CN105543166A - Manufacturing method of culture dish for culturing stem cell aggregate - Google Patents
Manufacturing method of culture dish for culturing stem cell aggregate Download PDFInfo
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- CN105543166A CN105543166A CN201511008331.0A CN201511008331A CN105543166A CN 105543166 A CN105543166 A CN 105543166A CN 201511008331 A CN201511008331 A CN 201511008331A CN 105543166 A CN105543166 A CN 105543166A
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0662—Stem cells
- C12N5/0667—Adipose-derived stem cells [ADSC]; Adipose stromal stem cells
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- C12N2533/00—Supports or coatings for cell culture, characterised by material
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- C12N2533/80—Hyaluronan
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Abstract
The invention discloses a manufacturing method of a culture dish for culturing stem cell aggregate. According to the anti-cell adhesion principle, a hydrophilic material single-component or multicomponent crosslinking system is coated onto a common culture dish surface in different coating modes, and different drying processes are performed to obtain the culture dish for culturing stem cell aggregate. The culture dish can stop the adherent adhesion of cells, so that the stem cells spontaneously aggregate to form the stem cell aggregate; the formed stem cell aggregate contains abundant cell-cell interactions, simulates the microenvironment of the in-vivo cells, and has excellent dryness maintenance capacity and induced differentiation capacity. The culture dish prepared by the method can be directly used for culturing the stem cell aggregate, is convenient to operate, and is efficient and controllable, so that the stem cell aggregate can be more widely used for cytological research, stem cell therapy or tissue engineering.
Description
Technical field
The invention belongs to bio-regeneration medicine technology field, be specifically related to the making method of a kind of cultural method of stem cell aggregate and the culture dish for culturing stem cells aggregate.
Background technology
Stem cell is a class is the multipotential cell with the of self-replication capacity, can be induced to differentiate into bone, cartilage, neural, and the cells such as fat, can be applied to organizational project as seed cell, be subject to extensive concern since self-discovery.
Traditional stem cell training method is generally carry out two-dimension single layer adherent culture on culture dish.Studies have found that at present, compared to the stem cell of traditional two-dimension single layer adherent culture, the stem cell that stem cell three-dimensional aggregates is cultivated has following advantage: there is cell-ECM interphase interaction widely in stem cell aggregate, simulate the microenvironment that cell is residing in vivo better, make the differentiation-inducing ability of stem cell and dryness maintain ability stronger, cytoactive is higher.
The methods such as the cultural method of current cell aggregation has the suspension culture of Keep agitation, Hanging drop culture, centrifugal and magnetic nanoparticle mediation.Utilize these methods all can form cell aggregation, but or complicated operation, or need special device, time and effort consuming and be unfavorable for that technician operates and applies on a large scale.Desirable cell aggregation cultural method convenient, efficiently, controllably can realize cell aggregation to be formed, and technician can be facilitated again to collect and application cell aggregate simultaneously.In a word, the cultural method of current cell aggregation is desirable not enough.
Summary of the invention
In order to solve the culture technique problem of existing stem cell aggregate, the object of the invention is to the deficiency overcoming prior art existence, provide a kind of making method of culture dish of culturing stem cells aggregate, this culture dish can be directly used in culturing stem cells aggregate.The single-component of hydrophilic material or polycomponent, according to Anti cell adhesion principle, are cross-linked by the present invention, apply all kinds of coating method and are coated on Nostoc commune Vanch ware surface, then made the culture dish being used for stem cell aggregate and cultivating by different drying meanss.This culture dish can be used for the cultivation of stem cell aggregate on a large scale, and simple operation, efficient, controlled, enables stem cell aggregate broadly for cytology research, stem-cell therapy or organizational project.
Create object for reaching foregoing invention, the present invention adopts following technical proposals:
A kind of making method of the culture dish for culturing stem cells aggregate, adopt single-component hydrophilic material to carry out dissolving or polycomponent hydrophilic material carry out dissolving crosslinked after the material system for preparing as coating material, and the mass concentration controlling the solution of coating material is 1-5wt%, then coating material is spread evenly across conventional culture dish and forms wet film on the surface, pass through drying means again, wet film on culture dish is solidificated in culture dish on the surface, drying and forming-film, prepares the culture dish for culturing stem cells aggregate.
As the preferred technical scheme of the present invention, described hydrophilic material preferably adopts any one material in chitosan, polyacrylic acid, polyoxyethylene glycol, L-glutamic acid, sodium alginate, hyaluronic acid, dextran and hyaluronic acid or any different materials, and wherein the deacetylation of chitosan is 60 ~ 90%.
As the preferred further technical scheme of such scheme of the present invention, the substrate material of described culture dish is plastics.Substrate material more preferably polypropylene or the polystyrene of described culture dish.
