CN101543644B - Constructing method of bracket-free engineering cartilaginous tissue and product thereof - Google Patents
Constructing method of bracket-free engineering cartilaginous tissue and product thereof Download PDFInfo
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- CN101543644B CN101543644B CN2008101028422A CN200810102842A CN101543644B CN 101543644 B CN101543644 B CN 101543644B CN 2008101028422 A CN2008101028422 A CN 2008101028422A CN 200810102842 A CN200810102842 A CN 200810102842A CN 101543644 B CN101543644 B CN 101543644B
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Abstract
The invention discloses a constructing method of a bracket-free engineering cartilaginous tissue and a product therefrom. The constructing method comprises the following steps: inducing and differentiating human umbilical mesenchymal stem cells into cartilaginous cells and then carrying out aggregation inducing culture so that the cartilaginous cells excrete stroma towards the outside of cells and form membranoid substance; continuously inducing and culturing the membranoid substance in a cell culture box and then transferring the membranoid substance into a centrifugal tube for inducing culture to form loose cartilaginous tissue agglomerate; and continuously inducing and culturing the loose cartilaginous tissue agglomerate in a rotating wall type bioreactor to obtain dense cartilaginous-like tissue. The cytology, the histology or the immunohistochemistry dyeing shows that the constructed engineering tissue has a normal cartilaginous cell type. The constructed bracket-free engineering cartilaginous tissue does not relate to the composite problem of the cells and an exogenous bracket, is absent from the biocompatibility problem between the cells and the bracket, is safe to human body, has even cell planting density and simple clinical application operation and can be applied to the treatment or the repair of cartilage loss.
Description
Technical field
The present invention relates to a kind of construction method of engineered tissue, relate in particular to construction method of bracket-free engineering cartilaginous tissue and products thereof, belong to regenerative medicine and field of tissue engineering technology.
Background technology
It is the focus of joint surgery research that the cartilage injury repairs always.Abroad (autologouschondrocyte implantation ACI) has obtained clinical efficacy preferably, but must second operation, increases patient's misery from the body chondrocyte cell transplantation.At present, the seed cell that is used to make up engineered cartilage tissue can derive from mesenchymal stem cells MSCs (bonemarrow stromal cells, BMSCs), but stem cell population and multiplication capacity significantly descend with age.The structure that autologous fat stem cell is used for the articular cartilage tissue engineering also has report, but these all exist and need the patient to carry out the problem of second operation.(embryonic stem cells ESCs) because the immunophenotype ateliosis can be used for heteroplastic transplantation, but faces ethics, legal problem, also has the back of transplanting to form teratomatous report to embryonic stem cell, and application is restricted.Therefore, seek new source of human stem cell and more and more receive various countries scholar's concern.Scholar separation and Culture mescenchymal stem cell (mesenchymal stem cells from puerperal obsolete material umbilical cord or Placenta Hominis is arranged recently; MSCs); And carry out phenotypic evaluation biology such as morphology, differentiation function and surface marker, for the multidirectional differentiation of stem cell and MSCs provide new seed cell source and experiment basis in Clinical Application.
Placenta Hominis and umbilical cord belong to the embryo to be organized outward, has the stem cell of a large amount of multidirectional differentiation.Adopt collagenase digestion can obtain the stem cell of a large amount of fibroblast appearance forms fast.In operating process, careful separation cord vessels tissue and umbilical cord adventitial tissue can obtain umbilical cord Wharton glue; Because umbilical cord only contains 3 blood vessels, does not contain blood capillary, can obtain the higher interstital stem cell of purity.The former time of supporting of being commissioned to train of umbilical cord Wharton glue interstital stem cell is about 3～5 days, and former being commissioned to train of BMSCs supported about 8～10 days of time; The CFU-F frequency is about 3: 10 in the umbilical cord Wharton glue interstital stem cell incubation
4, and BMSCs CFU-F frequency is merely 1: 10
4～10
5Flow cytometer detects umbilical cord Wharton glue stem cell positive expression CD44, CD105, CD271, and this is the surface marker of mescenchymal stem cell; Express CD34, CD45 hematopoietic stem cell sign and do not contain, this shows from the isolating stem cell of umbilical cord mesenchymal Wharton glue and all belongs to MSCs, and does not contain hematopoietic stem cell, and therefore, the purity of separating MSCs from umbilical cord Wharton glue is higher than BMSCs, and method is easier.
