CN1746295A - A kind of tissue engineered cartilage based on placenta-derived mesenchymal stem cells and its manufacturing method - Google Patents
A kind of tissue engineered cartilage based on placenta-derived mesenchymal stem cells and its manufacturing method Download PDFInfo
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Abstract
本发明是一种基于胎盘来源的间充质干细胞的组织工程化软骨及其制造方法，属组织工程领域。本发明采用胎盘来源的间充质干细胞作为种子细胞，与可降解的多孔生物材料复合构建组织工程化软骨。采用该方法构建的软骨由于不受细胞来源限制，因而不需要对患者造成二次创伤，也无需经历较长的细胞培养时间，并且更容易控制质量，减小批次间或个体间效果差异，临床应用更为方便。本发明可实现关节软骨损伤的功能性修复，具有明显的临床应用前景。The invention relates to tissue engineered cartilage based on placenta-derived mesenchymal stem cells and a manufacturing method thereof, belonging to the field of tissue engineering. The invention adopts placenta-derived mesenchymal stem cells as seed cells, which are combined with degradable porous biomaterials to construct tissue-engineered cartilage. Because the cartilage constructed by this method is not limited by the source of cells, it does not need to cause secondary trauma to the patient, nor does it need to undergo a long cell culture time, and it is easier to control the quality and reduce the difference in effects between batches or individuals. Application is more convenient. The invention can realize functional repair of articular cartilage damage, and has obvious clinical application prospect.
Description
Invention field
The present invention relates to a kind of organization engineered cartilage and manufacture method thereof, belong to field of tissue engineering technology based on placenta derived mesenchymal stem cell.
Background technology
The joint cartilage trauma repair is still a clinical medical huge challenge at present.Existing treatment plan comprises cartilaginous tissue or bone-cartilage tissue transplantation and joint prosthesis.From the body cartilage is best graft materials, but because of the Worker's Stadium limited, and usually need be in the operation of second position to obtain enough cartilaginous tissues that is used to transplant, therefore unsatisfactory with regard to application.And the substituting reparation of joint prosthesis still is difficult to reach functional repairing effect up to now, and regular meeting is with the generation of various complication.Although people have carried out unremitting effort for many years in this field, but still need to seek effective methods of treatment that a kind of articular cartilage damage is repaired, suitable physiological reparation is provided, thereby avoids collecting on one's body in a large number from the body cartilaginous tissue, with another wound of a kind of trauma repair from the patient again.
The rise of tissue engineering technique is that the physiological reparation of cartilaginous tissue has brought hope.Domestic and foreign literature reported in succession and adopted this technique construction cartilage, nude mice with have the example of achieving success in the animal bodies such as mouse, rat, rabbit, pig of immunizing power.But the cell that these institutes adopt is from body or allosome chondrocyte.And chondrocyte itself source less, lose phenotype when the difficulty of drawing materials, amplification in vitro easily.For overcoming the deficiency of chondrocyte as cartilage tissue engineered seed cell, people generally bet attention on the adult tissue stem cell of mescenchymal stem cell one class in recent years, wherein the mescenchymal stem cell of derived from bone marrow is because of it is drawn materials relatively easily, can keep phenotype in the amplification in vitro process, keep multidirectional differentiation capability and obtain broad research, and also in having animal models such as the rat of immunizing power, dog, sheep, pig, obtained success, demonstrated mesenchymal stem cells MSCs and have the potential potential applicability in clinical practice.Yet, from marrow, obtain mescenchymal stem cell and still have tangible deficiency.1) extracts patient's marrow and need cause operation wound, be not easy to be accepted by the patient; 2) extract the marrow amount seldom at every turn, 5-10ml only, and the content of mescenchymal stem cell is extremely low in the marrow, only is 1/100000 of karyocyte, so the cell harvesting amount is minimum; 3) though mescenchymal stem cell can carry out external continuous passage amplification, and at present extensive amplification technique is still immature, and time and effort consuming, with high costs, use needs of patients from drawing materials to and wait for the long period; 4) because the mescenchymal stem cell of derived from bone marrow has immunogenicity, so resource can't be shared between the allosome.Above factor shows that mesenchymal stem cells MSCs is not the ideal source of cartilage tissue engineered seed cell.
Current research is found, is had in the placenta tissue and the similar attached cell of mesenchymal stem cells MSCs.Report such as external Jaroscak (2000) separation and Culture from frozen placenta tissue goes out heterogeneous attached cell.Report such as domestic Zhang Yi adopts enzyme digestion to obtain the sustenticular cell of attached cell as hemopoietic stem cell from placenta tissue.Therefore, placenta tissue is expected to become the abundant source of potential of mescenchymal stem cell.The a small amount of that only has at present both at home and abroad fragmentary document to relate to placenta source attached cell is separated, and is mainly used in hematopoiesis support research, the technical data that mass-producing separation, purifying and the amplification method of placenta derived mesenchymal stem cell then lacked.The contriver finds out the separation method of the mescenchymal stem cell in placenta source through research practice repeatedly, and confirm that such cell has and derived from bone marrow mescenchymal stem cell similar biological, can be divided into the various kinds of cell type that comprises the chondrocyte under suitable condition.Because placenta tissue is discarded tissue, the source is abundant, the cell harvesting amount is big, so the mescenchymal stem cell in placenta source is expected to become cartilage tissue engineered ideal seed cell.
