CN106350478A - Stem cell culture system - Google Patents
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- CN106350478A CN106350478A CN201610931809.5A CN201610931809A CN106350478A CN 106350478 A CN106350478 A CN 106350478A CN 201610931809 A CN201610931809 A CN 201610931809A CN 106350478 A CN106350478 A CN 106350478A
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- C12N2533/00—Supports or coatings for cell culture, characterised by material
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Abstract
The invention discloses a stem cell culture system which comprises a serum-free culture medium and a stent material, wherein the stent material comprises a framework prepared from chitosan and gelatin, and lecithin and polylysine coated on the framework. By using the chitosan and gelatin with favorable biocompatibility to make the framework, the lecithin and polylysine are coated outside the framework, so that the stem cells can be adsorbed to the stent material more easily. The fiber connexin is added to reinforce the connection, so that the stent material has favorable cell adsorptivity, and the safety of the stem cells and stent material is enhanced.
Description
Technical field
The present invention relates to technical field of life science, particularly to a kind of stem cell cultivating system.
Background technology
Stem cell is the pluripotent cell that a class has the of self-replication capacity, under certain condition it can be divided into multiple
Functioning cell. stem cell can be mixed with timbering material, make stem cell as the seed cell in organizational project by a certain percentage
Attach to formation cell-scaffold material composite on timbering material, and differentiation of stem cells in vitro, then by this complex
The implantation tissue of collective or lesions position. gradually degrade in body with timbering material and absorb, implantation differentiated
Stem cell constantly breeds and extracellular matrix secretion in body, ultimately forms corresponding tissue or organ, thus reaching reparation
Wound and the purpose of Reconstruction of The Function.The three dimensional structure that biomaterial scaffolds are formed obtains nutrition, growth and metabolism for cell and carries
Supply a good environment.
During stem cell in-vitro multiplication and differentiation, general employing adds hyclone, tire Sanguis Bovis seu Bubali in culture medium
It is clearly conventional natural medium in a kind of cell culture, containing abundant nutritional labeling, be usually used in the external training of zooblast
Support, be cell attachment, spread over the source of the required factor in plastic culture substrate.But hyclone has the disadvantage in that one
It is to contain unknown composition in hyclone, may interfere with propagation and the differentiation of stem cell, two is containing micro- in hyclone
The hemoglobin of amount and endotoxin, can disturb growth and the propagation of stem cell.
For solving the above problems, the Chinese patent for cn105112365a for the application publication number " do by a kind of human umbilical cord mesenchymal
Serum-free medium of cell and preparation method thereof " disclose a kind of serum-free medium of human umbilical cord mesenchymal stem cells and its
Preparation method, including dmem basal medium, also comprises the following components: Recombinant Insulin Human, human serum albumin, turns ferrum egg
In vain, fibronectin, vitamin c, biotin, stem cell factor and stem cell factor.
But the serum-free medium of this human umbilical cord mesenchymal stem cells has the disadvantage in that using above-mentioned culture medium culturing
When stem cell and timbering material, due to there is no hyclone, lack cell attachment in culture medium and be attached on the institute on timbering material
Need the factor, lead to stem cell in incubation to be difficult to attach on timbering material.
Content of the invention
It is an object of the invention to provide a kind of stem cell cultivating system, there is stem cell and easily attach on timbering material
Effect.
The above-mentioned technical purpose of the present invention has the technical scheme that a kind of stem cell cultivating system,
Including serum-free medium, timbering material, skeleton that described timbering material includes being made by shitosan and gelatin, it is coated in
Lecithin on described skeleton and poly-D-lysine.
By adopting technique scheme, by culturing stem cells in serum-free medium, medium component determines, does not have
There are hemoglobin and creotoxin, culture organization security out is reliable.
