CN109172869A - A kind of human milk tooth stem cell microcapsules of cell surface marker specific enrichment and preparation method thereof - Google Patents
A kind of human milk tooth stem cell microcapsules of cell surface marker specific enrichment and preparation method thereof Download PDFInfo
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Abstract
The invention mainly relates to human milk tooth stem cell microcapsules of a kind of cell surface marker specific enrichment and preparation method thereof, and the microcapsules include microballoon kernel and shell.Invention further relates to preparation method, biological efficacy detection method.The present invention also relates to the purposes that the cell microcapsules that will be prepared through the invention are used for regenerative medicine.
Description
Technical field
The invention mainly relates to a kind of human milk tooth stem cell microcapsules of cell surface marker specific enrichment and its preparations
Method, the microcapsules include microballoon kernel and shell.Invention further relates to preparation method, biological efficacy detection method.This
The cell microcapsules that invention is also related to preparing through the invention are used for the purposes of regenerative medicine.
Background technique
Mescenchymal stem cell is a kind of multipotential stem cell with the of self-replication capacity and multi-lineage potential, in body
It plays an important role in histoorgan stable state (Homeostasis) maintenance process after developing and reaching maturity.Its this characteristic makes
It becomes the ideal seed cell of regenerative medicine, can be used for the injury repair of Various Tissues organ.
In repair process, being implanted into stem cell long-term surviving and maintaining biological characteristics is the key that guarantee its curative effect.To the greatest extent
Pipe mescenchymal stem cell immunogenicity is low, and has immunoregulatory activity, but results of animal is shown, the mesenchyma of transplanting is dry
Cell is not able to maintain long-term surviving.
In order to solve this problem, stem cell can be wrapped up through biomaterial, microcapsules is made.When being implanted into internal,
Stem cell spatially can keep isolation with the immunocyte of body and inflammatory environment.Meanwhile oxygen, nutriment and metabolism are useless
Object can effectively be exchanged by the biomaterial with semi-transparent membrane property.In this way, under the premise of not using immunosuppressor,
The stem cell of implantation long-term surviving and can function.
The biomaterial is selected from natural macromolecular material, collagen, alginates, chitosan, one of hyaluronic acid
Or it is a variety of.
Alginates are the salts of alginic acid.Alginic acid is a kind of poly uronic acid extracted from kelp, not soluble in water, but
Its sodium salt is soluble easily in water.Alginate soln passes through and bivalent cation, such as Ca2+Crosslinking can form gel.Alginate jelly is nontoxic,
It will not cause to be immunoreacted after being injected in vivo, but the disadvantage is that hole is greatly and unstable.
Chitosan is the derivative of chitin, covalent cross-linking can occur with Geniposide, have nontoxic, bio-compatible after crosslinking
The features such as property is good, anti-inflammatory.
One layer of chitosan film is wrapped up outside alginate jelly, the chitosan film of covalent cross-linking can provide better stability.
Stable microcapsules can extend the residence time of cell in vivo, improve its validity.
The size of microcapsules has larger impact to Cell viability therein is wrapped up.The diameter of microcapsules is too big (> 500 μm),
The cellular invasion at nutrients and oxygen to microcapsules center is insufficient, and metabolic waste cannot be discharged in time, will lead under Cell viability
Drop.Micro-fluidic technologies can prepare that particle diameter distribution is uniform, and microcapsules of the diameter less than 500 μm.
Deciduous teeth stem cell is one kind of mescenchymal stem cell, and passes through the subgroup of cell surface marker specific enrichment, excellent
Select CD18+Deciduous teeth stem cell subgroup has stronger clonality, more preferably Osteoblast Differentiation ability, have superior scientific research and
Potential applicability in clinical practice.
Summary of the invention
The present invention provides a kind of human milk tooth stem cell microcapsules of cell surface marker specific enrichment and its preparation sides
Method, the microcapsules include microballoon kernel and shell.Specifically, being achieved by the following technical solution:
This method uses containing there are many combinations of digestive ferment and is digested to human milk dental tissue, obtains individual cells suspension.Including
But be not limited to clostridiopetidase A, dispase, hyaluronidase, pancreatin, EDTA etc., the combination of these digestive ferments is greatly improved obtaining for cell
Rate.
