CN115074316A - Preparation method of human periodontitis gingival tissue single cell suspension - Google Patents
Preparation method of human periodontitis gingival tissue single cell suspension Download PDFInfo
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Abstract
The invention discloses a preparation method of a human periodontitis gingival tissue single-cell suspension, which comprises the following steps: washing, shearing and centrifuging human periodontitis gingival tissues to remove supernatant to obtain tissue precipitates; performing enzymolysis digestion on the tissue precipitate by adopting collagenase I and Dispase II, stopping digestion, filtering by adopting a filter screen, collecting filtrate, and centrifuging to obtain a first cell precipitate; adding erythrocyte lysate into the first cell sediment, standing, centrifuging to remove supernatant, cleaning, and centrifuging to obtain a second cell sediment; adding dead cells into the second cell sediment to remove magnetic beads, and incubating to obtain a cell suspension containing the magnetic beads; and (3) placing the suspension containing the magnetic beads into a magnetic column for filtration, collecting filtrate, centrifuging to obtain a third cell precipitate, and re-suspending to obtain the single cell suspension of the gingival tissue. The method does not need specialized equipment and skills, and can obtain a large amount of high-activity single-cell suspension in short time.
Description
Technical Field
The invention relates to the technical field of cell suspension culture, in particular to a preparation method of a human periodontitis gingival tissue single cell suspension.
Background
Periodontitis is a chronic inflammatory disease that occurs in periodontal tissues mediated by bacteria. It is clinically manifested as gingival inflammation, periodontal pocket formation, and alveolar bone resorption. If left untreated, it ultimately results in tooth loss. The fourth national oral health survey result in 2017 shows that 90% of adults in China have periodontal diseases, and the prevalence rates of periodontitis of populations aged 35-44 years, 55-64 years and 65-74 years are 52.8%, 69.3% and 64.6% respectively, which indicates that the prevention and control situation of periodontitis is severe.
In recent years, single-cell RNA sequencing technology has been widely used to study the number of different cell populations, gene expression levels and changes of related functions in tissues under disease and health conditions, and help researchers study the pathogenic mechanism of disease from single-cell level. The dissociation of tissue digestion into high quality single cell suspensions (cell viability above 80%) is the first prerequisite for single cell RNA sequencing. At present, most methods for separating the single cell suspension of the gingival tissue, such as a tissue block method and a collagenase digestion method, have the defects of long time consumption, small cell number, low cell activity, high cell agglomeration rate and the like, and are not suitable for single cell RNA sequencing.
Therefore, in order to solve the above technical problems, it is necessary to develop a method for preparing a single cell suspension of periodontium gingival tissue, which overcomes the disadvantages of the conventional method for preparing a single cell suspension of periodontium gingival tissue and facilitates the application of a single cell RNA sequencing technology in the research of periodontitis.
Disclosure of Invention
The invention aims to provide a preparation method of human periodontitis gingival tissue single cell suspension, which does not need specialized equipment and skills and can obtain a large amount of high-activity single cell suspension in short time.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect of the present invention, there is provided a method for preparing a single cell suspension of human periodontitis gingival tissue, the method comprising:
washing, shearing and centrifuging human periodontitis gingival tissue to remove supernatant to obtain tissue precipitate;
performing enzymolysis digestion on the tissue precipitate by adopting collagenase I and Dispase II to obtain a mixed solution, stopping digestion, filtering by adopting a filter screen, collecting filtrate, and centrifuging to obtain a first cell precipitate;
adding erythrocyte lysate into the first cell sediment, standing, centrifuging to remove supernatant, cleaning, and centrifuging to obtain a second cell sediment;
adding dead cells into the second cell sediment to remove magnetic beads, and incubating to obtain a cell suspension containing the magnetic beads; and (3) placing the suspension containing the magnetic beads into a magnetic column for filtration, collecting filtrate, centrifuging to obtain a third cell precipitate, and re-suspending to obtain the single cell suspension of the gingival tissue.
