer sample, second of 800g, 5min centrifugation
Remaining 4ml separating liquids are abandoned afterwards, and the cell precipitation of last tube bottom is the relatively single cell being enriched with；
4) cell precipitation is diluted to proper ratio with buffer solution, with oscillator mixing cell mass；
5) cell diluent is transferred on commercially available sedimentation pelleter, automatic film-making, pap staining；
Film-making dyeing, effect corresponding diagram 2 after the dyeing of embodiment one are completed according to above-mentioned steps.Separating liquid embodiment two：
100g separating liquids are configured to according to said components, upper separating medium is dissolved in ultra-pure water, viscosity-adjusting agent is added
Agent, preservative stir and evenly mix, and ultra-pure water supplies 100g.
Specific separation process：
1) centrifuge tube is added in the separating liquid of 4ml；
2) sample 8ml to be separated is slowly added to the separating liquid upper layer in 1) step；
3) two steps centrifuge, and are carefully inhaled after first time 200g, 2min centrifugation and abandon upper layer sample, second of 800g, 5min centrifugation
Remaining 4ml separating liquids are abandoned afterwards, and the cell precipitation of last tube bottom is the relatively single cell being enriched with；
4) cell precipitation diluted is to proper ratio, with oscillator mixing cell mass；
5) it and then inhales the cell liquid of the 0.5ml 1-2mm right over slide with the dropper of 1ml to locate, drop two drips cell liquid, is made
The cell sheet of one homogeneous diameter about 2cm；
6) after drying, 15min, pap staining are fixed with 95% ethyl alcohol.
Film-making dyeing, effect corresponding diagram 3 after the dyeing of embodiment two are completed according to above-mentioned steps.
Separating liquid embodiment three：
100g separating liquids are configured to according to said components, upper separating medium is dissolved in ultra-pure water, viscosity-adjusting agent is added
Agent, preservative stir and evenly mix, and ultra-pure water supplies 100g.
Specific separation process：
1) centrifuge tube is added in the separating liquid of 4ml；
2) sample 8ml to be separated is slowly added to the separating liquid upper layer in 1) step；
3) two steps centrifuge, and are carefully inhaled after first time 200g, 2min centrifugation and abandon upper layer sample, second of 500g, 5min centrifugation
Remaining 4ml separating liquids are abandoned afterwards, and the cell precipitation of last tube bottom is the relatively single cell being enriched with；
4) cell precipitation diluted is to proper ratio, with oscillator mixing cell mass；
5) it and then inhales the cell liquid of the 0.5ml 1-2mm right over slide with the dropper of 1ml to locate, drop two drips cell liquid, is made
The cell sheet of one homogeneous diameter about 2cm；
6) after drying, 15min, pap staining are fixed with 95% ethyl alcohol.
Film-making dyeing, effect corresponding diagram 4 after the dyeing of embodiment two are completed according to above-mentioned steps.
Separating liquid example IV：
| Component | Weight |
| Ficoll | 5.5g |
| Cardiografin | 5.00g |
| Sodium carboxymethylcellulose | 1.00g |
| Phenol | 0.10g |
| Potassium sorbate | 0.2g |
| Ultra-pure water | Supply 100g |
100g separating liquids are configured to according to said components, upper separating medium is dissolved in ultra-pure water, viscosity-adjusting agent is added
Agent, preservative stir and evenly mix, and ultra-pure water supplies 100g.
Specific separation process：
1) centrifuge tube is added in the separating liquid of 4ml；
2) sample 8ml to be separated is slowly added to the separating liquid upper layer in 1) step；
3) two steps centrifuge, and are carefully inhaled after first time 200g, 2min centrifugation and abandon upper layer sample, second of 500g, 5min centrifugation
Remaining 4ml separating liquids are abandoned afterwards, and the cell precipitation of last tube bottom is the relatively single cell being enriched with；
4) cell precipitation is diluted to proper ratio with buffer solution, with oscillator mixing cell mass；
5) cell diluent is transferred on commercially available sedimentation pelleter, automatic film-making, pap staining；It is complete according to above-mentioned steps
It is dyed at film-making, effect corresponding diagram 5 after the dyeing of embodiment two.