The culture dish for culturing stem cells aggregate that the present invention makes, before culturing stem cells aggregate, first the culture dish cultivated for stem cell aggregate is carried out disinfection and germicidal treatment, then stem cell is inoculated on clean culture dish, make stem cell self-assemble on culture dish, form the spherical micellar aggregates body that diameter is 50 ~ 200 μm, micelle formation time is less than 72h.The present invention utilizes Anti cell adhesion principle, the hydrophilic material be coated on conventional culture dish is utilized to block the adhesion of cell on prepared culture dish, allow cell self-assemble on prepared culture dish form the aggregate that diameter range is about 50-200 μm, concrete diameter regulates and controls by coated material.And the aggregate of diameter 50-200 μm is the aggregate cultivation size be comparatively suitable for, the survival ability of aggregate inner cell is strong, and under the effect of inducible factor, directed differentiation ability is high.
The stem cell applicable for the culture dish of stem cell aggregate cultivation prepared by the present invention can be following cell, include but not limited to: embryonic stem cell and mescenchymal stem cell, mescenchymal stem cell comprises fat stem cell, bone marrow stem cell and Synovial Mesenchymal Stem Cells.
As the preferred further technical scheme of such scheme of the present invention, preferably by preparing different hydrophilic material film coatings to prepare different culture dish on culture dish, and then carry out the diameter dimension that regulable control forms micellar aggregates body.
The present invention compared with prior art, has following apparent outstanding substantive distinguishing features and remarkable advantage:
1. the culture dish that prepared by the present invention can be directly used in the cultivation of stem cell aggregate, and simple operation, efficient, controlled, enables stem cell aggregate broadly for cytology research, stem-cell therapy or organizational project;
2. the conventional culture dish diameter range selected by the present invention is not limit, and is applicable to the culture dish of sizes, expands the range of application of culture dish and applicable biological field;
3. the adherent adhesion of culture dish cell capable of blocking prepared of the present invention, stem cell self-assemble is made to form stem cell aggregate, and in the stem cell aggregate formed, comprise a large amount of cell-ECM interactions, microenvironment residing for analogue body inner cell, shows excellent dryness and maintains ability and differentiation-inducing ability.
Accompanying drawing explanation
Fig. 1 is the aggregate photo of self-assemble formation on the culture dish that makes in the embodiment of the present invention one of the human adipose-derived stem cell of fluorescent microscope shooting.
Embodiment
Details are as follows for the preferred embodiments of the present invention:
embodiment one:
In the present embodiment, a kind of making method of the culture dish for culturing stem cells aggregate, be specially: under stirring at normal temperature, get homemade deacetylation be 80% Chitosan powder 0.06g be scattered in 6ml deionized water, drip vinegar acid for adjusting pH to 4-5, it is dissolved completely, and the concentration of chitosan solution is 1wt%.The diameter selected is the polypropylene culture dish of 120mm, the chitosan solution being 80% by this 6ml deacetylation is uniformly coated on conventional culture dish, under room temperature 25 DEG C of conditions after dry 48h, the obtained culture dish being coated with thin-film material is immersed in successively in gradient ethanol solution and removes residual acetic acid, and then dry 48h at ambient temperature, prepare the culture dish that can be used for culturing stem cells aggregate.
In the present embodiment, a kind of culture dish prepared by the present embodiment is used for the cultural method of stem cell aggregate, be specially: first uv sterilisation and germicidal treatment are carried out to the culture dish for culturing stem cells aggregate, then by after amplification in vitro to the human adipose-derived stem cell digestion collection of P3, adjustment cell concn is 5 × 10
5cells/ml, is inoculated on preparation-obtained culture dish, human adipose-derived stem cell self-assemble on culture dish after 72h, forms the aggregate that diameter is about 90-110 μm, see Fig. 1.Hydrophilic material adherent for Anti cell adhesion is coated on conventional culture dish by the present embodiment, then utilizes drying means, obtains Anti cell adhesion, stem cell self-assemble can be made to form the culture dish of aggregate.
embodiment two:
The present embodiment is substantially identical with embodiment one, and special feature is:
In the present embodiment, a kind of making method of the culture dish for culturing stem cells aggregate, be specially: under stirring at normal temperature, get homemade deacetylation be 70% Chitosan powder 0.06g be scattered in 6ml deionized water, drip vinegar acid for adjusting pH to 4-5, it is dissolved completely, and the concentration of chitosan solution is 1wt%.The diameter selected is the polypropylene culture dish of 120mm, the chitosan solution being 70% by this 6ml deacetylation is uniformly coated on conventional culture dish, under room temperature 25 DEG C of conditions after dry 48h, the obtained culture dish being coated with thin-film material is immersed in successively in gradient ethanol solution and removes residual acetic acid, and then dry 48h at ambient temperature, prepare the culture dish for culturing stem cells aggregate.