Summary of the invention
The objective of the invention is for overcoming the deficiency in the above-mentioned cartilage tissue engineered research, it is the organization engineered cartilage and the manufacture method thereof of seed cell that a kind of mescenchymal stem cell with the placenta source is provided.Its method is as follows:
1) separation of placenta derived mesenchymal stem cell, cultivation and amplification
A. the fresh placenta of giving birth to is carried out bloodletting, perfusion immediately, to remove hemocyte and fragment of tissue;
B. continue perfusion, or placental lobules is shredded, use collagenase digesting, the collecting cell composition;
C. adopt immunomagnetic beads absorption method or Percoll density gradient centrifugation to collect mononuclearcell;
D. measure cell doubling time and growth curve;
E. the cell that obtains is carried out freezing preservation;
E. adopt the cell of bio-reactor, comprise that the cell of recovering behind the cryopreservation carries out the mass-producing amplification being obtained.
2) structure of organization engineered cartilage
A. timbering material pre-treatment timbering material can be degradable multiporous bioceramic material, as α-or bata-tricalcium phosphate; Also can be collagen, polymer class.Preferred bata-tricalcium phosphate porous bio-ceramic among the present invention.The disk shape of prefabricated one-tenth all size, or be prefabricated into specified shape, 60gray as required
60The Co irradiation also can be 75% sterilization in alcohol-pickled 10 minutes～1 hour.With serum free medium or PBS damping fluid alcohol is cemented out then to improve wetting ability with preceding usefulness 95% is alcohol-pickled, the sterilization dry gauze blots liquid.The collagenic material of having sterilized can directly be used.
B. the structure of organization engineered cartilage makes 1～2 * 10 with fresh separated or through amplification or through the placenta derived mesenchymal stem cell of freezing preservation
7The cell suspension of/ml drips on timbering material gently, and material overturning gently, makes cell distribution as far as possible evenly.Cell-porous support materials complex body is put into incubator, 37 ℃, 5%CO
2, left standstill under the saturated humidity 6～10 hours, cell is attached in the hole of material and surperficial.Be transferred to then and carry out the 3 D stereo cultivation in the rotary bio-reactor.Speed of rotation 15 ~ 50rpm.
C. the organization engineered cartilage that builds of the preservation of organization engineered cartilage can adopt cryogenic freezing preservation technology to store and transport.Freezing and storing method can be to be the routine preservation technology of cryoprotectant with dimethyl sulfoxide (DMSO) (DMSO), also can be to be that protectant vitrifying does not have ice crystal preservation technology with VS55.
Beneficial effect
Organization engineered cartilage of the present invention be mescenchymal stem cell with placenta source as seed cell, cell is taken from the depleted placenta tissue, the source is abundant, harvest yield is big, no immunological rejection.Because this cell can freezingly be preserved, and shares between the crowd, therefore can realize ready access upon use, need not to wait for, thereby make things convenient for clinical application.On the other hand, the degradable multiporous bioceramic scaffold material good biocompatibility that uses, the physical strength height, microstructure is reasonable, be suitable for growing into and extrtacellular matrix deposition of chondrocyte, its degradation rate also newborn speed of cartilaginous tissue is coordinated mutually, the final cartilaginous tissue that function is arranged normally that forms in vivo, realize the regeneration of functional cartilage, the regenerated cartilaginous tissue can be integrated naturally with surrounding tissue, and have bigger final strength and stability, damage or damaged realization physiological are repaired.Because degradable multiporous biological ceramics complete processing maturation can be produced in batches, the mescenchymal stem cell in placenta source is drawn materials easily, is not had immunological rejection, so the organization engineered cartilage of manufacturing of the present invention has broad clinical application prospect.
Embodiment
The acquisition of embodiment 1 people's placenta derived mesenchymal stem cell
Place aseptic pallet or container after placenta is given birth to immediately, inject through Umbilical artery and contain antithrombotics and antibiotic balanced salt solution, bloodletting, and begin perfusion.Antithrombotics can be the heparin of 1 ~ 100u/mL, and microbiotic can be a mycillin.Collect perfusion liquid, centrifugal acquisition cellular constituent; Or the aseptic clip placental lobules in preliminary perfusion back, with trypsinase or collagenase digesting placenta tissue, obtain cell suspension, again cell suspension is crossed the cell sieve, remove fragment of tissue and hemocyte, the cell that obtains suspends again, with density is the percoll of 1.073g/mL, with 500g centrifugal force centrifugal 30 minutes, and results interface cellular layer, PBS washing back inoculation culture bottle or culture plate, or be directly used in other purpose.Also can separate the mescenchymal stem cell that has certain special marking with the immunomagnetic beads of D7-FIB or NGFR mark.Adopt the phenotype of flow cytometer detection placenta derived mesenchymal stem cell, as CD29, CD34, CD44, CD45, CD105, CD166, HLA-DR etc.Detect the cell cycle simultaneously, determine to be in G
0-G
1The ratio of phase cell.