Further, the washing is carried out by using a precooled calcium-magnesium-free PBS solution containing 5% of penicillin and streptomycin; shearing the mixture to 0.2-0.4 mm in the shearing process 3 Tissue patch of (2).
Further, the collagenase I and the Dispase II are dissolved in an alpha-mem culture medium in advance to obtain an enzyme-containing culture medium, and the working concentrations of the collagenase I and the Dispase II are 5-7 mg/ml and 7-9 mg/ml respectively; and carrying out enzymolysis digestion on the tissue sediment by adopting the enzyme-containing culture medium.
The reason that the working concentrations of the collagenase I and the Dispase II are 5-7 mg/ml and 7-9 mg/ml respectively is that the digestion efficiency is high, and if other working concentrations are selected, the digestion efficiency is low, and the adverse effect of incomplete digestion is caused;
further, the volume ratio of the tissue pellet to the enzyme-containing medium is 1: 9 to 11.
Further, in the enzymolysis digestion, the mixture is digested in a water bath at 37 +/-0.5 ℃ for 50-80 min, and the tissue sediment is resuspended by shaking every 5min during the digestion.
Further, in the termination of digestion, an α -mem medium containing 10% fetal bovine serum, 1% penicillin and streptomycin was used in a volume 3 to 5 times the volume of the mixed solution.
Further, the cell filter mesh has a pore size of 40 microns.
Further, the ratio of the volume of the erythrocyte lysate to the volume of the first cell sediment is 95-105: 1, the ratio of the volume of the dead cell removal magnetic beads to the volume of the second cell sediment is 18-22: 1, adding dead cells to remove magnetic beads, and incubating for 10-30 min.
One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:
the invention provides a preparation method of human periodontitis gingival tissue single cell suspension, which combines two digestive enzymes of I-type collagenase and Dispase II to fully digest and crack tissues while fully shearing tissue blocks, removes residual tissue fragments through cell filter screen filtration, and obtains a large amount of high-activity single cell suspension after removing dead cells by using dead cell removing magnetic beads. The preparation method does not need specialized equipment and skills, can obtain a large amount of high-activity single-cell suspension in short time, is convenient for researchers to operate, and has good application value.
Drawings
In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on the drawings without creative efforts.
FIG. 1 is a schematic diagram of single cell suspension counting, viability detection and cell dispersion degree detection obtained in example 1 of the present invention;
FIG. 2 is a schematic diagram of single cell suspension counting, viability detection and cell dispersion degree detection obtained in example 2 of the present invention;
FIG. 3 is a schematic diagram of single cell suspension counting, viability detection and cell dispersion degree detection obtained in example 3 of the present invention;
FIG. 4 is a schematic diagram of single cell suspension counting, viability detection and cell dispersion degree detection obtained in example 4 of the present invention;
FIG. 5 is a flow chart of a method for preparing a single cell suspension of human periodontitis gingival tissue according to an embodiment of the present invention.
Detailed Description
The present invention will be described in detail below with reference to specific embodiments and examples, and the advantages and various effects of the present invention will be more clearly apparent therefrom. It will be understood by those skilled in the art that these specific embodiments and examples are for the purpose of illustrating the invention and are not to be construed as limiting the invention.
Throughout the specification, unless otherwise specifically noted, terms used herein should be understood as having meanings as commonly used in the art. Accordingly, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. If there is a conflict, the present specification will control.
Unless otherwise specifically stated, various raw materials, reagents, instruments, equipment and the like used in the present invention are commercially available or can be obtained by an existing method.
The following will describe in detail a method for preparing a single cell suspension of human periodontitis gingival tissue according to the present application with reference to examples, comparative examples and experimental data.
