CN111647549A - 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 method for separating single cells in animals and plants. The method comprises 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, a lanthanum compound and 4-hydroxyethyl piperazine ethanesulfonic acid; filtering the soaked mixture with a filter membrane; placing the filter membrane and the precipitate above the filter membrane into the active solution for soaking and stirring, so that the single cells and the microorganisms are suspended in the active solution, and collecting the composite cell suspension; and (4) adding glass beads into the compounded cell suspension, oscillating and collecting the single cell suspension. The invention adopts active liquid to maintain the activity of cells, and adopts separating enzyme to separate jelly in a sample, thereby improving the cell yield and the cell purity.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a method for separating single cells in animals and plants.
Background
Biological resources are important resources for studying the origin, variation and development direction of organisms, and have important research value whether single-cell organisms or single cells isolated from biological tissues. Typical unicellular biological resources include bacteria, fungi, archaea, and the like, and these microorganisms are numerous in cell number and variety. The functional microorganisms of different species have wide application in the fields of food fermentation, organic matter degradation and the like. Many functional microbial unicells are present in animal and plant tissues and are mixed with other impurities such as proteins and fats, and it is difficult to separate the impurities.
Life materials such as animals and plants are high-grade life bodies composed of many single cells, and can perform complex life activities. Plant root, stem and leaves, as well as animal internal organs, are common samples of single cell isolation. The unicell separated from the animal and plant tissues is cultured, which is also of great significance for the research of animal and plant organ functions, genetic mutation of genes and the like. However, many cells and proteins in animal and plant tissues are adhered together, and the viability of single cells is far lower than that of microorganisms, and impurities are difficult to separate.
In summary, the prior art has the problems that most of single cells have the size of several microns to several tens of microns, the diameter is small, biological cells are difficult to separate from impurities, and the 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 method for separating single cells in animals and plants, which comprises the following steps:
pretreatment of a biological sample: adding active liquid and separating enzyme into a biological sample, and soaking for 1-2h to obtain a 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 ethanesulfonic acid, and distilled water with constant volume of 1L and pH of 7.2-7.4;
small molecule filtration: filtering the soaked mixture with a filter membrane with pore diameter of 0.22-0.45 μm;
filtering large particles: placing the filter membrane and the precipitate above the filter membrane into the active solution for soaking and stirring, so that the single cells and the microorganisms are suspended in the active solution, and collecting the composite cell suspension;
cell separation: and (3) adding glass beads with the diameter of 2-4mm into the composite cell suspension, oscillating for 20-30min, and collecting the single cell suspension.
Preferably, in the method for separating single cells from 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 method for separating single cells in animals and plants comprises adding 0.5-1g of pectinase and 0.5-1g of collagenase to each liter of active solution, wherein the pectinase and collagenase are a mixture.
Preferably, in the method for separating single cells from animals and plants, the material-to-liquid ratio of the biological sample to the active liquid is 10-20g:100 mL.
Preferably, in the method for separating a single cell from an animal or plant, the ratio of the glass beads to the combined cell suspension in the cell separation step is 1 g/10 mL.
Preferably, in the method for separating single cells in animals and plants, in order to further remove impurities, after the single cell suspension is collected in the cell separation step, the cell adsorption carrier is added into the single cell suspension for adsorption for 1-2 hours, then the cell adsorption carrier is taken out and soaked in water for desorption, so that the single cells are dissolved out, and the purified single cell suspension is collected again.
Preferably, in the method for separating single cells from animals and plants, 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.
Preferably, the desorption condition of the method for separating the single cells in the animals and plants is shaking for 10-15min at a shaking table at 220r/min or ultrasonic processing at 180W for 10-15 min.
Compared with the prior art, the method for separating the single cells in the animals and the plants has the following beneficial effects:
the invention adopts the active liquid to maintain the activity of cells and promote the separation of cells, wherein, the sodium chloride, the potassium chloride and the lanthanum compound can maintain the activity of cells. The pectinase and collagenase adopted by the invention degrade jelly in the sample, promote the cells to be dissolved out from the biological sample into water, and improve the yield and purity of the cells. The invention adopts the filter membrane with the aperture of 0.22-0.45 mu m for filtration, so that the micromolecule 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, the glass beads are adopted for single cell separation, so that the cell purity and the activity are high.
Detailed Description
In order that those skilled in the art will better understand the technical solutions of the present invention to practice, the present invention will be further described with reference to the following specific examples.
The following experimental data, if not indicated otherwise, apply the prior art to both the methods and reagents described.
