A solution for digesting alveolar bone tissue and its use and method in the isolation of alveolar bone cells

Abstract

The present application relates to the field of cell biology, in particular to a kind of alveolar bone tissue digestive juice and its use and method in alveolar bone cell separation, the alveolar bone tissue digestive juice includes basic culture medium, collagenase, DNA enzyme, BSA, EDTA, based on the total volume of alveolar bone tissue digestive juice, the final concentration of the collagenase is 200-400 U / ml, the final concentration of the DNA enzyme is 50-150 U / ml, the final concentration of the BSA is 0.1-2 mg / ml, and the final concentration of the EDTA is 0.5-1.5 mM.The separation method of the alveolar bone cell includes alveolar bone tissue in the alveolar bone tissue digestive juice is digested in several times, the alveolar bone tissue is grinded in the process, etc.Finally, alveolar bone cell is obtained.The alveolar bone tissue digestive juice and the separation method of alveolar bone cell of the present application realize the rapid and efficient extraction of alveolar bone tissue single cell suspension, and meet the high activity and high concentration required by single cell sequencing.

Description

Technical Field

[0001] The present invention relates to the field of cell biology, in particular to an alveolar bone tissue digestion fluid and its application and method in isolating alveolar bone cells. Background Art

[0002] Alveolar bone tissue is a special bone tissue located at the lower edge of the maxilla and the upper edge of the mandible surrounding the tooth roots. It includes three parts: cortical bone, cancellous bone, and proper alveolar bone. It can be seen that the structure of alveolar bone is complex. The cell populations contained in the alveolar bone tissue are extremely diverse and relatively small in number. They include stem cells with diverse differentiation potentials, osteoblasts, osteoclasts, and a variety of immune cells. These cell populations play a complex and coordinated role in maintaining the homeostasis of periodontal tissues and regulating the regeneration of periodontal tissues. However, due to the small size, low cell content, and high hard tissue content of the alveolar bone tissue itself, it is difficult to extract cells (i.e., alveolar bone cells) in the alveolar bone tissue, which in turn limits further related research on the alveolar bone.

[0003] Currently, alveolar bone cell extraction often involves prolonged digestion with digestive enzymes, which can lead to problems such as low cell yield, low cell viability, low purity, and a high proportion of bone debris. Furthermore, the alveolar bone cell suspension obtained in this way often lacks certain fragile and sensitive cell populations, such as stem cells and certain immune cell populations. These issues have significantly limited the research on alveolar bone tissue. Summary of the Invention

[0004] In view of the above-mentioned shortcomings of the prior art, the object of the present invention is to provide an alveolar bone tissue digestion fluid and its use and method in alveolar bone cell separation, so as to solve the problems in the prior art.

[0005] To achieve the above-mentioned purpose and other related purposes, the present invention provides an alveolar bone tissue digestion solution, which includes a basal culture medium, collagenase, DNA enzyme, BSA, and EDTA. Based on the total volume of the alveolar bone tissue digestion solution, the final concentration of the collagenase is 200-400 U / ml, the final concentration of the DNA enzyme is 50-150 U / ml, the final concentration of the BSA is 0.1-2 mg / ml, and the final concentration of the EDTA is 0.5-1.5 mM.

[0006] The present invention also provides use of the alveolar bone tissue digestion fluid in isolating alveolar bone cells.

[0007] The present invention provides a method for isolating alveolar bone cells, which comprises the following steps:

[0008] 1) placing the alveolar bone tissue in the alveolar bone tissue digestion solution for digestion;

[0009] 2) grinding the alveolar bone tissue during digestion;

[0010] 3) transferring the mixed system from step 2) to a shaker to continue digestion, and after digestion is complete, releasing the cells from the alveolar bone tissue into the digestive fluid;

[0011] 4) Transfer the cell-containing digestion solution to another container to terminate the digestion; add new alveolar bone tissue digestion solution to the alveolar bone tissue and continue digestion on a shaker;

[0012] 5) Repeat step 4) multiple times;

[0013] 6) combining the digestive fluid containing cells and alveolar bone tissue after multiple digestions;

[0014] 7) Removing bone debris from the mixed system of step 6) to obtain alveolar bone cells.

[0015] As described above, the alveolar bone tissue digestion solution of the present invention and its use and method for isolating alveolar bone cells have the following beneficial effects: rapid extraction of alveolar bone tissue single-cell suspensions is achieved, and the high activity and high concentration required for single-cell sequencing are met, which will facilitate the study of alveolar bone metabolism-related diseases at the single-cell level, as follows:

[0016] 1. Can be successfully used from a volume of about 15-20mm 3 30,000-50,000 alveolar bone tissue single cells were extracted from the alveolar bone tissue.