Example 1
The embodiment of the invention provides a preparation method of a human periodontitis gingival tissue single cell suspension, which comprises the following specific steps:
1. taking about 20mg of human periodontitis gingival tissue, placing the tissue in a sterile 6 cm culture dish, adding 4ml of calcium-magnesium-free PBS (phosphate buffer solution) containing 5% of penicillin and streptomycin, washing, and removing macroscopic blood on the surface of the tissue; after washing for three times, transferring the tissue to a sterile 6 cm culture dish, adding a small amount of calcium-magnesium-free PBS solution containing 5% penicillin and streptomycin to cover the tissue properly, and processing the tissue into fragments of about 0.3mm3 by using sterile tissue scissors; the mixed solution in the petri dish was transferred to a 15 ml centrifuge tube in its entirety. Centrifuging for 300X g min;
2. adding 1 ml of collagenase type I and 1 ml of Dispase II (preheated at 37 ℃) into a centrifuge tube, uniformly mixing, putting the mixture into a constant-temperature water bath box at 37 ℃, digesting for 60min, and thoroughly shaking the mixture every 5min to uniformly mix the suspension;
3. digestion was stopped by adding 8ml of alpha-mem medium containing 10% fetal bovine serum and 1% penicillin and streptomycin. Filtering the obtained mixed solution with a cell strainer with a pore size of 40 μm, centrifuging the filtrate at 4 deg.C of 300X g for 10min, carefully sucking off the supernatant;
4. adding 2ml erythrocyte lysate, standing for 2min at room temperature; 300X g centrifuging for 5 min; sucking off the supernatant, adding 2ml of PBS solution containing 0.04% BSA for resuspension and precipitation, centrifuging for 5min at 300X g, and sucking off the supernatant;
5. adding 100 microliters of dead cell removal magnetic beads into the cell sediment, resuspending the sediment, standing at room temperature for 15min, then rinsing the magnetic column with 500 microliters of binding buffer, adding 500 microliters of binding buffer into the cell suspension, transferring the cell suspension into the magnetic column, then adding 500 microliters of binding buffer into the magnetic column, thoroughly flushing the filtrate, repeating the steps for three times, and collecting the filtrate by using a 15-milliliter centrifuge tube. 300X g for 5min, the supernatant was aspirated and the cell pellet was resuspended in PBS containing 0.04% BSA;
the prepared single cell suspension was subjected to cell viability examination and cell counting by an automatic counter, and as shown in FIG. 1, the cell concentration of the cell suspension obtained by the above method was about 3.29X 10 6 The cell count/ml, the activity was 89.8%, and the cell dispersion degree was such that 93.4% of the cells were present as single cells.
Example 2
The embodiment of the invention provides a preparation method of a human periodontitis gingival tissue single cell suspension, which comprises the following specific steps:
1. taking about 5mg of healthy gingival tissue of human, placing the tissue in a sterile 6 cm culture dish, adding 4ml of calcium-magnesium-free PBS solution containing 5% of penicillin and streptomycin, washing, and removing macroscopic blood on the surface of the tissue; after washing for three times, transferring the tissue to a sterile 6 cm culture dish, adding a small amount of calcium-magnesium-free PBS solution containing 5% penicillin and streptomycin to cover the tissue properly, and processing the tissue into fragments of about 0.3mm3 by using sterile tissue scissors; the mixed solution in the petri dish was transferred to a 15 ml centrifuge tube in its entirety. Centrifuging for 300X g min;
2. adding 1 ml of collagenase type I and 1 ml of Dispase II (preheated at 37 ℃) into a centrifuge tube, uniformly mixing, putting the mixture into a thermostatic water bath box at 37 ℃, digesting for 60min, and thoroughly shaking the mixture every 5min to uniformly mix the suspension;
3. digestion was stopped by adding 8ml of alpha-mem medium containing 10% fetal bovine serum and 1% penicillin and streptomycin. Filtering the obtained mixed solution with a cell strainer with a pore size of 40 μm, centrifuging the filtrate at 4 deg.C of 300X g for 10min, carefully sucking off the supernatant;