The use concentration of clostridiopetidase A can be 1 mg/ml, 2 mg/ml, 3 mg/ml, 4 mg/ml, 5 mg/ in this method
Ml, 6 mg/ml, 7 mg/ml, 8 mg/ml, 9 mg/ml, 10 mg/ml.The use concentration of dispase can be 1 mg/ml, and 2
Mg/ml, 3 mg/ml, 4 mg/ml, 5 mg/ml, 6 mg/ml, 7 mg/ml, 8 mg/ml, 9 mg/ml, 10 mg/ml.
This method is by cell surface marker specific enrichment, and preferably CD18 is enriched with deciduous teeth stem cell subgroup, CD18
Fluorescent marker or marked by magnetic bead can be used.Correspondingly, accurate cell separation technology includes selected by flow cytometry apoptosis, or exempt from
Epidemic disease magnetic bead sorting.
The amplification in vitro basal medium of CD18 Positive Stem Cells can be α-MEM, DMEM, D/F-12 in this method,
RPMI-1640 etc..Complete medium is to add fetal calf serum, ascorbic acid 2- phosphate, paddy on the basis of basal medium
Glutamine, penicillin, streptomysin etc..
The concentration of fetal calf serum can be 5%, 10%, 15%, 20% in complete medium.The concentration of ascorbic acid 2- phosphate
It can be 50 μM, 100 μM, 150 μM, 200 μM.Other additive concentration are added according to art-recognized prioritization scheme.
The amplification passage ratio of CD18 Positive Stem Cells can be 1:3,1:4 or 1:5 in this method.It is used to prepare micro- glue
The cell of capsule was the 4th generation, the 5th generation or the 6th generation.
The alginate soln concentration that cell microsphere is used to prepare in this method can be 1%, 2% or 3%.Prepared solution
It need to be through 0.22 μm of membrane filtration degerming.
It is 1 × 10 that the concentration of cell, which is resuspended, in alginate soln in this method6/ ml, 1.5 × 106/ ml, or 2 × 106/ml。
The microfluidic device that cell microsphere size is controlled in this method is horizontal, vertical binary channels design, containing cell
Alginate soln is instilled straight down in the plate equipped with calcium chloride solution, forms cell microsphere.The flowing of soybean oil horizontal cross,
Alginate soln is truncated, controls the size of cell microsphere.The cell microsphere diameter prepared by the microfluidic device is less than 500 μ
m。
Calcium chloride solution concentration used in cell microsphere is prepared in this method can be 0.1M or 0.2M, prepared molten
Liquid need to be through 0.22 μm of membrane filtration degerming.
It need to be incubated for 15 minutes, 20 points in calcium chloride solution under the conditions of 37 DEG C by cell microsphere prepared by this method
Clock, 25 minutes, 30 minutes, 35 minutes, 40 minutes or 45 minutes.
The chitosan solution concentration that cell microcapsules are used to prepare in this method can be 0.1%, 0.2%, 0.3%, 0.4%,
0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%.Prepared solution need to be through 0.22 μm of membrane filtration degerming.
It is 15 minutes with time of chitosan solution coated cell microballoon, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40
Minute, 45 minutes.
It can be 1mg/ml, 2mg/ml, 3mg/ml for the concentration with chitosan crosslinked genipin solution in this method,
4mg/ml, 5mg/ml.Prepared solution need to be through 0.22 μm of membrane filtration degerming.
The condition of chitosan and genipin cross-linked is incubated for 1 hour under the conditions of being 37 DEG C in this method, and 2 hours, 3 hours, 4 is small
When, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours.
The present invention also provides a kind of detection methods of cell microcapsules exobiology effect.Specifically, being to pass through
What following technical solution was realized:
This method is using the external Osteoinductive differentiation ability of cell microcapsules as the side of assessment cell microcapsules biological efficacy
Method.
Osteogenic Induction Medium used is to add dexamethasone, vitamin C and β-phosphorus on the basis of basal medium
Acid glycerol etc..
The use concentration of dexamethasone can be 0.05mM, 0.1mM, 0.15mM, 0.2mM in Osteogenic Induction Medium,
0.25mM, 0.3mM, 0.35mM, 0.4mM, 0.45mM, 0.5mM.
It is ascorbic to can be 10mg/mL, 20mg/mL, 30mg/mL, 40mg/mL, 50mg/mL, 60mg/ using concentration
ML, 70mg/mL, 80mg/mL, 90mg/mL, 100mg/mL.
β-phosphoglycerol use concentration can be 5 mM, 6 mM, 7 mM, 8mM, 9 mM, 10 mM, 11 mM, 12 mM,
13 mM, 14 mM, 15 mM.
