CN109172869B

CN109172869B - Human milk dental stem cell microcapsule with enriched cell surface marker specificity and preparation method thereof

Info

Publication number: CN109172869B
Application number: CN201811126923.6A
Authority: CN
Inventors: 徐随民
Original assignee: Nanjing Taisheng Biological Technology Co ltd
Current assignee: Nanjing Taisheng Biological Technology Co ltd
Priority date: 2018-09-26
Filing date: 2018-09-26
Publication date: 2020-05-22
Anticipated expiration: 2038-09-26
Also published as: CN109172869A

Abstract

The invention mainly relates to a human tooth stem cell microcapsule with enriched cell surface marker specificity and a preparation method thereof. The invention also relates to a preparation method and a biological efficacy detection method thereof. The invention also relates to the use of the cell microcapsules prepared by the invention for regenerative medicine.

Description

Human milk dental stem cell microcapsule with enriched cell surface marker specificity and preparation method thereof

Technical Field

Background

Mesenchymal stem cells are pluripotent stem cells with self-replication capacity and multidirectional differentiation potential, and play an important role in the processes of development of organisms and maintenance of tissue organ Homeostasis (Homeostasis) after developmental maturation. Its characteristic makes it a seed cell ideal for regenerative medicine and can be used for repairing the damage of various tissues and organs.

In the repair process, the long-term survival and the maintenance of biological characteristics of the implanted stem cells are the key points for ensuring the curative effect of the implanted stem cells. Although mesenchymal stem cells have low immunogenicity and immunoregulatory activity, animal experimental results show that transplanted mesenchymal stem cells cannot keep alive for a long time.

To solve this problem, stem cells can be encapsulated with biomaterials to make microcapsules. When transplanted into the body, the stem cells can be spatially isolated from the body's immune cells and inflammatory environment. At the same time, oxygen, nutrients and metabolic waste products can be effectively exchanged through the biomaterial having the characteristics of the semi-permeable membrane. Thus, the implanted stem cells can survive and function for a long period of time without the use of immunosuppressive agents.

The biological material is selected from one or more of natural polymer materials, collagen, alginate, chitosan and hyaluronic acid.

Alginates are salts of alginic acid. Alginic acid is extracted from herba Zosterae MarinaePolyuronic acid, insoluble in water, but its sodium salt is readily soluble in water. Alginate solution is obtained by mixing with divalent cation, such as Ca²⁺Crosslinking may form a gel. Alginate gels are non-toxic and do not elicit an immune response after injection into the body, but have the disadvantage of being large and unstable.

The chitosan is a derivative of chitin, can be subjected to covalent crosslinking with genipin, and has the characteristics of no toxicity, good biocompatibility, anti-inflammation and the like after crosslinking.

The alginate gel is coated with a chitosan film, and the chitosan film which is covalently crosslinked can provide better stability. The stable microcapsule can prolong the retention time of cells in vivo and improve the effectiveness of the cells.

The size of the microcapsules has a large effect on the viability of the cells enclosed therein. Too large a diameter (>500 μm) of the microcapsules, insufficient cell diffusion of nutrients and oxygen to the center of the microcapsules, and non-timely discharge of metabolic waste products, can lead to a decrease in cell viability. The micro-fluid technology can prepare the micro-capsule with uniform particle size distribution and the diameter less than 500 μm.

Deciduous tooth stem cells are one of the mesenchymal stem cells, while the subset specifically enriched by cell surface markers, preferably CD18⁺The deciduous tooth stem cell subgroup has stronger clone forming capability and better osteogenic differentiation capability, and has excellent scientific research and clinical application prospects.

Disclosure of Invention

The invention provides a human tooth stem cell microcapsule with enriched cell surface marker specificity and a preparation method thereof. Specifically, the method is realized by the following technical scheme:

the method employs a combination of digestive enzymes to digest human milk dental tissue to obtain a single cell suspension. Including but not limited to collagenase, dispase, hyaluronidase, pancreatin, EDTA, etc., which in combination can greatly increase the yield of cells.