4. adding 2ml erythrocyte lysate, standing for 2min at room temperature; 300X g centrifuging for 5 min; sucking off the supernatant, adding 2ml of PBS solution containing 0.04% BSA for resuspension and precipitation, centrifuging for 5min at 300X g, and sucking off the supernatant;
5. adding 100 microliters of dead cell removal magnetic beads into the cell sediment, resuspending the sediment, standing at room temperature for 15min, then rinsing the magnetic column with 500 microliters of binding buffer, adding 500 microliters of binding buffer into the cell suspension, transferring the cell suspension into the magnetic column, then adding 500 microliters of binding buffer into the magnetic column, thoroughly flushing the filtrate, repeating the steps for three times, and collecting the filtrate by using a 15-milliliter centrifuge tube. 300X g for 5min, the supernatant was aspirated, and the cell pellet was resuspended in PBS containing 0.04% BSA;
the prepared single cell suspension was subjected to cell viability examination and cell counting by an automatic counter, and as shown in FIG. 1, the cell concentration of the cell suspension obtained by the above method was about 5.97X 10 5 The cell count/ml, the activity was 91.8%, and the degree of cell dispersion was such that 96.1% of the cells were present as single cells.
Example 3
The embodiment of the invention provides a preparation method of a human periodontitis gingival tissue single cell suspension, which comprises the following specific steps:
1. taking about 10mg of healthy gingival tissue of human, placing the tissue in a sterile 6 cm culture dish, adding 4ml of calcium-magnesium-free PBS solution containing 5% of penicillin and streptomycin, washing, and removing macroscopic blood on the surface of the tissue; after washing for three times, transferring the tissue to a sterile 6 cm culture dish, adding a small amount of calcium-magnesium-free PBS solution containing 5% penicillin and streptomycin to cover the tissue properly, and processing the tissue into fragments of about 0.3mm3 by using sterile tissue scissors; the mixed solution in the petri dish was transferred to a 15 ml centrifuge tube in its entirety. Centrifuging for 300X g min;
2. adding 1 ml of collagenase type I and 1 ml of Dispase II (preheated at 37 ℃) into a centrifuge tube, uniformly mixing, putting the mixture into a constant-temperature water bath box at 37 ℃, digesting for 60min, and thoroughly shaking the mixture every 5min to uniformly mix the suspension;
3. digestion was stopped by adding 8ml of alpha-mem medium containing 10% fetal bovine serum and 1% penicillin and streptomycin. Filtering the obtained mixed solution with a cell strainer with a pore size of 40 μm, centrifuging the filtrate at 4 deg.C of 300X g for 10min, carefully sucking off the supernatant;
4. adding 2ml erythrocyte lysate, standing for 2min at room temperature; 300X g centrifuging for 5 min; sucking off the supernatant, adding 2ml of PBS solution containing 0.04% BSA for resuspension and precipitation, centrifuging for 5min at 300X g, and sucking off the supernatant;
5. adding 100 microliters of dead cell removal magnetic beads into the cell sediment, resuspending the sediment, standing at room temperature for 15min, then rinsing the magnetic column with 500 microliters of binding buffer, adding 500 microliters of binding buffer into the cell suspension, transferring the cell suspension into the magnetic column, then adding 500 microliters of binding buffer into the magnetic column, thoroughly flushing the filtrate, repeating the steps for three times, and collecting the filtrate by using a 15-milliliter centrifuge tube. 300X g for 5min, the supernatant was aspirated and the cell pellet was resuspended in PBS containing 0.04% BSA;
the prepared single cell suspension was subjected to cell viability examination and cell counting by an automatic counter, and as shown in FIG. 1, the cell concentration of the cell suspension obtained by the above method was about 1.81X 10 6 One cell/ml, the activity was 89.2%, and 94.4% of the cells were present in the state of single cells as seen by the degree of cell dispersion.