Timbering material includes skeleton, lecithin and poly-D-lysine.The selection of timbering material is important in organizational project
Part, timbering material is except having good biocompatibility and biodegradability in addition it is also necessary to there be hydrophilic and good thin
Born of the same parents' adsorptivity.There is lecithin in skeleton expoeridium, lecithin has the phosphoric acid anion of nonpolar carbochain and high-polarity, energy concurrently
Disperse in solution and form micelle, nonpolar carbochain is gathered in the middle of micelle and one end of polarity is exposed in water, makes
Obtain timbering material and there is good hydrophilic, it is to avoid the phenomenon that solution is difficult on timbering material surface to spread occurs.
In view of there is no serum composition in serum-free medium, lack required for adherent growth during leading to stem cells hyperplasia
The factor, be also coated with poly-D-lysine on timbering material surface, poly-D-lysine has a good cell adsorptivity, can be substantially
Promote sticking of stem cell, and stem cell secretion substrate can be promoted, make the stem cell being attached on timbering material difficult for drop-off.
Skeleton in timbering material is made by shitosan and gelatin, and shitosan is natural polymolecular, is that chitin takes off
Acetylizad product, has good biological tissue's compatibility, can be degraded after implantation living organism.But shitosan make and
The support becoming enbrittle greatly, poor toughness, the slow shortcoming of degrading.It is fabricated to skeleton, gelatin by adding gelatin in shitosan
It is native biopolymer material, after adding gelatin, improves the toughness of skeleton, reduce the fragility of skeleton so that timbering material meets
The demand of organizational project.
In stem cell incubation, because stem cell constantly breeds and grows, lecithin and poly-D-lysine are once
Come off from skeleton, skeleton will exposed out, so skeleton itself also must have good cell adsorptivity.Framework material
In gelatin be a kind of amorphous material, there is good cell adsorptivity, be further ensured that the cell adsorptivity of timbering material.
Reach the effect that stem cell is easily adsorbed onto on timbering material
The further setting of the present invention is: described skeleton is obtained by following methods: a) Chitosan powder is dissolved in acetic acid molten
In liquid, after stirring, centrifugation, degassing, de- bundle, obtain chitosan solution；B) chitosan solution obtaining is added to mould
Interior, after pre-freeze is processed, then carry out lyophilizing, obtain lyophilizing frame；C) gelatin solution is added drop-wise on lyophilizing frame, aeration-drying；D) will
Lyophilizing frame is rinsed with sodium dihydrogen phosphate, after neutralizing unreacted acetic acid；E) lyophilizing frame is put into phosphinylidyne diimine rubber cross linker
After middle process, after soaking in ethanol solution, then use tri-distilled water washing by soaking, remove uncrosslinked phosphinylidyne diimine；F) lyophilizing
After process, obtain described skeleton.
By adopting technique scheme, shitosan is dissolved in formation chitosan solution after acetic acid, is fabricated in a mold
Shape required for organizational project.Lyophilizing frame is lyophilized after pre-freeze, forms spongy loose structure, Stem Cells adhesion is in support
The material secreted when on material can circulate in loose structure so that stem cell obtains effective material transmission, props up for attaching to
Stem cell on frame material provides an environment that can effectively carry out material and energy transmission.
Although the addition of gelatin can strengthen the toughness of timbering material, due to chitosan molecule mainly linear so that
The mechanical strength of timbering material is weaker, by phosphinylidyne diimine crosslinking Treatment so that chitosan molecule forms tridimensional network,
Improve the Mechanical Structure Strength of timbering material, the degradation speed of timbering material can also be slowed down simultaneously.
The further setting of the present invention is: by percentage to the quality, the concentration of described chitosan solution is 0.8%.
By adopting technique scheme, from the relatively low chitosan solution concentration of concentration so that being frozen after dry-cure
Skeletal internal structure is relatively rough, beneficial to the absorption of stem cell.
The further setting of the present invention is: is provided with cavity in described skeleton, is located at jelly in described cavity, described glue
Thing mixes by Collagen Type VI with for the inducible factor solution of differentiation of stem cells.
By adopting technique scheme, inducible factor refers to promote the exogenous inducing factors of stem cells hyperplasia differentiation, leads to
Cross inducible factor and Collagen Type VI are mixed into after jelly and be injected in cavity, the slow effect discharging inducible factor can be played.
