CN111647549B - Method for separating single cells in animals and plants - Google Patents
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Abstract
The invention belongs to the technical field of biology, and particularly relates to a single cell separation method in animals and plants. Comprising the following steps: adding active liquid and separating enzyme into a biological sample, and soaking to obtain a soaking mixture, wherein the active liquid comprises sodium chloride, potassium chloride, lanthanum compounds and 4-hydroxyethyl piperazine ethane sulfonic acid; filtering the soaked mixture with a filter membrane; soaking and stirring the filter membrane and the sediment above the filter membrane in the active liquid to enable single cells and microorganisms to be resuspended in the active liquid, and collecting composite cell suspension; glass beads were placed into the composite cell suspension, and single cell suspension was collected with shaking. The invention adopts the active liquid to keep the activity of the cells, adopts the separating enzyme to separate jelly in the sample, and improves the yield and purity of the cells.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a single cell separation method in animals and plants.
Background
Biological resources are important resources for studying the origin, variation and development of organisms, whether single-cell organisms or single cells isolated from biological tissues, and have important research values. Typical single-cell biological resources include bacteria, fungi, archaea, etc., which are numerous in number and variety of cells. Functional microorganisms of different species have wide application in the fields of food fermentation, organic matter degradation and the like. Many functional microorganisms exist in animal and plant tissues, and are mixed with other impurities such as proteins, fats and the like, so that the impurities are difficult to separate.
Living matter such as animals and plants is a high-grade living body composed of a plurality of single cells, and can perform complex vital activities. Plant roots, stems and leaves and animal viscera are common samples for single cell isolation. Single cells are separated from animal and plant tissues and cultured, and the method has important significance for researching animal and plant organ functions, genetic mutation and the like. However, a plurality of cells and proteins in animal and plant tissues adhere together, and the viability of single cells is far inferior to that of microorganisms, and it is also difficult to separate impurities.
In summary, the prior art has problems that most single cell sizes are between several micrometers to tens of micrometers, diameters are small, biological cells are difficult to separate from impurities, and cell purity is low.
Disclosure of Invention
In order to solve the technical problems, the invention provides a method for separating single cells in animals and plants.
The invention aims to provide a single cell separation method in animals and plants, which comprises the following steps:
pretreatment of biological samples: adding active liquid and separating enzyme into biological sample, soaking for 1-2h to obtain soaking mixture;
the active liquid is prepared according to the following method: 8-9g of sodium chloride, 0.5-1mg of potassium chloride, 0.01-0.05mg of lanthanum compound, 2.4g of 4-hydroxyethyl piperazine ethane sulfonic acid, and distilled water to a constant volume of 1L and a pH value of 7.2-7.4;
small molecule filtration: filtering the soaked mixture with a filter membrane with a pore size of 0.22-0.45 μm;
large particle filtration: soaking and stirring the filter membrane and the sediment above the filter membrane in the active liquid to enable single cells and microorganisms to be resuspended in the active liquid, and collecting composite cell suspension;
cell separation: and (3) putting glass beads with the diameter of 2-4mm into the compounded cell suspension, oscillating for 20-30min, and collecting single cell suspension.
Preferably, in the method for separating single cells in animals and plants, the biological sample is plant stem and leaf root system, animal epidermis or animal liver.
Preferably, in the method for separating single cells in animals and plants, the lanthanum compound is lanthanum nitrate or lanthanum chloride.
Preferably, the separation method of single cells in animals and plants comprises the step of adding 0.5-1g of pectase and 0.5-1g of collagenase into each liter of active liquid, wherein the separating enzyme is a mixture of pectase and collagenase.
Preferably, in the method for separating single cells in animals and plants, the feed liquid ratio of the biological sample to the active liquid is 10-20 g/100 mL.
Preferably, in the above method for separating single cells from animals and plants, the ratio of glass beads to the composite cell suspension in the cell separation step is 1 g/10 mL.
Preferably, in the above method for separating single cells from animals and plants, for further removing impurities, after single cell suspension is collected in the step of cell separation, a cell adsorption carrier is added thereto for adsorption for 1-2 hours, then the cell adsorption carrier is taken out and soaked in water for desorption, and then single cells are dissolved out, and the purified single cell suspension is collected again.
Preferably, in the method for separating single cells in animals and plants, the cell adsorption carrier is activated carbon particles with the particle size of 1-2mm or honeycomb ceramics with the pore diameter of 0.22-0.45 mu m.