Example 4
The embodiment of the invention provides a preparation method of a human periodontitis gingival tissue single cell suspension, which comprises the following specific steps:
1. taking about 10mg of inflammatory gingival tissue of human, placing the tissue in a sterile 6 cm culture dish, adding 4ml of calcium-magnesium-free PBS solution containing 5% of penicillin and streptomycin, washing, and removing macroscopic blood on the surface of the tissue; after washing for three times, transferring the tissue to a sterile 6 cm culture dish, adding a small amount of calcium-magnesium-free PBS solution containing 5% penicillin and streptomycin to cover the tissue properly, and processing the tissue into fragments of about 0.3mm3 by using sterile tissue scissors; the mixed solution in the petri dish was transferred to a 15 ml centrifuge tube in its entirety. Centrifuging for 300X g min;
2. adding 1 ml of collagenase type I and 1 ml of Dispase II (preheated at 37 ℃) into a centrifuge tube, uniformly mixing, putting the mixture into a constant-temperature water bath box at 37 ℃, digesting for 60min, and thoroughly shaking the mixture every 5min to uniformly mix the suspension;
3. digestion was stopped by adding 8ml of alpha-mem medium containing 10% fetal bovine serum and 1% penicillin and streptomycin. Filtering the obtained mixed solution with a cell strainer with a pore size of 40 μm, centrifuging the filtrate at 4 deg.C of 300X g for 10min, carefully sucking off the supernatant;
4. adding 2ml erythrocyte lysate, standing for 2min at room temperature; 300X g centrifuging for 5 min; sucking off the supernatant, adding 2ml of PBS solution containing 0.04% BSA for resuspension and precipitation, centrifuging for 5min at 300X g, and sucking off the supernatant;
5. adding 100 microliters of dead cell removal magnetic beads into the cell sediment, resuspending the sediment, standing at room temperature for 15min, then rinsing the magnetic column with 500 microliters of binding buffer, adding 500 microliters of binding buffer into the cell suspension, transferring the cell suspension into the magnetic column, then adding 500 microliters of binding buffer into the magnetic column, thoroughly flushing the filtrate, repeating the steps for three times, and collecting the filtrate by using a 15-milliliter centrifuge tube. 300X g for 5min, the supernatant was aspirated and the cell pellet was resuspended in PBS containing 0.04% BSA;
the prepared single cell suspension was subjected to cell viability examination and cell counting by an automatic counter, and as shown in FIG. 1, a cell real image derived from the automatic counter shows that the cell concentration of the cell suspension obtained by the above method was about 8.84X 10 5 One cell/ml, the activity was 89.2%, and 94.9% of the cells were present in the state of single cells as seen by the degree of cell dispersion.
