CN112080430A - Method for processing cell sample in single cell sorting process - Google Patents
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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Abstract
The invention provides a method for processing a cell sample in a single cell sorting process, which comprises the following steps in sequence: the method comprises the following steps: obtaining a desired cell sample; step two: paving the cell sample and a water retention substance for maintaining the cell activity on the surface of the sorting chip; step three: placing the chip on a sorting platform to sort target cells; step four: placing the sorted cells in a receiving substance in a receiving device; step five: and carrying out subsequent detection or culture operation on the sorted cells. The invention uses the substance with water retention function, can keep the original biological state and biological activity of the cells in the sorting process, and does not influence the detection and the expanded culture of the activity of the cells after sorting.
Description
Technical Field
The invention relates to a cell sample processing method, in particular to a cell sample processing method in a single cell sorting process.
Background
Cells are basic units of life, and because of phenotypic heterogeneity, research on the level of single cells is of great significance for analyzing deep operation mechanisms of life activities.
At present, only a few microorganisms in nature can be separated and cultured, and the research on the life activity rule of the microorganisms and the development of microorganism resources are seriously hindered.
To facilitate the study of microbial diversity, it is necessary to isolate and culture a small number of recalcitrant microorganisms from a complex sample. However, the existing environmental microorganisms are various in types and large in total number, and have different requirements on energy, nutrition and physicochemical conditions aiming at different microorganisms, the existing dilution coating plate method and plate marking method are difficult to separate and obtain the microorganisms which are difficult to culture, and the growth of a plurality of micro flora is limited in a complicated mixed sample due to the growth inhibition of dominant flora. At present, the microorganism can be separated in situ by applying a cell ejection technology and a microdissection technology, and a single cell can be obtained without using a conventional culture medium for coating and streaking and through growth, propagation and screening.
The single cell sorting method based on cell ejection technology and microdissection technology mainly aims at the combination of sorting, subsequent amplification and sequencing of microorganisms, and the main sample is an air-dried sample which has low activity or no biological activity when placed on a chip.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention provides a method for processing a cell sample in a single cell sorting process, which can maintain the cell activity of the cell sample in the single cell sorting process and effectively improve the sorting effect of a target single cell.
The technical scheme adopted by the invention for solving the technical problems is as follows: a method for processing a cell sample in a single cell sorting process comprises the following steps in sequence:
the method comprises the following steps: obtaining a desired cell sample;
step two: paving the cell sample and hydrogel water-retaining substances for keeping cell activity on the surface of the sorting chip;
step three: placing the chip on a sorting platform to sort target cells;
step four: placing the sorted cells in a receiving substance in a receiving device;
step five: and carrying out subsequent detection or culture operation on the sorted cells.
In a further preferred embodiment, the hydrogel water-retaining substance is a gel solution formed by uniformly mixing a collagen solution, a polyethylene glycol solution and an alginate solution.
As a further preferred embodiment, the collagen solution has a mass concentration of 0.5% to 10%; the volume concentration of the polyethylene glycol solution is 0.1-10%; the volume concentration of the alginate solution is 0.1-10%.
As a further preferred embodiment, the volume ratio of the collagen solution, the polyethylene glycol solution and the alginate solution in the gel solution is 1:1: 1.
As a further preferred embodiment, the kind of the cell sample in the first step is mammalian cell, plant cell or microbial cell.
As a further preferred embodiment, the specific operation of step two is: the hydrogel water-retaining substance is paved on the surface of the sorting chip to form a water-retaining thin layer with the thickness of 1-100 mu m, and then the cell sample is coated on the surface of the water-retaining thin layer.
As a further preferred embodiment, the method for sorting the target cells in step three is catapult sorting or microdissection.
As a further preferred embodiment, the receiving device in step four is a culture dish or a well plate.
As a further preferred embodiment, the receiving substance in step four is any one of a culture medium, water, PBS, and physiological saline.
The invention has the following positive effects: the invention uses the substance with water-retaining function, spreads the substance with water-retaining function on the chip, coats the cell sample on the chip to keep the cell in active state all the time, and then uses the single cell sorting method such as ejection sorting or laser microdissection to sort out the target single cell. The substance with the water retention function has the functions of keeping the water content of the cell sample (collagen is used as a framework, and the polyethylene glycol and alginate increase the moisture retention and viscosity) and delaying or preventing the water volatilization, so that the cell sample is wrapped in the gel droplets, and the sample can be stored on the sorting chip for a long time at the moment, and the cell activity is maintained, and the cell cannot be killed due to the fact that the cell is separated from the original inherent nutritional environment. The water-retaining substance is nontoxic and nondestructive to cell samples, can keep the original biological state and biological activity of cells in the sorting process, and does not influence the detection and the expanded culture of the activity of the cells after sorting.