Through the MSCs in people's umbilical cord Wharton glue is carried out histochemistry and immunohistochemical staining, find the Wharton glue MSCs extracellular matrix components similar with the chondrocyte tool.Bibliographical information: umbilical cord is the tissue that is rich in collagen and GAGs, and wherein collagen accounts for 50% of dry weight, and hyaluronic acid accounts for 70% of GAGs; Cartilaginous tissue also is rich in collagen and GAG, and collagen accounts for 50% of dry weight, and wherein the II Collagen Type VI accounts for 90%～95% of total collagen.The inventor finds that in former experiments umbilical cord MSC Toluidine blue staining, sarranine " O " dyeing and II Collagen Type VI immunohistochemical staining are the weak positive; Behind the chondrocyte induction culture medium inducing culture, Toluidine blue staining finds that extracellular matrix is rich in cartilage glycoprotein, sarranine " O " dyeing and finds that being rich in the aminopolysaccharide, the immunohistochemical staining that increase inside and outside the cell finds that extracellular matrix II Collagen Type VI increases.Umbilical cord MSC is carried out RT-PCR analyze discovery, natural expression Sox-9 of umbilical cord MSC and Col-2A1.The Sox-9 gene be positioned at No. 17 chromosomes of people long-armed on, play an important role at embryo gender decision, regulation and control cartilage differentiation and development.Sox-9 combines chondrocyte characteristic molecule I type i collagen, and aggrecan albumen enhancer element activates and expresses, thus the differentiation of regulation and control cartilage.Natural positive expression Sox-9 of umbilical cord MSC and Col-2A1 have disclosed umbilical cord MSC from molecular level and have had the characteristic of precartilage cell; Umbilical cord MSC is after chondrocyte induction is cultivated, and II Collagen Type VI immunohistochemical staining is strong positive, more approaches chondrocyte, more than show that all the isolated stem cell of people Wharton glue has the characteristics of nature to the cartilage differentiation.This also is it and adult's bone marrow stem cell, fat stem cell difference.
In sum; Umbilical cord Wharton glue stem cell has all confirmed to have the characteristics of MSCs from aspects such as morphology, multiplication characteristic, cell surface molecule sign and immunophenotypes; And confirmed to have the characteristic of precartilage cell, can better be converted into chondrocyte under certain condition.In addition, the huge advantage of umbilical cord also is: it is restricted hardly to draw materials; No ethics restriction; If can further show its immunosuppressant characteristic or reduce its antigenicity of removal, then can be used for heteroplastic transplantation, aspect the reparation of allogeneic articular cartilage broad prospect of application will arranged.
The reparation of articular cartilage damage is a clinical difficult problem, and seed cell is the subject matter that faces of articular cartilage damage reparation.Set up cartilage tissue engineered seed cell at present both at home and abroad and all derive from, and mostly the mode of external structure is cell compound cells support from body.Cell and above-mentioned timbering material compound comparatively loaded down with trivial details, the cell inoculation density unevenness, and exist in cell and support biocompatibility, the stake body catabolite that problems such as complicacy are examined in human safety, support clinical practice.
Summary of the invention
Technical problem to be solved by this invention is the deficiency that overcomes prior art, and a kind of construction method of unsupported cartilaginous tissue is provided.
Technical problem to be solved by this invention realizes through following technical scheme:
A kind of construction method of bracket-free engineering cartilaginous tissue comprises:
(1) (mesenchymal stem cells MSCs) is induced to differentiate into chondrocyte with human umbilical cord mesenchymal stem cells;
(2) chondrocyte is then carried out aggregation inducing culture; Make chondrocyte to cell exocrine substrate and form membranoid substance; Membranoid substance (this membranoid substance is made up of extracellular matrix and chondrocyte) is peeled off the back from the culture bottle wall in cell culture incubator, continue inducing culture; It is centrifugal afterwards culture to be transferred to centrifuge tube, under action of centrifugal force, cell is further assembled, and forms loose chondrocyte agglomerate;
(3) the loose cartilaginous agglomerate is rotated inducing culture in the rotation cell culture system, obtains fine and close class cartilaginous tissue.