Comparative example 1
The invention provides a preparation method of a human periodontitis gingival tissue single cell suspension, which comprises the following specific steps:
1. taking about 6mg of human periodontitis gingival tissue, placing the tissue in a sterile 6 cm culture dish, adding 4ml of calcium-magnesium-free PBS (phosphate buffer solution) containing 5% of penicillin and streptomycin, washing, and removing macroscopic blood on the surface of the tissue; after three repeated washes, the tissue was transferred to a sterile 6 cm petri dish, a small amount of a calcium-magnesium-free PBS solution containing 5% penicillin and streptomycin was added to just cover the tissue, and the tissue was processed to 0.3mm using sterile tissue scissors 3 Left and right fragments; the mixed solution in the petri dish was transferred to a 15 ml centrifuge tube in its entirety. Centrifuging for 300X g min;
2. adding 1 ml of collagenase type I (preheated at 37 ℃) into a centrifuge tube, uniformly mixing, putting the mixture into a constant-temperature water bath box at 37 ℃, digesting for 60min, and thoroughly shaking and uniformly mixing the suspension every 5 min;
3. digestion was stopped by adding 4ml of alpha-mem medium containing 10% fetal bovine serum and 1% penicillin and streptomycin. Filtering the obtained mixed solution with a cell strainer with a pore size of 40 μm, centrifuging the filtrate at 4 deg.C of 300X g for 10min, carefully sucking off the supernatant;
4. adding 2ml erythrocyte lysate, standing for 2min at room temperature; 300X g centrifuging for 5 min; sucking off the supernatant, adding 2ml of PBS solution containing 0.04% BSA for resuspension and precipitation, centrifuging for 5min at 300X g, and sucking off the supernatant;
5. adding 100 microliters of dead cell removal magnetic beads into the cell sediment, resuspending the sediment, standing at room temperature for 15min, then rinsing the magnetic column with 500 microliters of binding buffer, adding 500 microliters of binding buffer into the cell suspension, transferring the cell suspension into the magnetic column, then adding 500 microliters of binding buffer into the magnetic column, thoroughly flushing the filtrate, repeating the steps for three times, and collecting the filtrate by using a 15-milliliter centrifuge tube. 300X g for 5min, the supernatant was aspirated and the cell pellet was resuspended in PBS containing 0.04% BSA;
the prepared single cell suspension was examined for cell viability and counted by an automatic counter, and as shown in FIG. 1, the cell suspension obtained by the above method had a cell concentration of about 2.31X 10 5 One/ml, activity 63.11%, fineThe degree of cell dispersion was observed in the state where 90.44% of the cells were single cells.
Comparative example 2
The invention provides a preparation method of a human periodontitis gingival tissue single cell suspension, which comprises the following specific steps:
1. taking about 10mg of human periodontitis gingival tissue, placing the tissue in a sterile 6 cm culture dish, adding 4ml of calcium-magnesium-free PBS (phosphate buffer solution) containing 5% of penicillin and streptomycin, washing, and removing macroscopic blood on the surface of the tissue; after three repeated washes, the tissue was transferred to a sterile 6 cm petri dish, a small amount of a calcium-magnesium-free PBS solution containing 5% penicillin and streptomycin was added to just cover the tissue, and the tissue was processed to 0.3mm using sterile tissue scissors 3 The left and right fragments; the mixed solution in the petri dish was transferred to a 15 ml centrifuge tube in its entirety. Centrifuging for 300X g min;
2. adding 1 ml of Dispase II (preheated at 37 ℃) into a centrifuge tube, uniformly mixing, putting the mixture into a constant-temperature water bath box at 37 ℃, digesting for 60min, and thoroughly shaking and uniformly mixing the suspension every 5 min;
3. digestion was stopped by adding 4ml of alpha-mem medium containing 10% fetal bovine serum and 1% penicillin and streptomycin. Filtering the obtained mixed solution with a cell strainer with a pore size of 40 μm, centrifuging the filtrate at 4 deg.C of 300X g for 10min, carefully sucking off the supernatant;
4. adding 2ml erythrocyte lysate, standing for 2min at room temperature; 300X g centrifuging for 5 min; sucking off the supernatant, adding 2ml of PBS solution containing 0.04% BSA for resuspension and precipitation, centrifuging for 5min at 300X g, and sucking off the supernatant;
5. adding 100 microliters of dead cell removal magnetic beads into the cell sediment, resuspending the sediment, standing at room temperature for 15min, then rinsing the magnetic column with 500 microliters of binding buffer, adding 500 microliters of binding buffer into the cell suspension, transferring the cell suspension into the magnetic column, then adding 500 microliters of binding buffer into the magnetic column, thoroughly flushing the filtrate, repeating the steps for three times, and collecting the filtrate by using a 15-milliliter centrifuge tube. 300X g for 5min, the supernatant was aspirated and the cell pellet was resuspended in PBS containing 0.04% BSA;
cell viability examination of the prepared single cell suspensions using an automated counterAnd cell counting, as shown in FIG. 1, which is a physical map of cells derived from an automatic counter, it can be seen that the cell concentration of the cell suspension obtained by the above method is about 7.76X 10 5 The cell size per ml was 75.1%, and 61.39% of the cells were present as single cells, as seen by the degree of cell dispersion.