Example 1
A method for separating single cells in animals and plants, comprising:
step 1, pretreatment of a biological sample: adding active liquid and separating enzyme into a biological sample, and soaking for 1h to obtain a soaking 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 and 2.4g of 4-hydroxyethyl piperazine ethanesulfonic acid, wherein the volume of distilled water is fixed to 1L, and the pH value is 7.2;
the material-liquid ratio of the biological sample to the active liquid is 10g:100 mL;
the separating enzyme is a mixture of pectinase and collagenase, and each liter of active liquid is added with 0.5g of pectinase and 0.5g of collagenase;
step 2, small molecule filtration: filtering the soaked mixture with a filter membrane with the pore diameter of 0.22 μm;
and 3, filtering large particles: placing the filter membrane and the precipitate above the filter membrane into the active solution for soaking and stirring, so that the single cells and the microorganisms are suspended in the active solution, and collecting the composite cell suspension;
step 4, cell separation: adding glass beads with diameter of 2-3mm into the composite cell suspension, wherein the ratio of the glass beads to the cell suspension is 1g:10mL, shaking the shaking table at 220r/min for 30min, and collecting the single cell suspension by using a pipettor.
Example 2
A method for separating single cells in animals and plants, comprising:
step 1, pretreatment of a biological sample: adding active liquid and separating enzyme into a biological sample, and soaking for 1.5h to obtain a soaking 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 and 2.4g of 4-hydroxyethyl piperazine ethanesulfonic acid, and the volume of distilled water is fixed to 1L, and the pH value is 7.4;
the material-liquid ratio of the biological sample to the active liquid is 15g:100 mL;
the separating enzyme is a mixture of pectinase and collagenase, and each liter of active liquid is added with 0.75g of pectinase and 0.75g of collagenase;
step 2, small molecule filtration: filtering the soaked mixture with a filter membrane with the aperture of 0.45 μm;
and 3, filtering large particles: placing the filter membrane and the precipitate above the filter membrane into the active solution for soaking and stirring, so that the single cells and the microorganisms are suspended in the active solution, and collecting the composite cell suspension;
step 4, cell separation: adding glass beads with diameter of 3-4mm into the cell suspension, wherein the ratio of the glass beads to the composite cell suspension is 1g:10mL, shaking the shaking table at 220r/min for 20min, and collecting the single cell suspension by using a pipettor.
Example 3
A method for separating single cells in animals and plants, comprising:
step 1, pretreatment of a biological sample: adding active liquid and separating enzyme into a biological sample, and soaking for 2 hours to obtain a soaking 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 and 2.4g of 4-hydroxyethyl piperazine ethanesulfonic acid, wherein the volume of distilled water is fixed to 1L, and the pH value is 7.2;
the material-liquid ratio of the biological sample to the active liquid is 20g:100 mL;
the separating enzyme is a mixture of pectinase and collagenase, and 1g of pectinase 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 the pore diameter of 0.22 μm;
and 3, filtering large particles: placing the filter membrane and the precipitate above the filter membrane into the active solution for soaking and stirring, so that the single cells and the microorganisms are suspended in the active solution, and collecting the composite cell suspension;
step 4, cell separation: adding glass beads with diameter of 2-3mm into the cell suspension, wherein the ratio of the glass beads to the composite cell suspension is 1g:10mL, shaking the shaking table at 220r/min for 20min, and collecting the single cell suspension by using a pipettor.
Example 4
A method for separating single cells in animals and plants, comprising:
step 1, pretreatment of a biological sample: adding active liquid and separating enzyme into a biological sample, and soaking for 1h to obtain a soaking 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 and 2.4g of 4-hydroxyethyl piperazine ethanesulfonic acid, wherein the volume of distilled water is fixed to 1L, and the pH value is 7.2;
the material-liquid ratio of the biological sample to the active liquid is 10g:100 mL;
the separating enzyme is a mixture of pectinase and collagenase, and each liter of active liquid is added with 0.5g of pectinase and 0.5g of collagenase;
step 2, small molecule filtration: filtering the soaked mixture with a filter membrane with the pore diameter of 0.22 μm;
and 3, filtering large particles: placing the filter membrane and the precipitate above the filter membrane into the active solution for soaking and stirring, so that the single cells and the microorganisms are suspended in the active solution, and collecting the 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:10mL, shaking the table at 220r/min for 30min, and collecting the single cell suspension by using a pipettor;
and 5, adding activated carbon particles with the particle size of 1-2mm into the cell suspension obtained 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 10-15min by shaking in a shaking table at a speed of 220r/min, dissolving out single cells, and finally collecting the purified cell suspension by using a pipettor.
We performed the following experiments to demonstrate the effect of the present 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 the operation of example 1, except that: the active liquid in the step 1 is not added with a lanthanum compound, and the separating enzyme is collagenase.