Separating liquid embodiment five：
| Component | Weight |
| Percoll | 4.05g |
| Cardiografin | 7.00g |
| Sodium Amidotrizoate | 1.00g |
| Benzoic acid | 1.15g |
| Ultra-pure water | Supply 100g |
100g separating liquids are configured to according to said components, upper separating medium is dissolved in ultra-pure water, viscosity-adjusting agent is added
Agent, preservative stir and evenly mix, and ultra-pure water supplies 100g.
Specific separation process
1) centrifuge tube is added in the separating liquid of 4ml；
2) sample 8ml to be separated is slowly added to the separating liquid upper layer in 1) step；
3) two steps centrifuge, and are carefully inhaled after first time 200g, 2min centrifugation and abandon upper layer sample, second of 500g, 5min centrifugation
Remaining 4ml separating liquids are abandoned afterwards, and the cell precipitation of last tube bottom is the relatively single cell being enriched with；
4) cell precipitation is diluted to proper ratio with buffer solution, with oscillator mixing cell mass；
5) cell diluent is transferred on commercially available sedimentation pelleter, automatic film-making, pap staining；It is complete according to above-mentioned steps
It is dyed at film-making, effect corresponding diagram 6 after the dyeing of embodiment two.
Separating liquid embodiment six：
| Component | Weight |
| Ficoll | 5.75g |
| Cardiografin | 6.00g |
| Sodium Amidotrizoate | 1.00g |
| Methyl hydroxybenzoate | 0.20g |
| Ultra-pure water | Supply 100g |
100g separating liquids are configured to according to said components, upper separating medium is dissolved in ultra-pure water, viscosity-adjusting agent is added
Agent, preservative stir and evenly mix, and ultra-pure water supplies 100g.
Specific separation process：
1) centrifuge tube is added in the separating liquid of 4ml；
2) sample 8ml to be separated is slowly added to the separating liquid upper layer in 1) step；
3) two steps centrifuge, and are carefully inhaled after first time 200g, 2min centrifugation and abandon upper layer sample, second of 500g, 5min centrifugation
Remaining 4ml separating liquids are abandoned afterwards, and the cell precipitation of last tube bottom is the relatively single cell being enriched with；
4) cell precipitation diluted is to proper ratio, with oscillator mixing cell mass；
5) it and then inhales the cell liquid of the 0.5ml 1-2mm right over slide with the dropper of 1ml to locate, drop two drips cell liquid, is made
The cell sheet of one homogeneous diameter about 2cm；
6) after drying, 15min, pap staining are fixed with 95% ethyl alcohol.
Film-making dyeing, effect corresponding diagram 7 after the dyeing of embodiment six are completed according to above-mentioned steps.
Above-described embodiment and its design sketch can see：Greatly shorten the time of sample-adding and cell separation, easy operation stream
Journey；Separating liquid liquid level is not easy to be destroyed；It is good to remove inflammatory cell, mucus, cell fragment effect, film-making clean background；It can be compatible
Phenomena such as a variety of liquid-based tabletting technologies are not in cell detachment；Cell can be protected, cell is made not become in separating liquid
The problems such as shape, distortion, swelling；Separating effect is stable, reliable, can be enriched with the aim cell for having diagnostic significance of high concentration.
Although specifically showing and describing the present invention in conjunction with preferred embodiment, those skilled in the art should be bright
In vain, it is not departing from the spirit and scope of the present invention defined by the appended claims, in the form and details to this hair
It is bright to make a variety of changes, it is protection scope of the present invention.
Claims (13)
1. a kind of for detaching and the separating liquid of enrichment of cell, which is characterized in that including the separating liquid includes that separation is situated between
Matter, viscosity modifier, preservative and ultra-pure water.