Experimental example 1
Statistics of cell concentration and activity of the cell suspensions obtained in examples 1 to 4 and comparative examples 1 to 2 are shown in table 1.
TABLE 1
As can be seen from Table 1:
in comparative example 1-comparative example 2, the single cell suspension concentration was low and the cell survival rate was low;
the preparation method of the human periodontitis gingival tissue single cell suspension provided by the embodiment of the invention does not need specialized equipment and skills, can obtain a large amount of high-activity single cell suspensions in short time, and specifically comprises the following steps: the cell survival rate reaches 89.2-91.8%; the concentration of single cell suspension can be up to 8.84X 10 5 ～3.29×10 6 。
Finally, it should also be noted that the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
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 preparing a single cell suspension of human periodontitis gingival tissue, which comprises the following steps:
washing, shearing and centrifuging human periodontitis gingival tissues to remove supernatant to obtain tissue precipitates;
performing enzymolysis digestion on the tissue precipitate by adopting collagenase I and Dispase II to obtain a mixed solution, stopping digestion, filtering by adopting a filter screen, collecting filtrate, and centrifuging to obtain a first cell precipitate;
adding erythrocyte lysate into the first cell sediment, standing, centrifuging to remove supernatant, cleaning, and centrifuging to obtain a second cell sediment;
adding dead cells into the second cell sediment to remove magnetic beads, and incubating to obtain a cell suspension containing the magnetic beads; and (3) placing the suspension containing the magnetic beads into a magnetic column for filtration, collecting filtrate, centrifuging to obtain a third cell precipitate, and re-suspending to obtain the single cell suspension of the gingival tissue.
2. The method for preparing the single cell suspension of human periodontitis according to claim 1, wherein the rinsing is performed with a pre-cooled calcium-magnesium-free PBS solution containing 5% penicillin and streptomycin; shearing the mixture to 0.2-0.4 mm in the shearing process 3 Tissue patch of (2).
3. The method for preparing the single cell suspension of human periodontitis gingival tissue according to claim 1, wherein the collagenase type I and the Dispase II are pre-dissolved in an alpha-mem culture medium to obtain an enzyme-containing culture medium, and the working concentrations of the collagenase type I and the Dispase II are 5-7 mg/ml and 7-9 mg/ml respectively; and carrying out enzymolysis digestion on the tissue sediment by adopting the enzyme-containing culture medium.
4. The method for preparing the human periodontitis gingival tissue single-cell suspension according to claim 3, wherein a volume ratio of the tissue sediment to the enzyme-containing medium is 1: 9 to 11.
5. The method for preparing the human periodontitis single-cell suspension of the gingival tissue according to claim 1, wherein in the enzymolysis digestion, the water bath digestion at 37 ± 0.5 ℃ is adopted for 50-80 min, and the resuspension of the tissue sediment is performed at intervals of 5min with shaking.
6. The method of claim 1, wherein the stopping digestion is performed by using an α -mem culture medium containing 10% fetal bovine serum, 1% penicillin and streptomycin, and the volume of the α -mem culture medium is 3 to 5 times the volume of the mixed solution.
7. The method of claim 1, wherein the cell filter mesh has a pore size of 40 μm.
8. The method for preparing the human periodontitis single-cell suspension of the gingival tissue, according to claim 1, wherein a ratio of a volume of the erythrocyte lysate to a volume of the first cell precipitate is 95-105: 1.
9. the method of claim 1, wherein the ratio of the volume of the dead cell-depleted magnetic beads to the volume of the second cell pellet is 18-22: 1, adding dead cells to remove magnetic beads, and incubating for 10-30 min.
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