The cell microcapsules of external evoked culture 4 weeks are made histotomy by this method, are detected by Alizarin red staining
The Osteoblast Differentiation ability of cell in microcapsules, to assess the biological efficacy of cell microcapsules.
Cell microcapsules prepared by the present invention can be directly used for regenerative medicine.
Detailed description of the invention
Fig. 1 CD18 Positive Stem Cells subgroup
Fig. 2 CD18 Positive Stem Cells subgroup phenotypic analysis
Fig. 3 microcapsules Preparation equipment
Fig. 4 CD18 Positive Stem Cells microcapsules
The analysis of Fig. 5 proliferative capacity.
Specific embodiment
The separation and culture of 1 CD18 positive deciduous teeth stem cell of embodiment
Materials and methods
1, the children's deciduous incisors normally to fall off is collected, penicillin containing 50-300U/mL, the 50-300 μ g/ of 4 DEG C of pre-coolings are immediately placed on
In the α-MEM culture medium of mL streptomysin and 1-5 μ g/mL amphomoronal, it is transported to Clean Operating Lab under the conditions of 2 DEG C -8 DEG C, from receipts
Collect the time interval for starting to extract deciduous teeth stem cell to be no more than 72 hours；
2, it is transferred to Biohazard Safety Equipment, takes out deciduous incisors to culture dish, the sterile phosphate buffer being pre-chilled with 4 DEG C cleans, i.e.,
PBS cleans its outer surface；
3, pulp tissue is isolated from the corona of remaining；
4, pulp tissue is placed in the PBS of the collagenase type I of mg/ml containing 1-10 and 1-10 mg/ml dispase, 37 DEG C of digestion 1
Hour；
5, digestive juice is filtered by 70 microns of cell sieve, obtains the single cell suspension containing human milk tooth stem cell, 900rpm
It is centrifuged 5min, it is primary to wash cell with PBS；
6, cell is resuspended with PBS, CD18 monoclonal antibody is added, mixes, room temperature is protected from light incubation 30 minutes；
7, after washing, 900rpm is centrifuged 5min, and cell is resuspended with PBS；
8, by selected by flow cytometry apoptosis or immunological magnetic bead sorting, obtain CD18 Positive Stem Cells subgroup.
The results show that the cell of culture is spindle, in circinate growth (Fig. 1), cell phenotype CD18 is positive (Fig. 2)；
9, change liquid within every 3-4 days, after the fusion of cell 90%, 1:3 passage, preparing cell used in microballoon is forth generation.
The preparation of 2 cell microcapsules of embodiment
Materials and methods
1. alginates are dissolved in PBS, it is made into the alginate soln of 1-3%, through 0.22 μm of membrane filtration, packing is saved；
2. the CD18 positive deciduous teeth stem cell of culture to forth generation is digested through pancreatin, 900rpm is centrifuged 5min, is washed carefully with PBS
Born of the same parents are primary；
3. the CD18 positive deciduous teeth stem cell of harvest is resuspended with alginate soln, adjustment cell concentration is 1-2 × 106/ml；
4. by horizontal, vertical binary channels microfluidic device as shown in Figure 3, by alginate soln containing cell through channel 1
It instills in the plate equipped with 0.1-0.2M calcium chloride solution straight down, forms cell microsphere.Soybean oil is through 2 horizontal cross of channel
Flowing is truncated alginate soln, controls the size of cell microsphere；
5. microballoon is incubated for 15-45 minutes under the conditions of 37 DEG C, to fully crosslinked formation；
6. alginates cell microsphere is coated with 15-45 minutes in 0.1-1% chitosan solution；
It is incubated for the crosslinking of completion in 1-10 hours 7. being subsequently placed in 1-5 mg/ml Geniposide aqueous solution under the conditions of 37 DEG C, prepares cell
Microcapsules.
8. cell microcapsules are cleaned and collected with α-MEM, and it is inoculated in fetal calf serum containing 10%-20%, 50 μ
α-MEM the culture of M-200 μM of ascorbic acid 2- phosphate, 2mM glutamine, 100U/mL penicillin, 100 μ g/mL streptomysins
In base, it is placed in 37 DEG C, 5% CO2Incubator in cultivate；
9. using the shape and diameter of the cell microcapsules that optical microphotograph sem observation is formed.
The results show that the cell microcapsules of preparation are in spherical, it is uniform in size.Cell is uniformly distributed wherein, rounded (figure
4).