The collagenase may be used in the present method at a concentration of 1mg/ml, 2mg/ml, 3mg/ml, 4mg/ml, 5mg/ml, 6 mg/ml, 7 mg/ml, 8 mg/ml, 9 mg/ml, 10 mg/ml. The concentration of the dispersing enzyme used may be 1mg/ml, 2mg/ml, 3mg/ml, 4mg/ml, 5mg/ml, 6 mg/ml, 7 mg/ml, 8 mg/ml, 9 mg/ml, 10 mg/ml.

The method is used for enriching the milk tooth stem cell subset by specifically enriching cell surface markers, preferably CD18, and CD18 can be labeled by fluorescent markers or magnetic beads. Accordingly, accurate cell separation techniques include flow cytometry sorting, or immunomagnetic bead sorting.

The basic culture medium for the in vitro amplification of the CD18 positive stem cells in the method can be α -MEM, DMEM, D/F-12, RPMI-1640 and the like, and the complete culture medium is prepared by adding fetal bovine serum, ascorbic acid 2-phosphate, glutamine, penicillin, streptomycin and the like on the basis of the basic culture medium.

The concentration of fetal bovine serum in the complete medium may be 5%, 10%, 15%, 20%. The concentration of ascorbic acid 2-phosphate may be 50. mu.M, 100. mu.M, 150. mu.M, 200. mu.M. Other additive concentrations are added according to art-recognized optimization schemes.

The expansion passage ratio of the CD18 positive stem cells in the method can be 1: 3,1: 4, or 1: 5. the cells used to prepare the microcapsules are passage 4, passage 5, or passage 6.

The concentration of alginate solution used to prepare the cell microspheres in the present method may be 1%, 2%, or 3%. The prepared solution needs to be filtered and sterilized by a filter membrane with the diameter of 0.22 mu m.

The alginate solution in this method had a cell resuspension concentration of 1X 10⁶/ml，1.5×10⁶Per ml, or 2X 10⁶/ml。

In the method, the microfluid device for controlling the size of the cell microsphere is designed into a horizontal channel and a vertical channel, and the alginate solution containing the cells is vertically dropped into a plate filled with a calcium chloride solution downwards to form the cell microsphere. The soybean oil flows horizontally and transversely, cuts off the alginate solution and controls the size of the cell microspheres. The cell microspheres prepared by the microfluidic device have a diameter of less than 500 μm.

The concentration of the calcium chloride solution used for preparing the cell microspheres in the method can be 0.1M or 0.2M, and the prepared solution needs to be filtered and sterilized by a filter membrane of 0.22 mu M.

The cell microspheres prepared by the method need to be incubated in a calcium chloride solution at 37 ℃ for 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, or 45 minutes.

The concentration of the chitosan solution used to prepare the cell microcapsules in the present method may be 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%. The prepared solution needs to be filtered and sterilized by a filter membrane with the diameter of 0.22 mu m.

The time for coating the cell microspheres with the chitosan solution was 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes.

The concentration of the genipin solution used for crosslinking with chitosan in the present method may be 1mg/ml, 2mg/ml, 3mg/ml, 4mg/ml, 5 mg/ml. The prepared solution needs to be filtered and sterilized by a filter membrane with the diameter of 0.22 mu m.

In the method, the chitosan and the genipin are incubated for 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours and 10 hours at the temperature of 37 ℃.

The invention also provides a method for detecting the in vitro biological efficacy of the cell microcapsule. Specifically, the method is realized by the following technical scheme:

the method takes the in vitro osteogenesis inducing differentiation capability of the cell microcapsule as a method for evaluating the biological effectiveness of the cell microcapsule.

The osteogenesis induction culture medium is prepared by adding dexamethasone, vitamin C, β -phosphoglycerol, etc. on the basis of basic culture medium.

Dexamethasone can be used in the osteogenesis induction medium at a concentration of 0.05mM, 0.1mM, 0.15mM, 0.2mM, 0.25mM, 0.3mM, 0.35mM, 0.4mM, 0.45mM, 0.5 mM.