Group A: the biological sample was goat liver, and the cells were isolated by the method of example 1.
Group B: the biological sample was goat liver, and the cells were isolated by the method of example 2.
Group C: the biological sample was goat liver, and the cells were isolated by the method of example 3.
Group D: the biological sample was goat liver, and the cells were isolated by the method of example 4.
Group E: the biological sample was Elymus dahuricus roots (cut off grass leaves), and the cells were isolated by the method of example 1.
And F group: the biological sample was Elymus dahuricus roots (cut off grass leaves), and the cells were isolated by the method of example 4.
The main cellular objects isolated in the roots of group E and group F Alkale shavings are microorganisms that are parasitic on the 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 the operation of example 1, except that: the lanthanum compound was not added to the active solution of 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 the operation of example 1, except that: the isolated enzyme of step 1 is collagenase.
2. Statistical cell yield
The cell yield is total number of cells (number)/mass of biological sample (g) × 100%. Wherein the total cell number is counted by adopting a blood 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 results of cell yield of the above experimental groups are shown in Table 1. It can be seen from Table 1 that the cell yields are higher in the A-C group and the E group. For the same sheep liver samples, the A-C groups adopted the active solution containing lanthanum compound and the complex enzyme of collagenase and pectinase, so the single cell yield was higher than that of the blank group, the control group 1 and the control group 2. The F group had the lowest cell yield due to the addition of the purification step of activated carbon particles.
TABLE 1 cell yield for different experimental groups
3. Cell mass
The invention adopts the cell survival rate, the purity and the cell death rate after 10 generations to characterize the quality of the separated cells.
The cell viability rate is the number of viable cells/total number of cells × 100%.
The results in table 2 show that the blank control group has the lowest cell viability, purity and cell death rate after passage of 10, the groups a-C and E have higher cell death rate and purity after passage of 10 due to the addition of lanthanum compound, collagenase and pectinase, and the groups D and F have higher cell purity due to the addition of the activated carbon particle purification step.
TABLE 2 cell quality of different experimental groups
It should be noted that, in order to make the single cell separation speed faster, when the biological sample is the root system of plant stem leaves, the sample is cut into 3-5cm segments; 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 solution and the isolated enzyme are then added to the biological sample.
It should be noted that, when the present invention relates to a numerical range, it should be understood that two endpoints of each numerical range and any value between the two endpoints can be selected, and since the steps and methods adopted are the same as those in 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. Therefore, it is intended that the appended claims be interpreted as including 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 changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.
Claims (9)
1. A method for separating a single cell in an animal or plant, comprising:
pretreatment of a biological sample: adding active liquid and separating enzyme into a biological sample, and soaking for 1-2h to obtain a 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 ethanesulfonic acid, and distilled water with constant volume of 1L and pH of 7.2-7.4;
small molecule filtration: filtering the soaked mixture with a filter membrane with pore diameter of 0.22-0.45 μm;
filtering large particles: placing the filter membrane and the precipitate above the filter membrane into the active solution for soaking and stirring, so that the single cells and the microorganisms are suspended in the active solution, and collecting the composite cell suspension;
cell separation: and (3) adding glass beads with the diameter of 2-4mm into the composite cell suspension, oscillating for 20-30min, and collecting the single cell suspension.
2. The method for separating single cells from animals and plants according to claim 1, wherein the biological sample is plant stem and leaf root system, animal epidermis or animal liver.
3. The method for separating the single cells in the animal and plant according to claim 2, wherein the lanthanum compound is lanthanum nitrate or lanthanum chloride.
4. The method of claim 3, wherein the separating enzyme is a mixture of pectinase and collagenase, and 0.5-1g of pectinase and 0.5-1g of collagenase are added per liter of the active solution.
5. The method for separating single cells in an animal or plant according to claim 4, wherein the feed-liquid ratio of the biological sample to the active liquid is 10-20g:100 mL.
6. The method of claim 2, wherein the ratio of the glass beads to the cell suspension is 1g:10 mL.
7. The method for separating single cells from animals and plants according to claim 2, wherein the single cell suspension is collected in the cell separation step, then a cell adsorption carrier is added to the single cell suspension for adsorption for 1-2h, and then the cell adsorption carrier is taken out and soaked in water for desorption, so that the single cells are dissolved out, and the purified single cell suspension is collected again.
8. The method for separating single cells in animals and plants according to claim 7, wherein the cell adsorbing 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.
9. The method for separating single cells from animals and plants according to claim 8, wherein the desorption conditions are shaking for 10-15min at 220r/min or 180W ultrasound for 10-15 min.
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