The detection of 3 cell microcapsules exobiology effect of embodiment
Materials and methods
1. collecting cell microcapsules, and it is inoculated in the ascorbic acid 2- phosphoric acid of fetal calf serum containing 10%-20%, 50 μM -200 μM
Ester, 2mM glutamine, 100U/mL penicillin, 100 μ g/mL streptomysins α-MEM culture medium in, be placed in 37 DEG C, 5% CO2's
It is cultivated in incubator；
2. after culture 24-48 hours, being replaced with Osteogenic Induction Medium (containing 10% fetal calf serum, 100U/mL penicillin, 100 μ
G/mL streptomysin, 0.05-0.5mM dexamethasone, 10-100 mg/mL vitamin C and 5-15 mM β-phosphoglycerol
α-MEM culture medium)；
3. every 3-4 days change liquid；
4. after Fiber differentiation 4 weeks, fixing cell microcapsules 30 minutes under room temperature with 4% paraformaldehyde；
5. setting in PBS 15 minutes；
6. dehydration, transparent, paraffin embedding are sliced, dewaxing, rehydration；
7. cell microcapsules histotomy is placed in 2% alizarin red aqueous solution, room temperature dyes 5min；
8. after rinsing with ruinning water, using haematoxylin redyeing；
9. mounting microscopy.
The results show that passing through Alizarin red staining (Fig. 5), it is seen that in microcapsules in vitro after osteogenic induction culture in 3-4 weeks
Iuntercellular has mineral deposition, shows CD18 in microcapsules+Deciduous teeth stem cell can not influence to osteoblast differentiation, encapsulation
Its biological characteristics.
Claims (11)
1. a kind of human milk tooth stem cell microcapsules by cell surface marker specific enrichment, it is characterised in that:
The stem cell is people's deciduous teeth stem cell, and the man-year age is 5 to 8 years old, and the cell surface marker is selected from CD18,
STRO-1, CD106/VCAM-1, CD146/MUC-18, HOP-26, CD49A/ integrin β_1, one in SB-10/CD166 or
It is multiple；
The microcapsules include microballoon kernel and shell, and the microsphere diameter is less than 500 μm；
The microencapsulation material is selected from collagen, alginates, chitosan, one of hyaluronic acid or a variety of.
2. human milk tooth stem cell microcapsules described in claim 1, which is characterized in that using CD18 as cell surface marker, institute
It states CD18 and uses fluorescent marker or marked by magnetic bead.
3. human milk tooth stem cell microcapsules as claimed in claim 2, which is characterized in that the microcapsules inner nuclear material is selected from alginic acid
Salt.
4. human milk tooth stem cell microcapsules as claimed in claim 3, which is characterized in that the microcapsule shell material is poly- selected from shell
Sugared film, the chitosan film are covalent cross-linking.
5. the preparation method of human milk tooth stem cell microcapsules described in a kind of claim 4, which is characterized in that the method it is specific
Steps are as follows:
Step 1, alginates are dissolved in PBS, are made into the alginate soln of 1-3%, through 0.22 μm of membrane filtration, packing is protected
It deposits；
Step 2, the CD18 positive deciduous teeth stem cell by culture to forth generation digests through pancreatin, and 900rpm is centrifuged 5min, is washed with PBS
Cell is primary；
Step 3, be resuspended the obtained CD18 positive deciduous teeth stem cell of step 2 with alginate soln, adjustment cell concentration be (1-2) ×
106/ml；
Step 4, by horizontal, vertical binary channels microfluidic device, alginate soln containing cell is dripped downward into through channel
In plate equipped with 0.1-0.2M calcium chloride solution, cell microsphere is formed；It is molten that alginates are truncated through channel cross-flow in soybean oil
Liquid controls the size of cell microsphere；
Step 5, microballoon is incubated for 15-45 minutes under the conditions of 37 DEG C, to fully crosslinked formation；
Step 6, alginates cell microsphere is coated with 15-45 minutes in (0.1-1) % chitosan solution；
Step 7, it is subsequently placed in 1-5 mg/ml Geniposide aqueous solution under the conditions of 37 DEG C and is incubated for the crosslinking of completion in 1-10 hours, preparation
Cell microcapsules；
Step 8, cell microcapsules are cleaned and are collected with α-MEM, and be inoculated in fetal calf serum containing 10%-20%, 50 μM-
200 μM of ascorbic acid 2- phosphate, 2mM glutamine, 100U/mL penicillin, 100 μ g/mL streptomysins α-MEM culture medium
In, it is placed in 37 DEG C, 5% CO2Incubator in cultivate；
Step 9, the shape and diameter of the cell microcapsules formed using optical microphotograph sem observation.