The differentiation mode of inducing described in the step (1) can adopt monolayer inducing culture mode; Can induce differentiation (for example: Wang HS with reference to the disclosed mode of relevant document; Hung SC, Peng S T, et al.Mesenchymalstem cells in the Wharton ' s jelly of the human umbilical cord.Stem Cells; 2004,22 (7): 1330); Preferred, the differentiation of inducing described in the step (1) is carried out according to following cultivating system and condition of culture: inducing culture liquid is: DMEM (20%FBS) adds 10ng/ml hTGF-β 1,10ng/ml hFGF, and volume fraction is 1%ITS and 1.0 * 10
-8The mol/L dexamethasone; Condition of culture: cultivation temperature is 37 ℃, at 5%CO
2Incubator in cultivate, changed liquid once in per 3 days.
The inducing culture liquid of the aggregation inducing culture described in the step (2) and described inducing culture liquid of the same step of condition of culture (1) and condition of culture; Also can carry out (B.Johnstone, T.M.Hering, A.I.Caplan according to the condition of the disclosed aggregation inducing culture of document; V.M.Goldberg; J.U.Yoo, In vitro chondrogenesis ofbone marrow-derived mesenchymal progenitor cells, Exp.Cell Res.238 (1998) 265-272).
Described centrifuge tube is 15 milliliters conical centrifuge tube preferably; Saidly centrifugally preferably carry out centrifugal in following condition: centrifugal rotational speed is 1000rpm-2000rpm, and centrifugation time is 10 minutes.
In the step (3) with the loose cartilaginous agglomerate in the rotation cell culture system during inducing culture; The same step of described inducing culture liquid (1); Be that DMEM adds 20%FBS, add 10ng/ml hTGF-β 1,10ng/mlhFGF, volume fraction again and be 1% ITS and 1.0 * 10
-8The mol/L dexamethasone; Condition of culture is following: cultivation temperature is 37 ℃; The rotating speed proceed step by step adjustment of rotation cell culture system; That is: (1) is controlled to be 12 rev/mins with the starting velocity of rotation, and rotating and culturing was had a rest 30 minutes after 30 minutes; Repeat aforesaid operations again and afterwards change rotary speed into 16 rev/mins 2 times; Whenever continue rotation and changed inducing culture liquid once in three days, general continuing cultivated 10-20 days, was preferably 15 days (but the tissue size of what and required formation of those skilled in the art's visual cell and appropriate change or set incubation time).
The present invention at first is induced to differentiate into chondrocyte with human umbilical cord mesenchymal stem cells, and chondrocyte is carried out aggregation inducing culture, obtains a large amount of membranoid substances of being made up of extracellular matrix and chondrocyte; Be placed in the centrifuge tube after membranoid substance peeled off; Under the action of centrifugal force of centrifuge, membranoid substance is piled up, and takes out and piles up membranoid substance; In cell culture incubator, continue one week of inducing culture; Along with the prolongation of incubation time, continue excretory extracellular matrix membranoid substance is integrated, form the loose cartilaginous agglomerate; At last the cartilaginous tissue agglomerate is transferred in the rotation cell culture system and carries out inducing culture; Under the effect of rotation cell culture system, the loose cartilaginous agglomerate is under the stimulation of mechanics, and cell continues propagation; Justacrine substrate; Form the bigger agglomerate of organizing, tissue becomes fine and close, smooth and has gloss, and the tissue after feasible the inducing is near cartilaginous tissue.
The present invention is by the membrane structure of people's umbilical cord MSCs through forming behind the intensive inducing culture; In conjunction with the external evoked cultivation of bioreactor technology; The extracellular matrix (ECM) of emiocytosis has the composition of similar cartilage cell epimatrix after inducing; Be the good natural support of chondrocyte, make up and obtain unsupported engineered cartilage tissue.Show that through cytology, histology, immunohistochemical staining the constructed engineered tissue of the present invention has normal cartilage cell characteristics.