[0017] 2. The extracted cells have an activity of up to 90%.

[0018] 3. The extracted alveolar bone single-cell suspension can be successfully used in single-cell sequencing experiments, enabling detailed analysis of cell populations within the alveolar bone tissue. This overcomes the high activity and high concentration requirements of single-cell sequencing for cells from diverse tissues, such as pancreas, myocardium, and bone, laying the foundation for single-cell sequencing studies of bone tissue, particularly alveolar bone-related diseases.

[0019] 4. The extracted single-cell suspension of alveolar bone tissue can also be used to extract corresponding cells through corresponding cell surface markers for subsequent research. It is particularly suitable for the extraction of human alveolar bone stem cells, and can solve the problems of alveolar bone cell extraction, low cell yield, low cell activity, and difficulty in conducting subsequent experiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Shown is a diagram showing the results of flow cytometry sorting of cells separated using the method of the present invention.

[0021] Figure 2 Shown is a graph showing the cell activity ratio obtained by detection and sorting.

[0022] Figure 3 A graph showing the population diversity of cells obtained by detection and sorting.

[0023] Figure 4 Shown is the single-cell sequencing method used to detect various immune cell subsets in the alveolar bone. DETAILED DESCRIPTION

[0024] The present invention first provides an alveolar bone tissue digestion solution, which includes a basal culture medium, collagenase, DNA enzyme, BSA, and EDTA. Based on the total volume of the alveolar bone tissue digestion solution, the final concentration of the collagenase is 200-400 U / ml, the final concentration of the DNA enzyme is 50-150 U / ml, the final concentration of the BSA is 0.1-2 mg / ml, and the final concentration of the EDTA is 0.5-1 mM.

[0025] In one embodiment, the basal culture medium is selected from α-MEM, BME, DMEM, HAM F12, RPMI1640 or 199 medium. In a preferred embodiment, the basal culture medium is α-MEM.

[0026] The collagenase is selected from a single type of collagenase or a mixed type of collagenase. The single type of collagenase is selected from type I, type II, type III, type IV, or type V collagenase. In a preferred embodiment, the collagenase is selected from type I collagenase. The mixed type of collagenase is a combination of several types of types I, II, III, IV, and V.

[0027] The final concentration of the collagenase is selected from any of the following ranges: 200-250 U / ml, 250-270 U / ml, 270-290 U / ml, 290-300 U / ml, 300-310 U / ml, 310-330 U / ml, 330-350 U / ml, or 350-400 U / ml. Preferably, the final concentration of the collagenase is 295-305 U / ml. More preferably, the final concentration of the collagenase is 300 U / ml.

[0028] The DNA enzyme is DNase I, also known as Deoxyribonuclease I. DNase I is an endonuclease that can digest single-stranded or double-stranded DNA to produce monodeoxynucleotides or single-stranded or double-stranded oligodeoxynucleotides. The products of DNase I hydrolysis of single-stranded or double-stranded DNA have a phosphate group at the 5' end and a hydroxyl group at the 3' end.

[0029] The final concentration of the DNA enzyme is selected from any of the following ranges: 50-70 U / ml, 70-90 U / ml, 90-100 U / ml, 100-110 U / ml, 110-130 U / ml, or 130-150 U / ml. Preferably, the final concentration of the DNA enzyme is 90-110 U / ml. More preferably, the final concentration of the DNA enzyme is 100 U / ml.

[0030] BSA refers to bovine serum albumin. The final concentration of BSA is selected from any of the following ranges: 0.1-0.5 mg / ml, 0.5-0.7 mg / ml, 0.7-0.9 mg / ml, 0.9-1.1 mg / ml, 1.1-1.3 mg / ml, 1.3-1.5 mg / ml, or 1.5-2 mg / ml. Preferably, the final concentration of BSA is 0.9-1.1 mg / ml. More preferably, the final concentration of BSA is 1 mg / ml.

[0031] The EDTA is ethylenediaminetetraacetic acid. The final concentration of the EDTA is selected from any of the following ranges: 0.5-0.7 mM, 0.7-0.9 mM, 0.9-1.1 mM, 1.1-1.3 mM, and 1.3-1.5 mM. Preferably, the final concentration of the EDTA is 0.9-1.1 mM. More preferably, the final concentration of the EDTA is 1 mM.