Drawings
FIG. 1 is a diagram showing a state of a gel droplet of a hydrogel water-retaining substance described in example 1;
FIG. 2 is a diagram showing a state where a gel droplet is coated with a single bacterium according to example 1;
FIG. 3 is a graph of cell staining for assessing cell viability when cells are treated with a non-water-retaining substance;
FIG. 4 is a graph showing cell staining evaluation of cell viability when cells are treated with the hydrogel water-retaining substance described in example 1;
FIG. 5 is the state of the bacteria cultured after sorting on the hydrogel coating in example 1.
Detailed Description
Preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings.
Example 1
The preferred embodiment 1 of the present invention provides a method for processing a cell sample in a single cell sorting process, wherein the cell sample is a microbial cell sample, and the method specifically comprises the following steps performed in sequence:
step one, obtaining a required cell sample, and specifically operating as follows:
and (3) soil sample culture: obtaining a soil sample from the environment, and then placing the soil sample in a culture medium for culture, wherein the culture time is about 10 days;
sample preparation: collecting 1ml of the cultured soil sample, and filtering the soil sample through a filter membrane of 40 mu m to remove impurities in the soil. ② centrifuging for 5min at the rotating speed of 5000rpm to obtain the fine bacteria precipitation. ③ adding 1ml of sterilized ultrapure water into the bacterial sediment, washing for 5 times, centrifuging for 5min at the rotating speed of 5000rpm after each washing, and collecting the bacterial sediment. And fourthly, adding 1ml of ultrapure water, fully shaking and uniformly mixing to obtain a microbial cell sample for later use.
Step two: the cell sample and the hydrogel water-retaining substance for keeping the cell activity are paved on the surface of the sorting chip together, and the specific operation is as follows: the hydrogel water-retaining substance was spin-coated on the sorting chip to form a 50 μm-thick water-retaining thin layer, and then 1.2 μ l of the microbial cell sample was pipetted and dropped on the surface of the water-retaining thin layer, as shown in FIG. 2, and the state of bacteria was observed to be good.
The hydrogel water-retaining substance is a gel solution formed by uniformly mixing a collagen solution, a polyethylene glycol solution and an alginate solution, and the preparation method comprises the following specific steps:
(1) preparing a collagen solution with the mass concentration of 2.5%;
(2) preparing a polyethylene glycol solution with the volume concentration of 2%;
(3) preparing an alginate solution with the volume concentration of 1%;
(4) the collagen solution, the polyethylene glycol solution and the alginate solution are weighed and uniformly mixed according to the volume ratio of 1:1:1 to form a gel solution, and the state of gel liquid drops is shown in figure 1.
The activity of cells treated with the water-retaining substance and the non-water-retaining substance was maintained normal (viable cells were not stained as shown in FIG. 4) as shown in FIGS. 3 and 4, for example, in the case where cells not treated with the water-retaining substance were inactivated (dead cells were stained as shown in FIG. 3) and in the case where cells treated with the water-retaining substance of this example were not stained as shown in FIG. 4
Step three: placing the chip on a sorting platform for sorting target cells, which specifically comprises the following steps: determining target microbial cells according to the shape and size of bacteria, and then sorting the target microbial cells;
step four: receiving the sorted cells in a receiving substance in a receiving device;
step five: the sorted cells are placed in an incubator for subsequent culture operations, as shown in fig. 5, and can be cultured into clonal colonies.
Example 2
The preferred embodiment 2 of the present invention provides a method for processing a cell sample in a single cell sorting process, wherein the cell sample is a bacillus subtilis cell sample, and the method specifically comprises the following steps performed in sequence:
step one, obtaining a required cell sample, and specifically operating as follows:
sample culture: the sample to be separated is inoculated in LB culture medium and shake cultured overnight at 37 ℃.
Sample treatment: collecting 1ml of overnight cultured bacterial liquid in a 1.5ml centrifuge tube, and centrifuging for 5min at the rotating speed of 5000rpm to obtain bacterial sediment. ② washing 4 times by adding 1ml of sterilized ultrapure water, centrifuging for 5min at 5000rpm after each washing, and collecting bacterial precipitation. ③ adding 1ml of ultrapure water, fully shaking and mixing uniformly to obtain a cell sample for later use.
Step two: the cell sample and a water retention substance for maintaining the cell activity are paved on the surface of the sorting chip together, and the specific operation is as follows: the hydrogel water-retaining substance is spin-coated on the sorting chip to form a water-retaining thin layer with the thickness of 30 mu m, then 1.0 mu l of microbial cell sample is sucked and dripped on the surface of the water-retaining thin layer, and the good bacterial state is observed.