Preferably, the separation method of single cells in animals and plants comprises shaking the animal and plant with shaking table at 220r/min for 10-15min or 180W ultrasonic for 10-15min.
Compared with the prior art, the method for separating single cells in animals and plants has the following beneficial effects:
the invention adopts active liquid to maintain cell activity and promote cell separation, wherein sodium chloride, potassium chloride and lanthanum compounds can maintain cell activity. The pectase and collagenase adopted by the invention degrade jelly in the sample, promote cells to dissolve out of biological samples into water, and improve the yield and purity of the cells. The invention adopts a filter membrane with the aperture of 0.22-0.45 mu m to filter, so that small molecular substances such as protein, fat and the like are discarded along with the filtrate, single cells, microorganisms and large particle impurities are remained on the upper layer of the filter membrane, then the large particle impurities are removed, and finally glass beads are adopted to separate single cells, so that the cell purity is high and the activity is high.
Detailed Description
The present invention will be further described with reference to specific examples in order to enable those skilled in the art to better understand the technical aspects of the present invention.
The following experimental data, unless otherwise indicated, are for both the methods and reagents described using prior art techniques.
Example 1
A method for isolating single cells in animals and plants, comprising:
step 1, pretreatment of biological samples: adding active liquid and separating enzyme into a biological sample, and soaking for 1h to obtain a soaked mixture;
the active liquid is prepared according to the following method: 9g of sodium chloride, 0.5mg of potassium chloride, 0.01mg of lanthanum nitrate, 2.4g of 4-hydroxyethyl piperazine ethane sulfonic acid, and distilled water to a constant volume of 1L and a pH value of 7.2;
the feed liquid ratio of the biological sample to the active liquid is 10g to 100mL;
the separating enzyme is a mixture of pectase and collagenase, and 0.5g of pectase and 0.5g of collagenase are added into each liter of active liquid;
step 2, small molecule filtration: filtering the soaked mixture with a filter membrane with a pore size of 0.22 μm;
step 3, large particle filtration: soaking and stirring the filter membrane and the sediment above the filter membrane in the active liquid to enable single cells and microorganisms to be resuspended in the active liquid, and collecting composite cell suspension;
step 4, cell separation: glass beads with the diameter of 2-3mm are placed into the compounded cell suspension, the ratio of the glass beads to the cell suspension is 1g to 10mL, shaking is carried out for 30min by a 220r/min shaking table, and a liquid transfer device is used for collecting single cell suspension.
Example 2
A method for isolating single cells in animals and plants, comprising:
step 1, pretreatment of biological samples: adding active liquid and separating enzyme into a biological sample, and soaking for 1.5h to obtain a soaked mixture;
the active liquid is prepared according to the following method: 8.5g of sodium chloride, 0.7mg of potassium chloride, 0.05mg of lanthanum nitrate, 2.4g of 4-hydroxyethyl piperazine ethane sulfonic acid, and distilled water to a constant volume of 1L and a pH value of 7.4;
the feed liquid ratio of the biological sample to the active liquid is 15g to 100mL;
the separating enzyme is a mixture of pectase and collagenase, and 0.75g of pectase and 0.75g of collagenase are added into each liter of active liquid;
step 2, small molecule filtration: filtering the soaked mixture with a filter membrane with a pore size of 0.45 μm;
step 3, large particle filtration: soaking and stirring the filter membrane and the sediment above the filter membrane in the active liquid to enable single cells and microorganisms to be resuspended in the active liquid, and collecting composite cell suspension;
step 4, cell separation: glass beads with the diameter of 3-4mm are placed into the cell suspension, the ratio of the glass beads to the compounded cell suspension is 1g to 10mL, shaking is carried out for 20min by a 220r/min shaking table, and a liquid transfer device is used for collecting single cell suspension.
Example 3
A method for isolating single cells in animals and plants, comprising:
step 1, pretreatment of biological samples: adding active liquid and separating enzyme into a biological sample, and soaking for 2 hours to obtain a soaked mixture;
the active liquid is prepared according to the following method: 9g of sodium chloride, 0.5mg of potassium chloride, 0.03mg of lanthanum chloride, 2.4g of 4-hydroxyethyl piperazine ethane sulfonic acid, and distilled water to a constant volume of 1L and a pH value of 7.2;
the feed liquid ratio of the biological sample to the active liquid is 20g to 100mL;
the separating enzyme is a mixture of pectase and collagenase, and 1g of pectase and 1g of collagenase are added into each liter of active liquid;
step 2, small molecule filtration: filtering the soaked mixture with a filter membrane with a pore size of 0.22 μm;
step 3, large particle filtration: soaking and stirring the filter membrane and the sediment above the filter membrane in the active liquid to enable single cells and microorganisms to be resuspended in the active liquid, and collecting composite cell suspension;
step 4, cell separation: glass beads with the diameter of 2-3mm are placed into the cell suspension, the ratio of the glass beads to the compounded cell suspension is 1g to 10mL, shaking is carried out for 20min by a 220r/min shaking table, and a liquid transfer device is used for collecting single cell suspension.