2. according to claim 1 a kind of for detaching and the separating liquid of enrichment of cell, which is characterized in that the separation
Each component quality accounting in liquid：Separating medium 1%-10%, viscosity modifier 1%-10%, preservative 0.1%-5%, ultra-pure water supply institute
Need grammes per square metre.
3. according to claim 1 or 2 a kind of for detaching and the separating liquid of enrichment of cell, which is characterized in that described
Separating medium uses sucrose, ficoll or Percoll（Silica colloidal suspension liquid）.
4. according to claim 1 or 2 a kind of for detaching and the separating liquid of enrichment of cell, which is characterized in that described
Viscosity modifier uses two kinds of combinations in cardiografin, Sodium Amidotrizoate and sodium carboxymethylcellulose.
5. according to claim 4 a kind of for detaching and the separating liquid of enrichment of cell, which is characterized in that described is described
Viscosity modifier in two kinds combination proportionings 1：5-7.
6. according to claim 1 or 2 a kind of for detaching and the separating liquid of enrichment of cell, which is characterized in that described
The preservative uses phenol, benzoic acid, potassium sorbate or methyl hydroxybenzoate.
7. a kind of for detaching and the kit of enrichment of cell, which is characterized in that the kit contains in claim 1-6
Any separating liquid.
8. a kind of method of separation and enrichment of cell, which is characterized in that using described in claim 1-6 in the method
Kit described in separating liquid or use claim 7.
9. the method for a kind of separation and enrichment of cell according to claim 8, which is characterized in that include the following steps：
S1：Centrifuge tube is added in separating liquid；
S2：Sample to be separated is slowly added to the separating liquid upper layer in S1；
S3：It centrifuges for the first time, is inhaled after centrifugation and abandon upper layer sample；It is centrifuged into second, remaining separating liquid, tube bottom is abandoned after centrifugation
Cell precipitation be enriched with single cell；
S4：Cell precipitation in S3 passes through liquid based cytology film-making.
10. the method for a kind of separation and enrichment of cell according to claim 9, which is characterized in that the liquid in the S4
Basal cell's length of schooling piece is using sedimentation method of tableting or drop piece method.
11. the method for a kind of separation and enrichment of cell according to claim 10, which is characterized in that the sedimentation film-making
Method includes the following steps：
S11:Cell precipitation in S3 is diluted with buffer solution, with oscillator mixing cell mass;
S12:Cell diluent is transferred on sedimentation pelleter, automatic film-making, dyeing.
12. the method for a kind of separation and enrichment of cell according to claim 10, which is characterized in that the drop piece method packet
Include following steps：
S21：Cell precipitation in S3 is diluted with buffer solution, with oscillator mixing cell mass;
S22：S21 treated cell diluents places 1-2mm right over slide are inhaled with dropper, drop two drips cell liquid, is made one
Open uniform cell thin slice；
S23：After drying, 15min is fixed with 95% ethyl alcohol, is dyed.
13. the method for a kind of separation and enrichment of cell according to claim 9, which is characterized in that in the S3 twice
The condition of centrifugation is：It centrifuges for the first time：100-600g, 1-10min, the condition centrifuged for the second time are：200-2000g, 1-
20min。
Priority Applications (1)
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| CN201810359903.7A CN108517311A (en) | 2018-04-20 | 2018-04-20 | For detaching and the separating liquid of enrichment of cell, kit and its application |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810359903.7A CN108517311A (en) | 2018-04-20 | 2018-04-20 | For detaching and the separating liquid of enrichment of cell, kit and its application |
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Cited By (1)
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| CN114403130A (en) * | 2022-01-20 | 2022-04-29 | 灵知蓝诺(北京)生物技术有限公司 | Liquid-based cell preservation solution and preparation method and application thereof |
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| CN114403130A (en) * | 2022-01-20 | 2022-04-29 | 灵知蓝诺(北京)生物技术有限公司 | Liquid-based cell preservation solution and preparation method and application thereof |
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Application publication date: 20180911 |