The present invention (cell → cell aggregation becomes membranaceous → open texture piece → compact tissue piece → class chondroid tissue) in whole incubation does not relate to the compound problem of cell and exogenous support; Cell seeding density is even; The problem that does not have cell and support biocompatibility; To human body safety, simple to operate, clinical treatment or the reparation that can be applicable to articular cartilage defect.
Description of drawings
The phenotype of Fig. 1 mescenchymal stem cell.
The outward appearance of the no support cartilaginous tissue that Fig. 2 the present invention is constructed.
The HE coloration result of the no support cartilaginous tissue that Fig. 3 the present invention is constructed.
Stigma Croci " O " coloration result of the no support cartilaginous tissue that Fig. 4 the present invention is constructed.
The Toluidine blue staining result of the no support cartilaginous tissue that Fig. 5 the present invention is constructed.
The II Collagen Type VI immunohistochemical staining result of the no support cartilaginous tissue that Fig. 6 the present invention is constructed.
The RT-PCR testing result of the no support cartilaginous tissue that Fig. 7 the present invention is constructed ((Sox-9 and Col-2A1 are positive); 1:Marker; 2,7: β-actin; 3,5:Sox-9; 4,6:Col-2A1.2,3,4 are the result before inducing, and 5,6,7 are the result after inducing.
The specific embodiment
Further describe the present invention below in conjunction with specific embodiment, advantage of the present invention and characteristics will be more clear along with description.But these embodiment only are exemplary, scope of the present invention are not constituted any restriction.It will be understood by those skilled in the art that and down can make amendment with form or replace without departing from the spirit and scope of the present invention, but these modifications and replacing all fall in protection scope of the present invention the details of technical scheme of the present invention.
One, material and reagent
PE-CD44, PE-HLA-ABC, FITC-HLA-DPDQDR, FITC-CD34, FITC-CD45 and FITC-IgG1 and PE-IgG1 antibody (BD company; USA), and PE-CD105 antibody (Sant Crus company, USA); PE-CD271 antibody (Miltenyi Biotec GmbH, Germany); The DMEM culture medium (dulbecco ' smodified Eagle ' s medium; DMEM), hyclone (fetal bovine serum; FBS), insulin-transferrins-selenium (insulin-transferrin-selenium; ITS), pancreatin, dexamethasone, II Collagen Type VI monoclonal antibody (Sigma company, USA); Penicillin, streptomycin (North China pharmaceutical Co. Ltd); People's transforming growth factor B1 (human transforming growth factor β-1, hTGF-β 1, Pepro Tech EC company; USA), human fibroblastic growth factor (human fibroblast growth factor; HFGF, Pepro Tech EC company, USA).Trizol Reagent (Invitrogen company, USA), RT-PCR reagent (QIAGEN company, Germany), agarose (Promega company, USA).
Two, experimental facilities
CO
2Incubator (Hereus B5060 type, Germany); Inversion differ optical microscope and imaging system (OLYMPUS, IX70, Japan) and optical microscope and imaging system (OLYMPUS, BX51, Japan); Flow cytometer (BD company, USA); Gel analysis system (Beijing match intelligence foundation company limited, Champgel
TM2000 types); The rotation cell culture system (SYNTHECON company, USA).
Three, the preparation of D-hank ' s balance liquid
Get 8.0g NaCl, 0.4g KCl, 0.06g Na
2HPO
4.H
2O, 0.06g KH
2PO
4, add water 200ml dissolving, add phenol red mother solution 1ml, be settled to 1000ml, divide to install in the 250ml bottle, disinfection with high pressure steam, the time spent is used NaHCO
3About adjustment pH value to 7.2.
