CN109402049A - A kind of preparation method for the tissue engineering fat stem cell lamella with cartilage differentiation potential can be used for cartilage damage reparation - Google Patents
A kind of preparation method for the tissue engineering fat stem cell lamella with cartilage differentiation potential can be used for cartilage damage reparation Download PDFInfo
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Abstract
The invention discloses a kind of preparation methods of tissue engineering fat stem cell lamella with cartilage differentiation potential that can be used for cartilage damage reparation, the present invention is by preparing specific cultivating system, it is the complete laminated structure containing extracellular matrix using the ASCs lamella that cell sheets culture technique obtains, and has cartilage to differentiation potential.It is intracorporal only by the tissue of pure cell composition due to being transplanted to, avoid the use and its brought adverse reaction of the carriers such as bracket.And ASCs lamella remains extracellular matrix, can preferably keep the harmonious of function containing important cell surface protein, iuntercellulars such as ion channel, growth factor receptors and connection albumen, has the advantages that traditional tissue engineering technique is incomparable.Complete lamellar structure is conducive to cell retaining in transplantation site, substantially increases the utilization rate of cell and the activity of metastatic cells.
Description
Technical field
The invention belongs to cell transplantations and organizational project to repair field, more particularly to a kind of for the regenerated tool of repair of cartilage
There is the preparation method of the cell sheets of cartilage differentiation potential.
Background technique
Cartilage is made of the extracellular matrix of the round or ellipse cartilage cell being dispersed in and surrounding densification, without blood vessel,
Nerve and lymphoid tissue structure.The extracellular matrix of cartilage cell is mainly made of II collagen type and glycoprotein, is cartilage
Tissue provides enough mechanical strengths.The structure feature of cartilage keeps its self-repairing capability limited.It is closed as caused by a variety of causes
Section cartilage damage will lead to degenerative osteoarthritis.Currently, the main method that organizational project uses is according to institutional framework, benefit
Prepare similar three-dimensional rack with degradable biomaterial, and plant seed cell on it, formed framework's compound with
It is implanted into afterwards and is used to replace lesion or damaged tissues in vivo.Currently, organizational project is in cartilage, the reparation of bone and blood vessel etc.
Field has obtained certain progress.However, there is also some problems for the application of organizational project.Firstly, need can for timbering material
The biofacies content leaned on, certain biomaterials will cause transplantation site tissue around and inflammatory, necrosis etc. occur.Secondly, bracket drops
The space left after solution would generally be filled by proliferation fibr tissue, generate the tissue of non-functional.Manufacture functional organization's engineering
Structure there are many challenges, including cell migration and be colonized into bracket, receptor inflammatory reaction, micro vascularization deficiency cause
The ability transplanted on a large scale is limited, scaffold degradation and cell proliferation rate are asynchronous, the method based on simple bracket combination cell
The functional organization with natural complex organization's structure can not be generated.Cell sheets technology remains cell connection, and endogenous is thin
Extracellular matrix, and cell micro-environment is simulated in terms of various machinery, chemistry and biology characteristic, become the new side of cell transplantation
Method.
Adipose-derived source mescenchymal stem cell (ASCs) is sufficient, is easily isolated acquisition and cultured and amplified in vitro, and nothing is exempted from
Epidemic disease rejection, compared to the dry thin of the cartilage cell in nasal septum source, the mescenchymal stem cell of derived from bone marrow and tendon source
The common seed cell such as born of the same parents, adipose-derived stem cell have the cartilage differentiation potential more having, it has also become use in organizational project
Ideal seed cell is repaired in cartilage damage.Due to lacking enough cell densities and signal factor, cell adherence rate is low, carefully
The disadvantages of born of the same parents' storage and application form problem, simple plantation stem cell is more difficult acquirement good result.ASCs suspension combines branch
The method of frame transplanting, cell restocking rate and cell density are low, it is difficult to form fine and close destination organization.Therefore, organizational engineering is wanted
It constructs with compact tissue and blood supply bulk tissue abundant, it is necessary to solve to ask existing for seed cell harvest and transfer aspect
Topic.Cell sheets technology has been successfully applied to mesenchymal stem cell, epithelial cell etc., but utilizes traditional cell sheets skill
The more difficult lamella for obtaining adipose tissue-derived mesenchymal stem cells of art, this makes the technical application of cell sheets and the method for repair of cartilage
At great limitation.Ascorbic acid is the vital common battalion of a kind of pair of human health as a kind of polyol
Element is supported, there is very strong inoxidizability, play a crucial role in the biosynthesis of collagen and other extracellular matrix components,
And confactor is served as in many biological respinses of entire human body.After ascorbic acid is handled in periodontal mescenchymal stem cell
Telomerase activation and reverse transcriptase of telomere protein level increase.Reverse transcriptase of telomere is control DNA metabolism and proliferation
The cell sheets of one of crucial nucleoprotein, ascorbic acid induction promote its self-renewing and differentiation potential by telomerase activation.