6. the preparation method of human milk tooth stem cell microcapsules described in a kind of claim 5, which is characterized in that the culture to the 4th
The preparation step of the CD18 positive deciduous teeth stem cell in generation is as follows:
Step 1, the children's deciduous incisors normally to fall off is collected, penicillin containing 50-300U/mL, the 50-300 of 4 DEG C of pre-coolings are immediately placed on
In the α-MEM culture medium of μ g/mL streptomysin and 1-5 μ g/mL amphomoronal, it is transported to Clean Operating Lab under the conditions of 2 DEG C -8 DEG C, from
The time interval for starting to extract deciduous teeth stem cell is collected into be no more than 72 hours；
Step 2, it is transferred to Biohazard Safety Equipment, takes out deciduous incisors to culture dish, the sterile phosphate buffer being pre-chilled with 4 DEG C is clear
It washes, i.e. PBS cleans its outer surface；
Step 3, pulp tissue is isolated from the corona of remaining；Step 4, pulp tissue is placed in the I type glue of mg/ml containing 1-10
In the PBS of protoenzyme and 1-10 mg/ml dispase, 37 DEG C digest 1 hour；
Step 5, digestive juice is filtered by 70 microns of cell sieve, obtains the single cell suspension containing human milk tooth stem cell,
900rpm is centrifuged 5min, and it is primary to wash cell with PBS；
Step 6, cell is resuspended with PBS, CD18 monoclonal antibody is added, mix, room temperature is protected from light incubation 30 minutes；
Step 7, after washing, 900rpm is centrifuged 5min, and cell is resuspended with PBS；
Step 8, by selected by flow cytometry apoptosis or immunological magnetic bead sorting, CD18 Positive Stem Cells subgroup is obtained；
Step 9, liquid is changed within every 3-4 days, after the fusion of cell 90%, 1:3 passage, preparing cell used in microballoon is forth generation.
7. the stemness detection method of human milk tooth stem cell microcapsules described in a kind of claim 1-4, which is characterized in that the method
Specific step is as follows:
Step 1, cell microcapsules are collected, and are inoculated in the ascorbic acid 2- phosphorus of fetal calf serum containing 10%-20%, 50 μM -200 μM
Acid esters, 2mM glutamine, 100U/mL penicillin, 100 μ g/mL streptomysins α-MEM culture medium in, be placed in 37 DEG C, 5% CO2
Incubator in cultivate；
Step 2, after cultivating 24-48 hours, it is replaced with Osteogenic Induction Medium, that is, contains 10% fetal calf serum, 100U/mL mould
Element, 100 μ g/mL streptomysins, 0.05-0.5mM dexamethasone, 10-100 mg/mL vitamin C and 5-15 mM β-phosphoric acid
α-MEM the culture medium of glycerol, changes liquid in every 3-4 days；
Step 3, after Fiber differentiation 4 weeks, cell microcapsules 30 minutes are fixed under room temperature with 4% paraformaldehyde；
Step 4, it sets in PBS 15 minutes；
Step 5, it is dehydrated, transparent, paraffin embedding, is sliced, dewaxing, rehydration；
Step 6, cell microcapsules histotomy is placed in 2% alizarin red aqueous solution, room temperature dyes 5min；
Step 7, after rinsing with ruinning water, haematoxylin redyeing is used；
Step 8, mounting microscopy.
8. the purposes of human milk tooth stem cell microcapsules described in a kind of claim 1-4, which is characterized in that the regeneration for bone disease
The preparation of medicine and tissue engineering material.
9. the purposes of human milk tooth stem cell microcapsules described in a kind of claim 1-4, which is characterized in that will by transgenic technology
CD18 gene is transferred to stroma stem cell, the preparation of regenerative medicine and tissue engineering material for bone disease.
10. the purposes of human milk tooth stem cell microcapsules described in claim 9, which is characterized in that the stromal stem cell source
Selected from tooth, fat, marrow, umbilical cord, placenta or bleeding of the umbilicus.
11. the purposes of human milk tooth stem cell microcapsules described in claim 9, which is characterized in that the transgenic technology is to pass through
The mode of retrovirus-mediated method.
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