The vitamin C may be used at a concentration of 10mg/mL, 20mg/mL, 30mg/mL, 40mg/mL, 50mg/mL, 60mg/mL, 70mg/mL, 80mg/mL, 90mg/mL, 100 mg/mL.

β Glycerol phosphate can be used in a concentration of 5mM, 6 mM, 7 mM, 8mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM.

The method comprises the steps of preparing tissue sections by using cell microcapsules cultured for 4 weeks in an in vitro induction manner, and detecting osteogenic differentiation capacity of cells in the microcapsules by alizarin red staining, so as to evaluate the biological efficacy of the cell microcapsules.

The cell microcapsule prepared by the invention can be directly used for regenerative medicine.

Drawings

FIG. 1 subpopulation of CD18 positive stem cells

FIG. 2 phenotypic analysis of CD18 Positive Stem cell subsets

FIG. 3 microcapsule preparation apparatus

FIG. 4 CD18 Positive Stem cell microcapsules

FIG. 5 proliferation potency assay.

Detailed Description

Example 1 isolation and culture of CD18 Positive deciduous tooth Stem cells

Materials and methods

1, collecting normally fallen deciduous teeth of children, immediately placing the deciduous teeth in α -MEM culture medium which is precooled at 4 ℃ and contains 50-300U/mL penicillin, 50-300 mu g/mL streptomycin and 1-5 mu g/mL amphotericin B, transporting the deciduous teeth to a clean laboratory at the temperature of 2-8 ℃, wherein the time interval from collection to extraction of deciduous tooth stem cells does not exceed 72 hours;

2, transferring to a biological safety cabinet, taking out the milk cutting teeth to a culture dish, and washing with a sterile phosphate buffer solution precooled at 4 ℃, namely washing the outer surface of the milk cutting teeth with PBS;

3, separating pulp tissues from the residual dental crowns;

4, placing the dental pulp tissue in PBS containing 1-10 mg/ml type I collagenase and 1-10 mg/ml dispase, and digesting the dental pulp tissue for 1 hour at 37 ℃;

5, filtering the digestive juice through a 70-micron cell sieve to obtain a single cell suspension containing human deciduous tooth stem cells, centrifuging at 900rpm for 5min, and washing the cells once by using PBS;

6, resuspending the cells by PBS, adding the CD18 monoclonal antibody, uniformly mixing, and incubating for 30 minutes at room temperature in a dark place;

7, after washing, centrifuging at 900rpm for 5min, and resuspending the cells with PBS;

and 8, obtaining the CD18 positive stem cell subset by flow cytometry sorting or immunomagnetic bead sorting.

The results showed that the cultured cells were spindle-shaped, growing in a vortex (fig. 1), the cell phenotype was CD18 positive (fig. 2);

and 9, changing the solution every 3 to 4 days, and after 90 percent of cells are fused, 1: passage 3, and the cell used for preparing the microsphere is the fourth passage.

EXAMPLE 2 preparation of cell microcapsules

Materials and methods

1. Dissolving alginate in PBS to obtain 1-3% alginate solution, filtering with 0.22 μm filter membrane, and packaging;

2. digesting the CD18 positive deciduous tooth stem cells cultured to the fourth generation by pancreatin, centrifuging at 900rpm for 5min, and washing the cells once by PBS;

3. resuspending the harvested CD18 positive deciduous tooth stem cells with alginate solution to adjust the cell concentration to 1-2X 10⁶/ml；

4. The alginate solution containing the cells was dropped vertically down through channel 1 into a dish containing 0.1-0.2M calcium chloride solution to form cell microspheres by a horizontal, vertical dual channel microfluidic device as shown in FIG. 3. Soybean oil horizontally and transversely flows through the channel 2, the alginate solution is cut off, and the size of the cell microspheres is controlled;

5. incubating the microspheres at 37 ℃ for 15-45 minutes until complete crosslinking is achieved;

6. coating alginate cell microspheres in 0.1-1% chitosan solution for 15-45 min;

7. then placing the mixture into 1-5 mg/ml genipin aqueous solution to incubate for 1-10 hours at 37 ℃ to complete crosslinking, and preparing the cell microcapsule.