[0032] The alveolar bone tissue digestion solution further comprises antibiotics. In one embodiment, the antibiotics are a penicillin-streptomycin mixture.

[0033] In one embodiment, based on the total volume of the alveolar bone tissue digestion fluid, the final concentration of penicillin is 50 U / ml to 150 U / ml, and / or the final concentration of streptomycin is 0.05 to 0.15 mg / ml.

[0034] The alveolar bone tissue digestion solution was stored at 4° C. and preheated at 37° C. for 10 minutes before use.

[0035] The present invention also provides use of the alveolar bone tissue digestion fluid in isolating alveolar bone cells.

[0036] The present invention provides a method for isolating alveolar bone cells, which comprises the following steps:

[0037] 1) placing the alveolar bone tissue in the alveolar bone tissue digestion solution for digestion;

[0038] 2) grinding the alveolar bone tissue during digestion;

[0039] 3) transferring the mixed system from step 2) to a shaker to continue digestion, and after digestion is complete, releasing the cells from the alveolar bone tissue into the digestive fluid;

[0040] 4) Transfer the cell-containing digestion solution to another container to terminate the digestion; add new alveolar bone tissue digestion solution to the alveolar bone tissue and continue digestion on a shaker;

[0041] 5) Repeat step 4) multiple times;

[0042] 6) combining the digestive fluid containing cells and alveolar bone tissue after multiple digestions;

[0043] 7) Removing bone debris from the mixed system of step 6) to obtain a single cell suspension of alveolar bone cells.

[0044] In one embodiment, the alveolar bone tissue obtained clinically is placed in a complete culture medium and cryopreserved before the cell separation experiment. The cryopreservation refers to storage at 0-4°C.

[0045] In one embodiment, the alveolar bone tissue is washed to remove complete culture medium prior to digestion. The washing may be performed with PBS or normal saline.

[0046] In one embodiment, the alveolar bone tissue digestion solution is preheated before use. Preferably, the alveolar bone tissue digestion solution is preheated to 37°C.

[0047] In one embodiment, the ratio of alveolar bone tissue to alveolar bone tissue digestion fluid in step 1) is 15-20 mm 3 For alveolar bone tissue, use 4 ml of alveolar bone tissue digestion solution.

[0048] In one embodiment, grinding the alveolar bone tissue during the digestion process can refine the bone tissue, increase the contact area between the alveolar bone tissue and the digestive fluid, and accelerate the digestion process.

[0049] In one embodiment, the digestion conditions in step 3) on a shaker are any of the following: 34-38°C, 100-140 rpm, and horizontal shaking. Digestion on a shaker can also increase the contact area between the alveolar bone tissue and the digestive fluid, accelerating the digestion process.

[0050] In one embodiment, digestion is stopped using α-MEM medium containing 10% serum (complete medium).

[0051] Digesting the alveolar bone tissue multiple times in steps 4) and 5) ensures sufficient digestion and extraction of cells from the alveolar bone tissue. In a preferred embodiment, the digestion in step 5) is repeated twice.

[0052] In one embodiment, step 7) further comprises removing red blood cells from the mixed system. In one embodiment, red blood cell lysis solution can be used to lyse and remove red blood cells.

[0053] In one embodiment, step 7) is as follows: the mixed system obtained in step 6) is filtered through a 40 μm filter, and the red blood cell-removed mixed system is allowed to stand at low temperature for 5 minutes to allow the bone debris to settle to the bottom. The supernatant is then removed to obtain a single-cell suspension of alveolar bone cells. The isolated alveolar bone cells are all single cells.

[0054] In step 7), filtering the mixed system with a filter can remove larger bone fragments, and allowing the mixture to stand at low temperature can remove bone fragments not filtered out by the filter. After filtering and standing, the final single cell suspension contains a relatively small amount of bone fragments.

[0055] The following describes the embodiments of the present invention through specific examples. Those skilled in the art will readily understand the other advantages and benefits of the present invention from the disclosure herein. The present invention may also be implemented or applied through various other specific embodiments, and the details in this specification may be modified or altered based on different viewpoints and applications without departing from the spirit of the present invention.

[0056] Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing specific specific embodiments rather than for limiting the scope of protection of the present invention; in the present specification and claims, unless otherwise expressly stated herein, the singular forms "a", "an" and "the" include plural forms.

[0057] When the embodiments provide numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the understanding of the prior art by those skilled in the art and the description of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention may also be used to implement the present invention.