When stem cell just starts to attach on timbering material, inducible factor does not also discharge, and stem cell constantly expands, and works as inducible factor
Gradually discharge, after the many hollow structures on support touch stem cell, promote stem cell to be divided into tissue-specific cells.Dry
Cell is implanted in living organism when undifferentiated has oncogenicity, there is potential safety hazard, by releasing from inside to outside in timbering material
Put inducible factor so that stem cell on timbering material for the absorption all can touch inducible factor, promote the differentiation of stem cell, protect
Card stem cell and timbering material implant the safety after living organism.
The further setting of the present invention is: also includes fibronectin in described stem cell cultivating system.
By adopting technique scheme, fibronectin is family's hmw protein, can promote cell and substrate
Between the physiological action that connects, stem cell can secrete one layer of cellular matrix being attached on timbering material in tactophily, in training
So that stem cell can be firmly adsorbed on timbering material after addition fibronectin in foster base.
The further setting of the present invention is: described concentration of fibronectin is 10 μ g/l-50 μ g/l.
So that fibronectin is maintained at suitable concentration, do not interfere with stem cell by using technique scheme
Normal proliferative.
The further setting of the present invention is: described concentration of fibronectin is 30 μ g/l.
By adopting technique scheme, the concentration of fibronectin is 30 μ g/l, has not both interfered with the increasing of stem cell
Grow, have and can make stem cell absorption firmly.
In sum, the method have the advantages that using the shitosan with good biocompatibility and gelatin
It is fabricated to skeleton, lecithin and poly-D-lysine are coated on outside skeleton, improve stem cell and be readily adsorbed on timbering material, its
Secondary, add fibronectin, reinforce and connect, reached timbering material cell excellent adsorption, improved stem cell and timbering material
The effect of safety.
Specific embodiment
Specific embodiment is only explanation of the invention, and it is not limitation of the present invention, those skilled in the art
As needed the present embodiment can be made after reading this specification does not have the modification of creative contribution, but as long as at this
All protected by Patent Law in bright right.
Embodiment 1: a kind of stem cell cultivating system, can be used for mammalian stem cell culture.Including serum-free medium
And timbering material.Serum-free medium can adopt the serum-free medium of commercialization it is also possible to prepare in the following proportions, including
α-mem culture medium adds 10mg/l bfgf, 10mg/l egf, 5g/l bsa, 1.5mg/l reduced glutathion, 0.1mmol/
L beta -mercaptoethanol, 1% (v/v) site, 10mg/l hydrocortisone, 10nmol/l dexamethasone and aminoacid concentrated solution.Xiang Wu
Add fibronectin in blood serum medium, and make concentration in serum-free medium for the fibronectin be 30 μ
g/l.
The preparation of timbering material, timbering material includes skeleton and is coated on ovum lipoid and the poly-D-lysine on skeleton surface.
Weigh Chitosan powder at room temperature to be added in acetic acid solution, after being sufficiently stirred for, centrifugation, degassing, de- slag, it is configured to quality hundred
Divide than the chitosan solution for 0.8%.It is added to molten for shitosan in mould, first in subzero 90 degree of pre-freezes 2 hours, then zero again
Lower 50 degree of frozen drieds 24 hours.Received with phosphoric acid hydrogen two again after frozen dried and rinse 5 minutes, to neutralize unreacted acetic acid,
Frozen dried 24 hours again under subzero 50 degrees Celsius, obtain lyophilizing frame.Form cavity in the middle part of lyophilizing frame and upper end is provided with out
Mouthful.Prepare the gelatin solution that mass percent is 2% to be added drop-wise on lyophilizing frame, after making lyophilizing frame Surface coating have one layer of gelatin,
Aeration-drying is processed 20 hours.Again lyophilizing frame is put in the phosphinylidyne diimine cross-linking agent of 50mmol/l and process 10 hours,
Alcohol solution dipping with 40% is processed 30 minutes, after repeating to soak 4 times, then with tri-distilled water washing by soaking 20 minutes, repetition 2
Secondary, after removing uncrosslinked phosphinylidyne diimine, again in subzero 50 degrees Celsius lower lyophilizing 24 hours, obtain skeleton.Skeleton is put
After entering embedding in lecithin soln, it is placed again into embedding in Poly-L-Lysine Solution, take out rear venting and process 15 minutes, obtain
Inside is provided with the timbering material of cavity.