Addition ascorbic acid can be used as growth promoter to increase cell Proliferation and DNA synthesis, extracellular matrix and sink in the medium
Product.Meanwhile having researches show that ascorbic acid can raise the expression of Oct4 gene, and Oct4 is for maintaining the mostly latent of embryonic stem cell
Energy property and self-renewing have extremely important effect.During cell sheets culture, glucose, pyruvic acid, isocitric acid, amber
Acid, serine and glutamine largely consume.Glucose, pyruvic acid, isocitric acid, succinic acid be glycolysis raw material and in
Between product, and main component of the serine as proteoglycans largely disappeared in the synthesis and secretion of inducing cell epimatrix
Consumption.According to the metabolic characteristic of cell sheets, can completely be obtained to a certain extent by specific culture technique containing extracellular
Matrix, growth factor receptors connect albumen, Cell tracking, ion channel and other important cells surface proteins it is complete thin
Born of the same parents' lamella, even with the stem cell lamella of specific differentiation potential.Further, cell sheets, which can be assembled into, is similar to naturally
The three-dimensional structure of tissue is simultaneously grafted directly to damage location.
Summary of the invention
In view of the above-mentioned deficiencies in the prior art, it is an object of the present invention to provide a kind of fat source with cartilage differentiation potential
The preparation method of property cell sheet engineering layer.The present invention is based on cellular layer lamella technologies, by preparing specific cultivating system
Fiber differentiation ASCs lamella, so that being easier to obtain ASCs lamella, and the cell sheets cultivated have cartilage differentiation potential, are conducive to
It forms chondroid tissue and with surrounding tissue ining conjunction in repair of cartilage, common to participate in repair tissue damage, promotes to regenerate,
And the influence generated by implantation material to damage location is reduced, cell micro-environment is simulated in terms of chemistry and biology characteristic, is improved
The Retention of cell and the activity of transplanted cells.
The purpose of the present invention is achieved through the following technical solutions: it is a kind of can be used for cartilage damage reparation have it is soft
The preparation method of the tissue engineering fat stem cell lamella of bone differentiation potential, comprising the following steps:
(1) adipose-derived mescenchymal stem cell lamella forms the preparation of cultivating system: volume is added in DMEM culture medium
The fetal calf serum and 50 μ g/ml serines that concentration is 12%, obtain the first culture medium, and a volumetric concentration that contains of another preparation is 12%
Fetal calf serum, 20 μ g/ml ascorbic acid and 50 μ g/ml serines the second culture medium, use preceding equal 37 DEG C of shaking tables preheating；
(2) adipose-derived mescenchymal stem cell passes on amplification cultivation: by adipose-derived primary mescenchymal stem cell 1ml
The trypsin treatment 1min that mass volume ratio is 0.25% becomes spherical, Cell tracking disconnection to cellular morphology, with containing body
The DMEM culture solution 3ml for the fetal calf serum that product concentration is 10% terminates trypsin acting, moves into centrifuge tube, 1500rpm centrifugation
5min abandons supernatant, and the DMEM culture solution that the fetal calf serum for being 10% containing volumetric concentration is added is obtained in 1:3 ratio secondary culture
First generation fat mesenchymal stem cell；According to said method continue secondary culture and obtains third generation fat mesenchymal stem cell；
(3) preparation of fat-derived stem cells lamella: the third generation fat mesenchymal stem cell that step (2) is obtained is used
1ml mass volume ratio is seeded in culture bottle after being 0.25% trypsin digestion 1min, and inoculum density is 5 × 103cells/
cm2, in the first culture medium, 37 DEG C of constant temperature, volumetric concentration 5%CO2It cultivates in saturated humidity incubator to 90% degrees of fusion
(confluent), it is changed to the second culture medium, to 21 days, cell formed a film-form layer structure in bottom for continuous culture, will
It separates the tissue engineering fat stem cell lamella for obtaining and having cartilage differentiation potential.