[0058] Example 1 Cell extraction from alveolar bone

[0059] 1. Remove alveolar bone fragments produced during clinical oral implant surgery, with a volume of approximately 15mm 3 Place in complete culture medium (10% FBS + 1% P / S + 89% α-MEM medium) and immediately place on ice.

[0060] 2. Rinse the bone fragments 2-3 times with PBS containing 1% P / S (penicillin-streptomycin mixture).

[0061] 3. Prepare alveolar bone digestion solution: Add 300 U / ml collagenase type I, 100 U / ml DNase I, 1% P / S, 1 mg / ml BSA, and 1 mM EDTA to α-MEM medium to make the digestion solution. Store at 4°C. Preheat to 37°C for 10 minutes before use.

[0062] 4. Place the alveolar bone fragments in 4 ml of digestive fluid and grind them carefully in a mortar to refine the bone tissue and increase the contact area between the bone tissue and the digestive fluid.

[0063] 5. Transfer the bone fragments and all digestion solutions to a 6 cm culture dish and shake horizontally at 37°C (120 rpm) for digestion. After digestion, single cells will be suspended in the digestion solution.

[0064] 6. Transfer the digestion solution containing single cells from step 5 to a new 50 ml centrifuge tube and add twice the volume of αMEM medium containing 10% FBS to the tube. Terminate the digestion and store on ice.

[0065] 7. Add 5 ml of new digestion solution to the bone fragments and continue digestion to gradually separate single cells from the bone fragments.

[0066] 8. Repeat steps 5-7 twice. After three cycles of digestion, transfer the digestion solution containing single cells and the bone fragments into the 50 ml centrifuge tube described above. Terminate the digestion process with complete culture medium.

[0067] 9. Filter the mixed solution obtained in step 8) through a 40 μm filter.

[0068] 10. Collect the cell pellet by centrifugation at 1000 rpm for 5 minutes.

[0069] 11. Add 1 ml of ACK lysis buffer to the cell pellet, mix thoroughly, and let it sit on ice for 5 minutes. Then add 15 ml of PBS to stop the lysis process.

[0070] 12. Centrifuge at 300g for 7 minutes to obtain the cell pellet.

[0071] 13. Resuspend the cell pellet in 2 ml of complete culture medium.

[0072] 14. Place on ice for 5 minutes to allow the bone fragments to settle to the bottom of the tube. Transfer 1.8 ml of the supernatant cell solution to a 5 ml flow cytometry tube.

[0073] 15. Centrifuge to obtain a cell pellet, resuspend in 200 μl PBS, add 2 μl of 2 mM Calcine solution, and incubate at 37°C for 30 minutes in the dark.

[0074] 16. After incubation, wash the single cell suspension twice with complete medium, resuspend after centrifugation, and sort the FITC-positive (Calcine-positive) cells on the machine. The results are as follows Figure 1 As shown, a group of living alveolar bone cells that are calcein-positive can be clearly separated, confirming that the isolated cells are the bone cells required for the experiment. Sorting the obtained cells can produce alveolar bone single cell suspension for subsequent experiments.

[0075] Example 2 Detection of the activity ratio of cells obtained by sorting

[0076] The alveolar bone cell samples obtained by sorting were stained for live and dead cells according to the following steps.

[0077] 1. Resuspend the obtained alveolar bone cells in PBS at a concentration of 5×10^5 cells / mL.

[0078] 2. Add 5 μl of Draq7 and 10 μl of calcein to each ml of cell suspension and incubate at 37°C in the dark for 20 minutes.

[0079] 3. After incubation, the cell suspension was analyzed for activity using a BD scanner (BD, USA).

[0080] The results are as follows Figure 2 As shown, after calcein staining (live cell staining), a large number of cells showed calcein-positive staining under the microscope (Figure A, white), indicating that the cells are live. Draq7 staining (dead cell staining) showed that Draq7-positive staining was almost invisible under the microscope (Figure B, white indicates positive staining). Calculating the proportion of calcein-positive cells to the total number of cells showed that the activity ratio of single alveolar bone cells obtained by this method can reach over 90% (N=5) (Figure C).

[0081] Example 3 Detection of Population Diversity of Sorted Cells

[0082] Flow cytometry analysis was performed on the harvested alveolar bone cells. The cell suspension was incubated with Thy1, PDPN, and CD45 flow cytometry antibodies at 4°C in the dark for half an hour before analysis by flow cytometry. The experimental procedures were identical to those in Example 1, except for the antibodies used.