In the present embodiment, a kind of culture dish prepared by the present embodiment is used for the cultural method of stem cell aggregate, be specially: first uv sterilisation and germicidal treatment are carried out to the culture dish cultivated for stem cell aggregate, then by after amplification in vitro to the human adipose-derived stem cell digestion collection of P3, adjustment cell concn is 5 × 10
5cells/ml, is inoculated on preparation-obtained culture dish, human adipose-derived stem cell self-assemble on culture dish after 72h, forms the aggregate that diameter is about 130-150 μm.
embodiment three:
The present embodiment and previous embodiment are substantially identical, and special feature is:
In the present embodiment, a kind of making method of the culture dish for culturing stem cells aggregate, be specially: under stirring at normal temperature, a certain amount of L-glutamic acid is dissolved in deionized water by the NaOH dripping 3mol/L, and regulate the pH of poly-L-glutamic acid acid solution to neutral, then quantitative NHS and EDC powder is added for activating side chain carboxyl group on L-glutamic acid to poly-L-glutamic acid acid solution order, control L-glutamic acid, the mol ratio of NHS and EDC is 1:1:5, after activation L-glutamic acid side chain carboxyl group 8h, add in mixed system the better self-control deacetylation of a certain amount of dissolving be 60% pH be the chitosan solution of 4-5, carry out chemically crosslinked, and the molar ratio that the carboxyl-content controlled on L-glutamic acid and deacetylation are the amino content of the chitosan of 60% is 1:1, and the polymer concentration controlling the two component compound system of whole L-glutamic acid/chitosan is 1-5wt%.Get the compound system coated materials after a certain amount of being cross-linked on conventional culture dish, wet film is formed on the surface at culture dish, then under room temperature 25 DEG C of conditions after dry 48h, the culture dish deionized water dialysis being coated with thin-film material again by obtained is clean, and then dry 48h at ambient temperature, prepare the culture dish that can be used for culturing stem cells aggregate.
In the present embodiment, a kind of culture dish prepared by the present embodiment is used for the cultural method of stem cell aggregate, be specially: first uv sterilisation and germicidal treatment are carried out to the culture dish for culturing stem cells aggregate, then by after amplification in vitro to the human adipose-derived stem cell digestion collection of P3, adjustment cell concn is 5 × 10
5cells/ml, is inoculated on preparation-obtained culture dish, human adipose-derived stem cell self-assemble on culture dish after 72h, forms the aggregate that diameter is about 100-120 μm.
By reference to the accompanying drawings the embodiment of the present invention is illustrated above; but the invention is not restricted to above-described embodiment; multiple change can also be made according to the object of innovation and creation of the present invention; change, the modification made under all spirit according to technical solution of the present invention and principle, substitute, combination or simplify; all should be the substitute mode of equivalence; as long as goal of the invention according to the invention; only otherwise deviate from making method and the inventive concept of the culture dish of culturing stem cells aggregate of the present invention, all protection scope of the present invention is belonged to.
Claims (5)
1. the making method for the culture dish of culturing stem cells aggregate, it is characterized in that: adopt the hydrophilic material of single-component or many components hydrophilic material to be cross-linked the material system of preparation as coating material, and the mass concentration controlling the solution of coating material is 1-5wt%, then coating material is spread evenly across conventional culture dish and forms wet film on the surface, pass through drying means again, wet film on culture dish is solidificated in culture dish on the surface, drying and forming-film, prepares the culture dish for culturing stem cells aggregate.
2. according to claim 1 for the making method of the culture dish of culturing stem cells aggregate, it is characterized in that: described hydrophilic material adopts any one material in chitosan, polyacrylic acid, polyoxyethylene glycol, L-glutamic acid, sodium alginate, hyaluronic acid, dextran and hyaluronic acid or any different materials, and wherein the deacetylation of chitosan is 60 ~ 90%.
3. according to claim 1 or 2 for the making method of the culture dish of culturing stem cells aggregate, it is characterized in that: the substrate material of described culture dish is plastics.
4. according to claim 3 for the making method of the culture dish of culturing stem cells aggregate, it is characterized in that: the substrate material of described culture dish is polypropylene or polystyrene.
5. according to claim 1 or 2, culture dish is used for stem cell aggregate cultural method, it is characterized in that: prepare different culture dish by preparing different hydrophilic material film coatings on culture dish, and then regulable control forms the diameter dimension of micellar aggregates body.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114686375A (en) * | 2020-12-30 | 2022-07-01 | 华熙生物科技股份有限公司 | Multi-well plate for culturing cell aggregates and preparation and use methods thereof |
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| CN114686375A (en) * | 2020-12-30 | 2022-07-01 | 华熙生物科技股份有限公司 | Multi-well plate for culturing cell aggregates and preparation and use methods thereof |
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