The beneficial effects of the present invention are: the present invention by preparing specific cultivating system, utilizes the success of cell sheets technology
The fat-derived mescenchymal stem cell lamella with cartilage differentiation potential is obtained, the ASCs lamella of harvest is containing extracellular base
The complete laminated structure of matter, and there is cartilage differentiation potential.It is intracorporal only by the tissue of pure cell composition due to being transplanted to, it keeps away
The use and its brought adverse reaction of the carriers such as bracket are exempted from.And ASCs lamella remains extracellular matrix, containing from
Important cell surface protein, the iuntercellulars such as subchannel, growth factor receptors and connection albumen can preferably keep function
It is harmonious, have the advantages that traditional tissue engineering technique is incomparable.Substantially increase the utilization rate and metastatic cells of cell
Activity.
Detailed description of the invention
Fig. 1 is the fat mesenchymal stem cell lamella figure by obtaining after specific cultivating system culture；
Fig. 2 is adipose-derived mescenchymal stem cell and fat-derived stem cells lamella related gene expression comparison diagram.
Specific embodiment
The present invention is described further combined with specific embodiments below, but protection scope of the present invention is not limited in
This:
A kind of tissue engineering fat with cartilage differentiation potential can be used for cartilage damage reparation provided by the invention is dry
The preparation method of cell sheets, comprising the following steps:
1, adipose-derived mescenchymal stem cell lamella forms the preparation of cultivating system: it is dense that volume being added in DMEM culture medium
The fetal calf serum and 50 μ g/ml serines that degree is 12%, obtain the first culture medium, and another preparation portion is 12% containing volumetric concentration
Second culture medium of fetal calf serum, 20 μ g/ml ascorbic acid and 50 μ g/ml serines is preheated using preceding equal 37 DEG C of shaking tables；
2, adipose-derived mescenchymal stem cell passes on amplification cultivation: by adipose-derived primary mescenchymal stem cell 1ml
The trypsin treatment 1min that mass volume ratio is 0.25% becomes spherical, Cell tracking disconnection to cellular morphology, with containing body
The DMEM culture solution 3ml for the fetal calf serum that product concentration is 10% terminates trypsin acting, moves into centrifuge tube, 1500rpm centrifugation
5min abandons supernatant, and the DMEM culture solution that the fetal calf serum for being 10% containing volumetric concentration is added is obtained in 1:3 ratio secondary culture
First generation fat mesenchymal stem cell；According to said method continue secondary culture and obtains third generation fat mesenchymal stem cell；
3, the preparation of fat-derived stem cells lamella: the third generation fat mesenchymal stem cell 1ml that step (2) is obtained
Mass volume ratio is seeded in culture bottle after being 0.25% trypsin digestion 1min, and inoculum density is 5 × 103cells/cm2,
In the first culture medium, 37 DEG C of constant temperature, volumetric concentration 5%CO2It cultivates in saturated humidity incubator to 90% degrees of fusion
(confluent), it is changed to the second culture medium, continuous culture was to 21 days, and cell forms a film-form layer structure in bottom, such as
Shown in Fig. 1, it is isolated the tissue engineering fat stem cell lamella for obtaining and there is cartilage differentiation potential.By responsive to temperature type
Culture surface is separated by temperature change and obtains cell sheets.
By PCR method detect ASCs cell sheets and ASCs osteogenic potential, Chondrogenesis and at rouge potential it is related
Difference in gene expression finds fat-derived mescenchymal stem cell lamella height expression Collagen II, Agg and Sox9, explanation
ASCs cell sheets have cartilage differentiation potential.
The lamella obtained to fat mesenchymal stem cell and culture carries out gene expression analysis, as shown in Fig. 2, discovery obtains
Induction after stem cell lamella cartilage differentiation potential (collagen II, Agg, Sox9) be better than filling between the fat that is not induced
Matter stem cell；And skeletonization, at the fat mesenchymal stem cell in rouge and fibrocartilage differentiation potential gene expression, not induced
Higher than the cell sheets after induction, illustrates that the fat mesenchymal cell sheets obtained by specific system culture are more suitable and be used for
The reparation of cartilage damage and defect.