[0083] The results are as follows Figure 3As shown, 15.7% of alveolar bone cells were positive for Thy1 (a marker of human skeletal precursor cells), 31.2% were positive for PDPN (a marker of human skeletal stem cells), and 0.45% were positive for CD45 (an immune cell marker). These results suggest that human alveolar bone cells extracted using this method can maintain good cell population diversity, meeting the requirements for single-cell sequencing.

[0084] Example 4 Single-cell sequencing of sorted cells

[0085] Single-cell sequencing is performed by a third-party company. The general steps are as follows: Single-cell suspensions of alveolar bone are collected and tested for viability, confirming that cell viability is greater than 80%. Each cell is captured using the BD sorting platform. RNA fragments within the cells are bound to magnetic beads. The bead-labeled RNA is reverse transcribed and introduced into CBs and UMIs. The resulting cDNA is amplified using random-primed PCR. The product is then sequenced using the Illumina HiSeq sequencing platform, generating 100GB of data per sample.

[0086] The results are as follows Figure 4 As shown, single-cell sequencing methods detected the presence of abundant immune cell subsets (T cells, B cells, monocytes, plasma cells, mast cells, neutrophils) as well as osteoblasts, mesenchymal cells, endothelial cells, perivascular cells, etc. in the alveolar bone.

[0087] Conclusion: The alveolar bone single cell suspension obtained by the method of the present invention can better ensure the cell activity and cell diversity of the alveolar bone.

[0088] The above examples are intended to illustrate the embodiments disclosed herein and are not to be construed as limiting the present invention. In addition, the various modifications listed herein and variations of the methods in the invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been specifically described in conjunction with various specific preferred embodiments of the present invention, it should be understood that the present invention should not be limited to these specific embodiments. In fact, various modifications apparent to those skilled in the art as described above to obtain the invention should be included within the scope of the present invention.

Claims

1. Use of alveolar bone tissue digestion fluid in isolating alveolar bone cells, characterized in that: The alveolar bone tissue digestion solution includes basal culture medium, collagenase, DNA enzyme, BSA, and EDTA. Based on the total volume of the alveolar bone tissue digestion solution, the final concentration of the collagenase is 200-400 U / ml, the final concentration of the DNA enzyme is 50-150 U / ml, the final concentration of the BSA is 0.1-2 mg / ml, and the final concentration of the EDTA is 0.5-1.5 mM. The collagenase is type I collagenase. The alveolar bone tissue digestion solution also includes antibiotics, and the DNA enzyme is DNAse I.

2. The use according to claim 1, characterized in that The basic culture medium is selected from α-MEM, BME, DMEM, HAMF12, RPMI1640 or 199 culture medium.

3. The use according to claim 1, characterized in that The antibiotic is a penicillin-streptomycin mixture. Based on the total volume of the alveolar bone tissue digestion fluid, the final concentration of the penicillin is 50 U / ml to 150 U / ml, and / or the final concentration of the streptomycin is 0.05 to 0.15 mg / ml.

4. A method for isolating alveolar bone cells, characterized in that: The separation method comprises the following steps: 1) digesting the alveolar bone tissue in the alveolar bone tissue digestion solution according to any one of claims 1 to 3; 2) grinding the alveolar bone tissue during digestion; 3) transferring the mixed system from step 2) to a shaker to continue digestion, and after digestion is complete, releasing the cells from the alveolar bone tissue into the digestive fluid; 4) Transfer the cell-containing digestion solution to another container to terminate the digestion; add new alveolar bone tissue digestion solution to the alveolar bone tissue and continue digestion on a shaker; 5) Repeat step 4) multiple times; 6) combining the digestive fluid containing cells and alveolar bone tissue after multiple digestions; 7) Removing bone debris from the mixed system of step 6) to obtain alveolar bone cells.

5. The separation method according to claim 4, characterized in that 15-20mm in step 1) 3 For alveolar bone tissue, use 4 ml of alveolar bone tissue digestion solution.

6. The separation method according to claim 4, characterized in that The conditions for continuing the digestion on a shaker in step 3) are any one of the following: 34-38° C., 100-140 rpm, and horizontal shaking.

7. The separation method according to claim 4, characterized in that Step 7) also includes removing red blood cells from the mixed system.

8. The separation method according to claim 4, characterized in that The steps of step 7) are as follows: the mixed system obtained in step 6) is filtered through a 40 μm filter, and the mixed system after removing red blood cells is placed at 0-4° C. to allow the bone debris to settle to the bottom, and the supernatant taken out is the alveolar bone cells.

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