By 10 μ g/ml insulins, 1 μm of ol/l dexamethasone, 0.5mmol/l ibmx, 0.1mmol/l indometacin and gelatin
After mix homogeneously, supplement the jelly of stem cell adipogenic induction between being formed for bone marrow with money, jelly is circulated in timbering material
Cavity in.
The preparation of stem cell, commercially available new zealand white rabbit, separates and culture obtains mesenchymal stem cells MSCs.In view of from rabbit
Internal separation obtains mesenchymal stem cells MSCs for prior art, is not described in detail here.By mesenchymal stem cells MSCs with 3 ×
105Cell density be inoculated on timbering material, put into culture in serum-free medium.
Cell adsorptivity detects: by hoechst-pi staining, with ultramicroscope observation of cell on timbering material
Adhesion, distribution situation.
Cell differentiation detects: behind cell culture 6 days, 12 days, 18 days, makes cell climbing sheet, observation of cell form, then enters
Row oil red o fat stains method, has seen whether orange red fat drips, if any orange red fat drips, has then shown mesenchymal stem cells MSCs
Adipogenic induction breaks up successfully.And experimental result is listed in the following table 1.
Embodiment 2: a kind of stem cell cultivating system, it is with the difference of embodiment 1, fiber in serum-free medium
Connection protein concentration is 10 μ g/l.And carry out the detection of cell adsorptivity and cell differentiation detection, testing result is arranged in Table 1
Go out.
Embodiment 3: a kind of stem cell cultivating system, it is with the difference of embodiment 1, fiber in serum-free medium
Connection protein concentration is 50 μ g/l.And carry out the detection of cell adsorptivity and cell differentiation detection, testing result is arranged in Table 1
Go out.
Comparative example 1: a kind of stem cell cultivating system, it is with the difference of embodiment 1, timbering material contains only bone
Frame.And carry out the detection of cell adsorptivity and cell differentiation detection, testing result is listed in Table 1.
Comparative example 2: a kind of stem cell cultivating system, it is with the difference of embodiment 1, do not contain in serum-free medium
There is fibronectin.And carry out the detection of cell adsorptivity and cell differentiation detection, testing result is listed in Table 1.
Comparative example 3: a kind of stem cell cultivating system, it is inside timbering material, do not have glue with the difference of embodiment 1
Shape thing.And carry out the detection of cell adsorptivity and cell differentiation detection, testing result is listed in Table 1.
Table 1
| Cell absorption, distribution | Cell differentiation detects | |
| Embodiment 1 | Cell adsorbs, and is attached at timbering material surface in flat | There are orange red fat drips |
| Embodiment 2 | Cell adsorbs, and is attached at timbering material surface in flat | There are orange red fat drips |
| Embodiment 3 | Cell adsorbs, and is attached at timbering material surface in flat | There are orange red fat drips |
| Comparative example 1 | Cell adsorbs, in certain shape of crispaturaing, non-full extension | There are orange red fat drips |
| Comparative example 2 | Cell adsorbs, in certain shape of crispaturaing, non-full extension | There are orange red fat drips |
| Comparative example 3 | Cell adsorbs, and is attached at timbering material surface in flat | No orange red fat drips |
Claims (6)
1. a kind of stem cell cultivating system, including serum-free medium, timbering material it is characterised in that: described timbering material bag
Include the skeleton being made by shitosan and gelatin, the lecithin being coated on described skeleton and poly-D-lysine.