The hydrogel water-retaining substance is a gel solution formed by uniformly mixing a collagen solution, a polyethylene glycol solution and an alginate solution, and the preparation method comprises the following specific steps:
(1) preparing a collagen solution with the mass concentration of 1%;
(2) preparing a polyethylene glycol solution with the volume concentration of 0.8%;
(3) preparing an alginate solution with the volume concentration of 3%;
(4) and (3) weighing the collagen solution, the polyethylene glycol solution and the alginate solution, and uniformly mixing according to the volume ratio of 1:1:1 to form a gel solution.
Step three: placing the chip on a sorting platform, and separating the bacillus subtilis by using a single-cell ejection sorter;
step four: placing the sorted cells in a receiving material in a receiving device, wherein the receiving device is a culture dish, and the receiving material is LB solid culture medium;
step five: the sorted cells are placed in an incubator at 37 ℃ for overnight culture, and colony formation is observed, so that clone colonies can be cultured.
Example 3
A preferred embodiment 3 of the present invention provides a method for processing a cell sample in a single cell sorting process, wherein the cell sample is a microbial cell sample, and the method specifically comprises the following steps performed in sequence:
step one, obtaining a required cell sample, and specifically operating as follows:
and (3) soil sample culture: obtaining a soil sample from the environment, and then placing the soil sample in a culture medium for culturing for about 15 days;
sample preparation: collecting 1ml of the cultured soil sample, and filtering the soil sample through a filter membrane of 40 mu m to remove impurities in the soil. ② centrifuging for 5min at the rotating speed of 5000rpm to obtain the fine bacteria precipitation. ③ adding 1ml of sterilized ultrapure water into the bacterial sediment, washing for 3 times, centrifuging for 5min at the rotating speed of 5000rpm after each washing, and collecting the bacterial sediment. And fourthly, adding 1ml of ultrapure water, fully shaking and uniformly mixing to obtain a microbial cell sample for later use.
Step two: the cell sample and the hydrogel water-retaining substance for keeping the cell activity are paved on the surface of the sorting chip together, and the specific operation is as follows: the hydrogel water-retaining substance is spin-coated on the sorting chip to form a water-retaining thin layer with the thickness of 80 mu m, then 1.5 mu l of microbial cell sample is sucked and dripped on the surface of the water-retaining thin layer, and the good bacterial state is observed.
The hydrogel water-retaining substance is a gel solution formed by uniformly mixing a collagen solution, a polyethylene glycol solution and an alginate solution, and the preparation method comprises the following specific steps:
(1) preparing a collagen solution with the mass concentration of 6%;
(2) preparing a polyethylene glycol solution with the volume concentration of 8%;
(3) preparing an alginate solution with the volume concentration of 0.5%;
(4) and (3) weighing the collagen solution, the polyethylene glycol solution and the alginate solution, and uniformly mixing according to the volume ratio of 1:1:1 to form a gel solution.
Step three: placing the chip on a sorting platform for sorting target cells, which specifically comprises the following steps: determining target microbial cells according to the shape and size of bacteria, and then sorting the target microbial cells;
step four: receiving the sorted cells in a receiving substance in a receiving device;
step five: and placing the sorted cells in an incubator for subsequent culture operation, and culturing the cells into clone colonies.
The above embodiments are only preferred embodiments of the present invention, and it should be understood that the above embodiments are only for assisting understanding of the method and the core idea of the present invention, and are not intended to limit the scope of the present invention, and any modifications, equivalents and the like made within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (9)
1. A method for processing a cell sample in a single cell sorting process is characterized by comprising the following steps in sequence:
the method comprises the following steps: obtaining a desired cell sample;
step two: paving the cell sample and hydrogel water-retaining substances for keeping cell activity on the surface of the sorting chip;
step three: placing the chip on a sorting platform to sort target cells;
step four: placing the sorted cells in a receiving substance in a receiving device;
step five: and carrying out subsequent detection or culture operation on the sorted cells.
2. The method of claim 1, wherein the method comprises: the hydrogel water-retaining substance is a gel solution formed by uniformly mixing a collagen solution, a polyethylene glycol solution and an alginate solution.
3. The method of claim 2, wherein the method comprises: the mass concentration of the collagen solution is 0.5-10%; the volume concentration of the polyethylene glycol solution is 0.1-10%; the volume concentration of the alginate solution is 0.1-10%.
4. The method of claim 3, wherein the method comprises: the volume ratio of the collagen solution, the polyethylene glycol solution and the alginate solution in the gel solution is 1:1: 1.