The structure and the evaluation of embodiment 1 no framework engineered cartilage tissue
1, separation and the cultivation of people's umbilical cord Wharton glue stem cell
Get the healthy puerpera's umbilical cord tissue of mature normal product from operating-table, the blood that D-Hank balance liquid thorough washing is residual is removed umbilical vein, tremulous pulse and umbilical cord adventitia, peels off Wharton glue, shreds the back with 37 ℃ of magnetic stirring apparatus effects of type i collagen enzyme digestion down.With postdigestive contain cell dissociation buffer with the D-Hank balance liquid clean, 1500 rev/mins of centrifugal 10min, after centrifugal 3 times, with the cell seeding of collecting in containing the DMEM culture bottle that volume fraction is 10%FBS, 37 ℃, 5%CO
2Incubator in cultivate, observe with inverted phase contrast microscope.
2, people's umbilical cord Wharton glue stem cell surface molecular marker detects and identifies
Collect second filial generation people umbilical cord Wharton glue stem cell; Add hematopoietic stem cell sign antibody FITC-CD34 and FITC-CD45 respectively; MSCs sign antibody PE-CD44, PE-CD105 and PE-CD271; Major histocompatibility antigen complex (major histocompability complex; MHC) sign antibody PE-HLA-ABC and FITC-HLA-DPDQDR, observer's umbilical cord stem cell hemopoietic sign, MSCs sign and MHC expression adopt PE-IgG1 and FITC-IgG1 as going together contrast simultaneously.People's umbilical cord stem cell and corresponding antibodies are hatched 30min in 4 ℃ of refrigerators, PBS washing and adjustment cell concentration are 1 * 10
6/ ml, flow cytometer detects.PE-CD44, PE-CD105 and PE-CD271 are positive as a result, and FITC-CD34 and FITC-CD45 are negative; HLA-ABC is positive, HLA-DPDQDR negative (Fig. 1).
3, the structure of cartilaginous tissue (inducing culture in aggregation inducing culture+rotation cell culture system):
(1) get highdensity second filial generation people's umbilical cord Wharton glue MSCs place to cartilage induced differentiation culture fluid (DMEM (20%FBS) adds 10ng/ml hTGF-β 1,10ng/ml hFGF, volume fraction is 1%ITS and 1.0 * 10
-8The mol/L dexamethasone); Condition of culture: 37 ℃, 5%CO
2Incubator in cultivate, changed liquid in per 3 days, visiblely induce descendant's umbilical cord Wharton glue MSCs to form membranaceous rapidly;
(2) along with aggregation inducing culture; The membranoid substance of forming is along with the prolongation of induction time, and the edge of film starts, and cell membrane is is carefully blown and beaten; Moving in the centrifuge tube at the 15ml point end at rotating speed is centrifugal 10min under the condition of 1000-2000rpm; Cell is further assembled, and makes membranoid substance be piled up in bottom the point centrifuge tube at the end the same step of inducing culture liquid (1) in the centrifuge tube; Take out membranoid substance then, in cell culture incubator, continue inducing culture, induced for 1 week after; Proceed inducing culture with careful will the moving to based on the loose agglomerate appearance tissue of cell membrane in the rotation cell culture system of suction pipe, the same step of inducing culture liquid (1) in the rotation cell culture system, cultivation temperature is 37 ℃; The rotating speed proceed step by step adjustment of rotation cell culture system, that is: (1) starting velocity is 12 rev/mins, rotating and culturing (rest) 30 minutes of stopping the rotation after 30 minutes; (2) step (1) is repeated 2 times again, rotary speed changes 16 rev/mins into afterwards, whenever continues rotation and changes inducing culture liquid once in three days; Continue to cultivate about 15 days, promptly get.
4, identify:
Tissue type of the having cartilage characteristics that form, outward appearance: tissue has gloss and elasticity, and diameter is 5mm (Fig. 2)
Histochemistry and immunocytochemistry are identified: chondrocyte is circular, is arranged in lacuna, sarranine " O ", Toluidine blue staining, the inside and outside all positives of II Collagen Type VI staining cell (Fig. 3, Fig. 4, Fig. 5, Fig. 6).
DNA identifies: RT-PCR detects, Sox-9 and Col-2A1 positive (Fig. 7).