8. The cell microcapsules were washed and collected with α -MEM, inoculated into α -MEM medium containing 10% -20% fetal bovine serum, 50. mu.M-200. mu.M ascorbic acid 2-phosphate, 2mM glutamine, 100U/mL penicillin, 100. mu.g/mL streptomycin, and placed at 37 ℃ with 5% CO₂Culturing in an incubator;

9. the shape and diameter of the formed cell microcapsules were observed using an optical microscope.

The results show that the prepared cell microcapsule is spherical and has uniform size. The cells were uniformly distributed therein, with a circular shape (FIG. 4).

Example 3 in vitro biological potency assay of cell microcapsules

Materials and methods

1. Collecting cell microcapsules, inoculating in α -MEM medium containing 10% -20% fetal calf serum, 50 μ M-200 μ M ascorbic acid 2-phosphate, 2mM glutamine, 100U/mL penicillin, 100 μ g/mL streptomycin, placing at 37 deg.C and 5% CO₂Culturing in an incubator;

2. after 24-48 hours of culture, the medium was changed to osteogenic induction medium (α -MEM medium containing 10% fetal bovine serum, 100U/mL penicillin, 100. mu.g/mL streptomycin, 0.05-0.5mM dexamethasone, 10-100 mg/mL vitamin C and 5-15 mM β -phosphoglycerate);

3. changing the liquid every 3-4 days;

4. after 4 weeks of induction culture, fixing the cell microcapsules for 30 minutes by using 4% paraformaldehyde at room temperature;

5. placing in PBS for 15 minutes;

6. dehydrating, transparentizing, embedding in paraffin, slicing, dewaxing and rehydrating;

7. placing the cell microcapsule tissue section in 2% alizarin red solution, and staining for 5min at room temperature;

8. after washing with tap water, counterstaining with hematoxylin;

9. and (6) performing mounting microscopic examination.

The results showed that after 3-4 weeks osteogenic induction culture in vitro, mineral deposition was visible between the cells in the microcapsules by alizarin red staining (FIG. 5), indicating CD18 in the microcapsules⁺The deciduous tooth stem cells can be differentiated into osteoblasts, and the biological characteristics of the deciduous tooth stem cells are not influenced by microcapsule coating.

Claims

1. A human dental stem cell microcapsule specifically enriched by cell surface markers, characterized by:

the stem cells are human deciduous tooth stem cells, the human years are 5-8 years old, CD18 is used as a cell surface marker, and CD18 is labeled by fluorescent markers or magnetic beads;

the deciduous teeth are selected from deciduous incisors of children who normally fall off;

the microcapsule comprises a microsphere inner core and a microsphere outer shell, and the diameter of the microsphere is less than 500 mu m;

the microcapsule core material is selected from alginate;

the microcapsule shell material is selected from chitosan film, and the chitosan film is covalent cross-linked.

2. A method for preparing the human deciduous tooth stem cell microcapsule according to claim 1, which comprises the following steps:

step 1, dissolving alginate in PBS to prepare 1-3% alginate solution, filtering through a 0.22 mu m filter membrane, and subpackaging and storing;

step 2, digesting the CD18 positive deciduous tooth stem cells cultured to the fourth generation by pancreatin, centrifuging at 900rpm for 5min, and washing the cells once by PBS;

step 3, resuspending the CD18 positive deciduous tooth stem cells obtained in step 2 with alginate solution, and adjusting the cell concentration to (1-2). times.10⁶/ml；