5. The method of claim 4, wherein the method comprises: the cell sample in the first step is animal cell, plant cell or microbial cell.
6. The method of claim 4, wherein the method comprises: the specific operation of the second step is as follows: the hydrogel water-retaining substance is paved on the surface of the sorting chip to form a water-retaining thin layer with the thickness of 1-100 mu m, and then the cell sample is coated on the surface of the water-retaining thin layer.
7. The method of claim 4, wherein the method comprises: the sorting method of the target cells in the third step is catapult sorting or microdissection.
8. The method of claim 4, wherein the method comprises: in the fourth step, the receiving device is a culture dish or a pore plate.
9. The method of claim 4, wherein the method comprises: in the fourth step, the receiving substance is any one of culture medium, water, PBS and physiological saline.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113073029A (en) * | 2021-03-17 | 2021-07-06 | 长春长光辰英生物科学仪器有限公司 | Infiltration modified cell sorting chip for laser induced transfer and sorting method |
| CN113502207A (en) * | 2021-08-18 | 2021-10-15 | 长春长光辰英生物科学仪器有限公司 | Multifunctional cell sorting device based on laser system and operation method |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103353452A (en) * | 2013-07-12 | 2013-10-16 | 北京惟馨雨生物科技有限公司 | Cell carrier chip and single cell rapid identifying or sorting method employing same |
| CN106065391A (en) * | 2016-07-20 | 2016-11-02 | 国家纳米科学中心 | For unicellular sorting and the micro-fluidic chip of unicellular whole genome amplification |
| CN109557068A (en) * | 2017-09-26 | 2019-04-02 | 中国科学院青岛生物能源与过程研究所 | A kind of integrated sorting unit for unicellular Raman Measurement and laser microprobe dating |
| CN109554333A (en) * | 2018-11-20 | 2019-04-02 | 上海药明生物技术有限公司 | A method of unicellular sorting is carried out using the unicellular separator of Namocell |
| CN109765287A (en) * | 2019-01-30 | 2019-05-17 | 长春长光辰英生物科学仪器有限公司 | A kind of microorganism rapid identification method of cell ejection sorting and mass spectrometry |
| CN111676132A (en) * | 2020-06-17 | 2020-09-18 | 长春长光辰英生物科学仪器有限公司 | Chip protective layer for laser-induced transfer and cell sorting method |
-
2020
- 2020-09-22 CN CN202011000900.8A patent/CN112080430A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103353452A (en) * | 2013-07-12 | 2013-10-16 | 北京惟馨雨生物科技有限公司 | Cell carrier chip and single cell rapid identifying or sorting method employing same |
| CN106065391A (en) * | 2016-07-20 | 2016-11-02 | 国家纳米科学中心 | For unicellular sorting and the micro-fluidic chip of unicellular whole genome amplification |
| CN109557068A (en) * | 2017-09-26 | 2019-04-02 | 中国科学院青岛生物能源与过程研究所 | A kind of integrated sorting unit for unicellular Raman Measurement and laser microprobe dating |
| CN109554333A (en) * | 2018-11-20 | 2019-04-02 | 上海药明生物技术有限公司 | A method of unicellular sorting is carried out using the unicellular separator of Namocell |
| CN109765287A (en) * | 2019-01-30 | 2019-05-17 | 长春长光辰英生物科学仪器有限公司 | A kind of microorganism rapid identification method of cell ejection sorting and mass spectrometry |
| CN111676132A (en) * | 2020-06-17 | 2020-09-18 | 长春长光辰英生物科学仪器有限公司 | Chip protective layer for laser-induced transfer and cell sorting method |
Non-Patent Citations (3)
| Title |
|---|
| YI WANG ET AL.: "Raman-activated sorting of antibiotic-resistant bacteria in human gut microbiota", 《ENVIRONMENTAL MICROBIOLOGY》 * |
| 刘程等: "智能水凝胶及其在生物方向的应用", 《中国材料进展》 * |
| 张敏等: "互穿网络聚合物水凝胶的制备及其吸附研究进展", 《化工进展》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113073029A (en) * | 2021-03-17 | 2021-07-06 | 长春长光辰英生物科学仪器有限公司 | Infiltration modified cell sorting chip for laser induced transfer and sorting method |
| CN113073029B (en) * | 2021-03-17 | 2023-03-21 | 长春长光辰英生物科学仪器有限公司 | Infiltration modified cell sorting chip for laser induced transfer and sorting method |
| CN113502207A (en) * | 2021-08-18 | 2021-10-15 | 长春长光辰英生物科学仪器有限公司 | Multifunctional cell sorting device based on laser system and operation method |
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Application publication date: 20201215 |