Claims (5)
1. the construction method of a bracket-free engineering cartilaginous tissue comprises:
(1) the human umbilical cord mesenchymal stem cells inducing culture is divided into chondrocyte;
(2) chondrocyte is carried out aggregation inducing culture; Make chondrocyte to cell exocrine substrate and form membranoid substance; Membranoid substance is continued inducing culture in cell culture incubator, afterwards culture is transferred in the centrifuge tube centrifugal, under action of centrifugal force; Cell is further reunited, form loose chondrocyte agglomerate;
(3) loose chondrocyte agglomerate is rotated inducing culture in the rotation cell culture system, obtains fine and close class cartilaginous tissue;
Rotation rotating speed described in the step (3) is regulated and control according to following steps: (1) is controlled to be 12 rev/mins with initial rotary speed, rotates 30 minutes, stops the rotation 30 minutes; (2) step (1) is repeated 2 times and later rotary speed is controlled to be 16 rev/mins, continue rotation 10-20 days.
2. according to the construction method of claim 1, it is characterized in that: the centrifuge tube described in the step (2) is 15 milliliters a conical centrifuge tube.
3. according to the construction method of claim 1, it is characterized in that: centrifugal rotation speed is 1000rpm-2000rpm described in the step (2), and centrifugation time is 10min.
4. according to the construction method of claim 1, it is characterized in that: the time of described lasting rotation is 15 days.
5. the resulting bracket-free engineering cartilaginous tissue of any one construction method of claim 1-4.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109152864A (en) * | 2015-12-30 | 2019-01-04 | 艾克斯赛尔治疗公司 | A method of being used to prepare three-dimensional cartilage organoid block |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102796701A (en) * | 2012-07-30 | 2012-11-28 | 成都恒春之源生物科技股份有限公司 | Method for amplifying human umbilical cord mesenchymal stem cells through in-vitro 3D stent-free suspension cultivation |
| CN102899287B (en) * | 2012-10-24 | 2013-08-21 | 天津和泽干细胞科技有限公司 | Method for inducing differentiation from mesenchymal stem cells to cartilage cells and application of mesenchymal stem cells in osteoarthritis |
| CN111139220B (en) * | 2020-01-08 | 2022-04-12 | 深圳市旷逸生物科技有限公司 | Three-dimensional culture method of umbilical cord mesenchymal stem cells |
| CN116688233A (en) * | 2022-02-25 | 2023-09-05 | 上海软馨生物科技有限公司 | Tissue engineering cartilage particle implant and preparation method thereof |
Citations (2)
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|---|---|---|---|---|
| CN1553951A (en) * | 2000-11-03 | 2004-12-08 | ���ﰲҽ�ƹɷ�����˾ | Human cord blood derived unrestricted somatic stem cells (USSC) |
| CN1630717A (en) * | 2002-02-19 | 2005-06-22 | 美迪宝斯特有限公司 | Isolation and culture-expansion methods of mesenchymal stem/progenitor cells from umbilical cord blood and differentation method of umbilical cord blood-derived mesenchymal stem/progenitor cells into |
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|---|---|---|---|---|
| CN1553951A (en) * | 2000-11-03 | 2004-12-08 | ���ﰲҽ�ƹɷ�����˾ | Human cord blood derived unrestricted somatic stem cells (USSC) |
| CN1630717A (en) * | 2002-02-19 | 2005-06-22 | 美迪宝斯特有限公司 | Isolation and culture-expansion methods of mesenchymal stem/progenitor cells from umbilical cord blood and differentation method of umbilical cord blood-derived mesenchymal stem/progenitor cells into |
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| Brian Johnstone, et al.In Vitro Chondrogenesis of Bone Marrow-Derived Mesenchymal Progenitor Cells.《EXPERIMENTAL CELL RESEARCH》.1998,第238卷第265-272页. * |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109152864A (en) * | 2015-12-30 | 2019-01-04 | 艾克斯赛尔治疗公司 | A method of being used to prepare three-dimensional cartilage organoid block |
| CN109152864B (en) * | 2015-12-30 | 2021-12-10 | 艾克斯赛尔治疗公司 | Method for preparing three-dimensional cartilage organoid block |
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