Step 4, through a horizontal and vertical double-channel microfluid device, an alginate solution containing cells is dripped downwards into a plate filled with 0.1-0.2M calcium chloride solution through a channel to form cell microspheres; soybean oil flows transversely through the channel, alginate solution is cut off, and the size of the cell microspheres is controlled;

step 5, incubating the microspheres at 37 ℃ for 15-45 minutes until complete crosslinking is achieved;

step 6, coating the alginate cell microspheres in (0.1-1)% chitosan solution for 15-45 minutes;

step 7, then putting the cell into 1-5 mg/ml genipin aqueous solution, incubating the cell for 1-10 hours at 37 ℃ to complete crosslinking, and preparing cell microcapsules;

step 8, washing and collecting the cell microcapsules with α -MEM, inoculating the microcapsules in α -MEM medium containing 10% -20% fetal bovine serum, 50. mu.M-200. mu.M ascorbic acid 2-phosphate, 2mM glutamine, 100U/mL penicillin, 100. mu.g/mL streptomycin, and placing the microcapsules at 37 ℃ in 5% CO₂Culturing in an incubator;

and 9, observing the shape and the diameter of the formed cell microcapsule by using an optical microscope.

3. A method for preparing the human deciduous tooth stem cell microcapsule according to claim 2, wherein the CD18 positive deciduous tooth stem cells cultured to the fourth generation are prepared by the following steps:

step 1, collecting normally fallen deciduous teeth of children, immediately placing the deciduous teeth into α -MEM culture medium which is precooled at 4 ℃ and contains 50-300U/mL penicillin, 50-300 mu g/mL streptomycin and 1-5 mu g/mL amphotericin B, transporting the deciduous teeth to a clean laboratory at the temperature of 2-8 ℃, wherein the time interval from collection to extraction of deciduous teeth stem cells is;

step 2, transferring to a biological safety cabinet, taking out the milk cutting teeth to a culture dish, and washing with a sterile phosphate buffer solution precooled at 4 ℃, namely washing the outer surface of the milk cutting teeth with PBS;

step 3, separating pulp tissues from the residual dental crowns; step 4, placing the dental pulp tissue in PBS containing 1-10 mg/ml type I collagenase and 1-10 mg/ml dispase, and digesting the dental pulp tissue for 1 hour at 37 ℃;

step 5, filtering the digestive juice through a 70-micron cell sieve to obtain a single cell suspension containing human deciduous tooth stem cells, centrifuging at 900rpm for 5min, and washing the cells once with PBS;

step 6, resuspending the cells with PBS, adding the CD18 monoclonal antibody, mixing uniformly, and incubating for 30 minutes at room temperature in the dark;

step 7, after washing, centrifuging at 900rpm for 5min, and resuspending the cells with PBS;

step 8, obtaining a CD18 positive stem cell subset through flow cytometry sorting or immunomagnetic bead sorting;

step 9, changing the liquid every 3 to 4 days, and after 90 percent of cells are fused, 1: passage 3, and the cell used for preparing the microsphere is the fourth passage.

4. The method for detecting the dryness of the human deciduous tooth stem cell microcapsule according to claim 1, which comprises the following specific steps:

step 1, collecting cell microcapsules, inoculating the microcapsules in 10-20% fetal calf serum and 50-200 μ M of ascorbic acid2-phosphate ascorbate, 2mM glutamine, 100U/mL penicillin, and 100. mu.g/mL streptomycin in α -MEM, and was incubated at 37 ℃ with 5% CO₂Culturing in an incubator;

step 2, after culturing for 24-48 hours, replacing the culture medium with an osteogenesis induction culture medium, namely a α -MEM culture medium containing 10% fetal calf serum, 100U/mL penicillin, 100 mu g/mL streptomycin, 0.05-0.5mM dexamethasone, 10-100 mg/mL vitamin C and 5-15 mM β -glycerol phosphate, and changing the culture medium every 3-4 days;

step 3, after the induction culture for 4 weeks, fixing the cell microcapsules for 30 minutes by using 4% paraformaldehyde at room temperature;

step 4, placing the mixture in PBS for 15 minutes;

step 5, dehydrating, transparentizing, embedding in paraffin, slicing, dewaxing and rehydrating;

step 6, placing the cell microcapsule tissue section in a 2% alizarin red solution, and dyeing for 5min at room temperature;

step 7, washing with tap water, and counterstaining with hematoxylin;

and 8, performing mounting microscopic examination.

5. Use of the human deciduous tooth stem cell microcapsule according to claim 1 for regenerative medicine for bone diseases and preparation of tissue engineering materials.