Example 4
A method for isolating single cells in animals and plants, comprising:
step 1, pretreatment of biological samples: adding active liquid and separating enzyme into a biological sample, and soaking for 1h to obtain a soaked mixture;
the active liquid is prepared according to the following method: 9g of sodium chloride, 0.5mg of potassium chloride, 0.01mg of lanthanum nitrate, 2.4g of 4-hydroxyethyl piperazine ethane sulfonic acid, and distilled water to a constant volume of 1L and a pH value of 7.2;
the feed liquid ratio of the biological sample to the active liquid is 10g to 100mL;
the separating enzyme is a mixture of pectase and collagenase, and 0.5g of pectase and 0.5g of collagenase are added into each liter of active liquid;
step 2, small molecule filtration: filtering the soaked mixture with a filter membrane with a pore size of 0.22 μm;
step 3, large particle filtration: soaking and stirring the filter membrane and the sediment above the filter membrane in the active liquid to enable single cells and microorganisms to be resuspended in the active liquid, and collecting composite cell suspension;
step 4, cell separation: putting glass beads with the diameter of 2-3mm into the cell suspension, wherein the ratio of the glass beads to the composite cell suspension is 1g to 10mL, shaking the cell suspension for 30min by a shaking table with 220r/min, and collecting single cell suspension by a liquid transfer device;
and 5, adding activated carbon particles with the particle size of 1-2mm into the cell suspension in the step 4, adsorbing for 1h, taking out the activated carbon particles (or filtering and collecting the activated carbon particles), soaking the activated carbon particles in water for 220r/min shaking table oscillation for 10-15min, dissolving out single cells, and finally collecting the purified cell suspension by using a liquid transfer device.
We have carried out the following experiments to demonstrate the effect of the invention:
1. sample design
Blank group: the biological sample is sheep liver, and the method for separating single cells in animals and plants is basically the same as that of example 1, except that: the lanthanum compound is not added into the active liquid in the step 1, and the separating enzyme is collagenase.
Group A: the biological sample was sheep liver and cells were isolated by the method of example 1.
Group B: the biological sample was sheep liver and cells were isolated by the method of example 2.
Group C: the biological sample was sheep liver and cells were isolated by the method of example 3.
Group D: the biological sample was sheep liver and cells were isolated by the method of example 4.
Group E: the biological sample was grass roots (grass leaves cut) and cells were isolated by the method of example 1.
Group F: the biological sample was grass roots (grass leaves cut) and cells were isolated by the method of example 4.
The primary isolated cellular objects in the grass roots of groups E and F were microorganisms that were parasitic on the grass roots.
Control group 1: the biological sample is sheep liver, and the method for separating single cells in animals and plants is basically the same as that of example 1, except that: no lanthanum compound is added to the active solution in step 1.
Control group 2: the biological sample is sheep liver, and the method for separating single cells in animals and plants is basically the same as that of example 1, except that: the isolated enzyme of step 1 is collagenase.
2. Statistical cell yield
Cell yield = total cell number (number)/biological sample mass (g) ×100%. Wherein the total cell number is counted by adopting a blood cell counting plate counting method, dead cells and living cells are not distinguished, and the mass of the biological sample is weighed by adopting a balance with the precision of 0.01 g.
The cell yield results of the above experimental groups are shown in Table 1. It can be seen from Table 1 that the cell yields of groups A-C and E were higher. For the same sheep liver samples, the single cell yield is higher than that of the blank group, the control group 1 and the control group 2 because the active liquid containing the lanthanum compound and the compound enzyme of collagenase and pectase are adopted in the A-C group. Group F had the lowest cell yield due to the addition of the purification step of the activated carbon particles.
TABLE 1 cell yields for different experimental groups
3. Cell mass
The invention adopts the cell viability, purity and cell death rate after 10 generations to characterize the quality of the separated cells.
Cell viability = number of viable cells/total number of cells x 100%.