2. a kind of stem cell cultivating system according to claim 1 it is characterised in that: described skeleton is by following methods system
:
A) Chitosan powder is dissolved in acetic acid solution, after stirring, centrifugation, degassing, de- bundle, obtain chitosan solution；
B) chitosan solution obtaining is added in mould, after pre-freeze is processed, then carries out lyophilizing, obtain lyophilizing frame；
C) gelatin solution is added drop-wise on lyophilizing frame, aeration-drying；
D) lyophilizing frame is rinsed with sodium dihydrogen phosphate, after neutralizing unreacted acetic acid；
E) lyophilizing frame is put into after process in phosphinylidyne diimine rubber cross linker, after soaking in ethanol solution, then use tri-distilled water
Washing by soaking, removes uncrosslinked phosphinylidyne diimine；
F), after frozen dried, obtain described skeleton.
3. a kind of stem cell cultivating system according to claim 2 it is characterised in that: by percentage to the quality, described shell
The concentration of polysaccharide solution is 0.8%.
4. a kind of stem cell cultivating system according to claim 1 it is characterised in that: be provided with sky in described skeleton
Chamber, it is located at jelly in described cavity, described jelly mixes by gelatin with for the inducible factor solution of differentiation of stem cells
Conjunction forms.
5. a kind of stem cell cultivating system according to claim 1 it is characterised in that: in described stem cell cultivating system also
Including fibronectin.
6. a kind of stem cell cultivating system according to claim 5 it is characterised in that: described concentration of fibronectin is
10μg/l-50μg/l.
A kind of stem cell cultivating system according to claim 6 it is characterised in that: described concentration of fibronectin be 30
μg/l.
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| CN201610931809.5A CN106350478A (en) | 2016-10-25 | 2016-10-25 | Stem cell culture system |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107529547A (en) * | 2017-09-28 | 2018-01-02 | 吉林省拓华生物科技有限公司 | A kind of CD29 for improving skeletal muscle atrophy under high glucose and high fat environment+People's Mesenchymal Stem Cells from Umbilical Cord cultural method |
| CN108047482A (en) * | 2017-12-12 | 2018-05-18 | 华中科技大学鄂州工业技术研究院 | A kind of porous chitosan microcarrier and its preparation method and application |
| CN110819586A (en) * | 2019-11-26 | 2020-02-21 | 广州赛莱拉生物基因工程有限公司 | Culture medium for inducing differentiation from dental pulp stem cells to osteoblasts and preparation method and application thereof |
| CN114507916A (en) * | 2022-04-18 | 2022-05-17 | 中国科学院苏州纳米技术与纳米仿生研究所 | Chitosan microfiber with groove topological structure and preparation method and application thereof |
-
2016
- 2016-10-25 CN CN201610931809.5A patent/CN106350478A/en active Pending
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| CHENG YOU等: "hydrophilicity and adsorptivity of a novel scaffold twice embedded by lecithin and poly-L-lysine to human nasal septum chondrocytes", 《中国组织工程研究与临床康复》 * |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107529547A (en) * | 2017-09-28 | 2018-01-02 | 吉林省拓华生物科技有限公司 | A kind of CD29 for improving skeletal muscle atrophy under high glucose and high fat environment+People's Mesenchymal Stem Cells from Umbilical Cord cultural method |
| CN107529547B (en) * | 2017-09-28 | 2021-02-26 | 吉林省拓华生物科技有限公司 | CD29 for improving skeletal muscle atrophy in high-sugar and high-fat environment+Human umbilical cord source mesenchymal stem cell culture method |
| CN108047482A (en) * | 2017-12-12 | 2018-05-18 | 华中科技大学鄂州工业技术研究院 | A kind of porous chitosan microcarrier and its preparation method and application |
| CN110819586A (en) * | 2019-11-26 | 2020-02-21 | 广州赛莱拉生物基因工程有限公司 | Culture medium for inducing differentiation from dental pulp stem cells to osteoblasts and preparation method and application thereof |
| CN114507916A (en) * | 2022-04-18 | 2022-05-17 | 中国科学院苏州纳米技术与纳米仿生研究所 | Chitosan microfiber with groove topological structure and preparation method and application thereof |
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Application publication date: 20170125 |