The above description is only an embodiment of the present invention, is not intended to limit the scope of the invention.For institute of the present invention
For the those of ordinary skill for belonging to technical field, without departing from the inventive concept of the premise, it can also make and several simply push away
It drills or replaces, be regarded as protection scope of the present invention.
Claims (1)
1. a kind of preparation for the tissue engineering fat stem cell lamella with cartilage differentiation potential that can be used for cartilage damage reparation
Method, which comprises the following steps:
(1) adipose-derived mescenchymal stem cell lamella forms the preparation of cultivating system: volumetric concentration is added in DMEM culture medium
For 12% fetal calf serum and 50 μ g/ml serines, the first culture medium is obtained, another a tire for being 12% containing volumetric concentration of preparation
Second culture medium of cow's serum, 20 μ g/ml ascorbic acid and 50 μ g/ml serines is preheated using preceding equal 37 DEG C of shaking tables；
(2) adipose-derived mescenchymal stem cell passes on amplification cultivation: by adipose-derived primary mescenchymal stem cell 1ml mass
The trypsin treatment 1min that volume ratio is 0.25% becomes spherical, Cell tracking disconnection to cellular morphology, and with containing, volume is dense
Degree terminates trypsin acting for the DMEM culture solution 3ml of 10% fetal calf serum, moves into centrifuge tube, and 1500rpm is centrifuged 5min,
Supernatant is abandoned, the DMEM culture solution that the fetal calf serum for being 10% containing volumetric concentration is added obtains first in 1:3 ratio secondary culture
Fat subsitutes mescenchymal stem cell；According to said method continue secondary culture and obtains third generation fat mesenchymal stem cell；
(3) preparation of fat-derived stem cells lamella: the third generation fat mesenchymal stem cell 1ml matter that step (2) is obtained
Amount volume ratio is seeded in culture bottle after being 0.25% trypsin digestion 1min, and inoculum density is 5 × 103cells/cm2,
In first culture medium, 37 DEG C of constant temperature, volumetric concentration 5%CO2It cultivates in saturated humidity incubator to 90% degrees of fusion
(confluent), it is changed to the second culture medium, to 21 days, cell formed a film-form layer structure in bottom for continuous culture, will
It separates the tissue engineering fat stem cell lamella for obtaining and having cartilage differentiation potential.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111870741A (en) * | 2020-08-06 | 2020-11-03 | 厦门大学附属中山医院 | Application of morroniside combined stem cells in preparation of cartilage repair material |
| CN117683711A (en) * | 2024-02-01 | 2024-03-12 | 哈尔滨龙慧干细胞生物科技有限公司 | Use of stem cells or transgenic stem cells |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030103947A1 (en) * | 2001-02-23 | 2003-06-05 | Urlich Noth | In vitro engineered cartilage constructs produced by coating biodegradable polymer with human mesenchymal stem cells |
| CN103773734A (en) * | 2014-01-24 | 2014-05-07 | 浙江大学 | Preparation method of tissue engineering cell sheet with osteogenic differentiation and vasculogenesis |
| CN106350483A (en) * | 2016-10-14 | 2017-01-25 | 中卫华医（北京）生物科技有限公司 | Culture method for inducing adipose tissue-derived stromal cells to differentiate to chondrocyte |
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- 2018-09-18 CN CN201811090433.5A patent/CN109402049B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030103947A1 (en) * | 2001-02-23 | 2003-06-05 | Urlich Noth | In vitro engineered cartilage constructs produced by coating biodegradable polymer with human mesenchymal stem cells |
| CN103773734A (en) * | 2014-01-24 | 2014-05-07 | 浙江大学 | Preparation method of tissue engineering cell sheet with osteogenic differentiation and vasculogenesis |
| CN106350483A (en) * | 2016-10-14 | 2017-01-25 | 中卫华医（北京）生物科技有限公司 | Culture method for inducing adipose tissue-derived stromal cells to differentiate to chondrocyte |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111870741A (en) * | 2020-08-06 | 2020-11-03 | 厦门大学附属中山医院 | Application of morroniside combined stem cells in preparation of cartilage repair material |
| CN117683711A (en) * | 2024-02-01 | 2024-03-12 | 哈尔滨龙慧干细胞生物科技有限公司 | Use of stem cells or transgenic stem cells |
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