The results in Table 2 show that the cell viability, purity and cell death rate after 10 passages of the blank group are all the lowest, and the cell death rate and purity after 10 passages of the cells are higher in the A-C group and the E group due to the addition of the lanthanum compound, collagenase and pectinase, and the cell purity is higher in the D group and the F group due to the addition of the activated carbon particle purification step.
TABLE 2 cell mass of different experimental groups
In order to make the single cell separation speed faster, when the biological sample is a plant stem leaf root system, the sample is cut into small sections of 3-5 cm; when the biological sample is animal epidermis or animal liver, firstly cutting the sample into meat blocks with the length of 1-2cm, the width of about 1cm and the thickness of about 1 cm; the active liquid and the isolated enzyme are then added to the biological sample.
It should be noted that, when numerical ranges are referred to in the present invention, it should be understood that two endpoints of each numerical range and any numerical value between the two endpoints are optional, and because the adopted step method is the same as the embodiment, in order to prevent redundancy, the present invention describes a preferred embodiment. While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. It is therefore intended that the following claims be interpreted as including the preferred embodiments and all such alterations and modifications as fall within the scope of the invention.
It will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention also include such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof.
Claims (6)
1. A method for separating sheep liver single cells, which is characterized by comprising the following steps:
pretreatment of biological samples: adding active liquid and separating enzyme into biological sample, soaking for 1-2h to obtain soaking mixture;
the active liquid is prepared according to the following method: 8-9g of sodium chloride, 0.5-1mg of potassium chloride, 0.01-0.05mg of lanthanum compound, 2.4g of 4-hydroxyethyl piperazine ethane sulfonic acid, and distilled water to a constant volume of 1L and a pH value of 7.2-7.4;
small molecule filtration: filtering the soaked mixture with a filter membrane with a pore size of 0.22-0.45 μm;
large particle filtration: soaking and stirring the filter membrane and the sediment above the filter membrane in the active liquid to enable single cells and microorganisms to be resuspended in the active liquid, and collecting composite cell suspension;
cell separation: putting glass beads with the diameter of 2-4mm into the composite cell suspension, oscillating for 20-30min, and collecting single cell suspension by using a liquid transfer device;
the lanthanum compound is lanthanum nitrate or lanthanum chloride;
the separating enzyme is a mixture of pectase and collagenase, and 0.5-1g of pectase and 0.5-1g of collagenase are added into each liter of active liquid.
2. The method for separating sheep liver single cells according to claim 1, wherein the ratio of the biological sample to the active liquid is 10-20 g/100 mL.
3. The method according to claim 1, wherein the ratio of glass beads to the composite cell suspension in the cell separation step is 1 g/10 mL.
4. The method according to claim 1, wherein after the single-cell suspension is collected in the cell separation step, a cell adsorption carrier is added thereto for adsorption for 1 to 2 hours, and then the cell adsorption carrier is taken out and immersed in water for desorption, so that the single-cell is eluted, and the purified single-cell suspension is collected again.
5. The method for separating sheep liver single cells according to claim 4, wherein the cell adsorption carrier is activated carbon particles with a particle size of 1-2mm or honeycomb ceramics with a pore size of 0.22-0.45 μm.
6. The method for separating sheep liver single cells according to claim 5, wherein the condition of desorption is 220r/min shaking table oscillation for 10-15min or 180W ultrasound for 10-15min.
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| WO2002053251A1 (en) * | 2000-12-29 | 2002-07-11 | Upfront Chromatography A/S | Extracorporeal capturing of specific bio-macromolecular entities from extracellular body fluids |
| JP6317353B2 (en) * | 2012-09-13 | 2018-04-25 | プレジデント・アンド・フェロウズ・オブ・ハーバード・カレッジ | Method for multiple analytical measurements in single cells of solid tissue |
| WO2017075293A1 (en) * | 2015-10-28 | 2017-05-04 | Silicon Valley Scientific, Inc. | Method and apparatus for encoding cellular spatial position information |
| US20190367964A1 (en) * | 2016-02-02 | 2019-12-05 | Dana-Farber Cancer Institute, Inc. | Dissociation of human tumor to single cell suspension followed by biological analysis |
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| CN102094003A (en) * | 2009-12-14 | 2011-06-15 | 中国科学院城市环境研究所 | Method for extracting DNA (deoxyribonucleic acid) of activated carbon biomembrane |
| CN102634480A (en) * | 2012-03-27 | 2012-08-15 | 中国农业大学 | Method for isolating and culturing liver primary cells |
| CN109161518A (en) * | 2018-10-10 | 2019-01-08 | 郑州大学第附属医院 | A method of being separately cultured primary hepatic cell |
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