2 one kinds of manufacture method of embodiment based on the organization engineered cartilage of the mescenchymal stem cell in placenta source
People's placenta derived mesenchymal stem cell of fresh separated or cryopreservation resuscitation is prepared into 1.5～2 * 10
7The cell suspension of/ml is inoculated on cylindric β-TCP support, makes cell distribution even as far as possible.β-TCP porous bio-ceramic material can be by the method and the explained hereafter of last sea cowry Ao Lu house journal, and material is through 60gray
60Be used for engineered joint cartilage behind the Co illumination-based disinfection and make up, in a preferred scheme, its microstructure is: aperture-100～300 μ m, porosity-52% connects footpath-30～100 μ m between the hole.After leaving standstill 6 hours, mixture is moved into RCCS (rotary bio-reactor, Synthecom company), cultivated 48 hours in containing the DMEM of 10%FCS, rotating speed is 30rpm.Mixture after the cultivation can be directly used in clinical repair patient articular cartilage defect, but also cryopreservation, as organization engineered cartilage tissue bank.
Claims (10)
1. organization engineered cartilage and manufacture method thereof based on the mescenchymal stem cell in placenta source.The mescenchymal stem cell that obtains with separation and Extraction from placenta tissue is as seed cell, with the compound structure organization engineered cartilage of degradable multiporous biologic bracket material.
2. the placenta described in the claim 1 is the placenta of leaving over after healthy puerpera's normal labor or c-section fetus are given birth to.Placenta with give bloodletting immediately after parent and fetus separate, and pour into.Perfusion liquid is aseptic balanced salt solution, wherein contains antithrombotics and microbiotic.
3. can external preservation after the placenta bloodletting described in the claim 2 2-24 hour.Storage temperature can be a room temperature, also can be 4 ℃.
4. the separation of the mescenchymal stem cell in the placenta described in the claim 1 source can be adopted perfusion, promptly continues perfusion and centrifugal collecting cell composition, and it is resuspended to concentrate the back, carries out density gradient centrifugation collecting interface cell layer with the percoll of 1.073g/ml; Also can adopt digestion method, placenta tissue be shredded the back digest with collagenase or pancreatin, the cell suspension that obtains carries out density gradient centrifugation collecting interface cell layer with the percoll of 1.073g/ml.
5. the interface cellular layer described in the claim 3 can be seeded in culturing bottle or the culture dish and cultivate, or compound with microcarrier, places amplification in the bio-reactor; Also can use the immunomagnetic beads isolation technique, separate certain specific cell subsets,, separate cell mass with corresponding phenotype as using the magnetic bead that coupling has NGFR antibody or D7-FIB antibody or Stro-1 antibody or CD105 antibody.
6. the degradable multiporous biologic bracket material described in the claim 1 can be a polymer-based material, as natural collagen, also can be the polymkeric substance of chemosynthesis.Preferable material is the porous bioceramic scaffold material among the present invention.
7. the porous bioceramic scaffold material described in the claim 6 is characterized in that it can being that (α-TCP) also can be bata-tricalcium phosphate (β-TCP) to type alpha tricalcium phosphate.Its moiety tricalcium phosphate is identical with the inorganic components of body bone, thereby has excellent biological compatibility and mechanics of machinery characteristic, can provide adapt circumstance and strong support for organizing new life.It is characterized in that having controlled special microstructure, the pass rule, the porosity height has tubulose to connect between the Kong Yukong.This special features of microstructures is that the aperture can be between 100～300 μ m, and porosity can be between 45%～52%, and connecting the footpath between the hole can be between 30～100 μ m.This structure not only can improve the growing into property of tissue and cell, can also be adjusted in intravital degradation rate whereby, so that it is coordinated mutually with the newborn speed of cartilaginous tissue.
8. compound described in the claim 1 can be the cell suspension of the mescenchymal stem cell in placenta source being made high density, is inoculated on the timbering material, forms cell/composite body.Complex body extracorporeal culture certain hour closely attaches cell and material.
9. the extracorporeal culture described in the claim 8 can be that static cultivation was directly used in reparation in 16-48 hour in culturing bottle or culture dish, also can transfer to after static cultivation finishes and continue in the bio-reactor to cultivate 24-48 hour.
10. the described organization engineered cartilage of claim 1, it is characterized in that to be used for the physiological reparation of the various articular cartilage damages that reasons such as wound, inflammation, tumour cause, the treatment that also can be used to improve looks, plastic surgery etc. needs cartilaginous tissue tissue substitute to be provided or to make.
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| CNA2004100095328A CN1746295A (en) | 2004-09-09 | 2004-09-09 | A kind of tissue engineered cartilage based on placenta-derived mesenchymal stem cells and its manufacturing method |
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| CNA2004100095328A CN1746295A (en) | 2004-09-09 | 2004-09-09 | A kind of tissue engineered cartilage based on placenta-derived mesenchymal stem cells and its manufacturing method |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102477419A (en) * | 2010-11-26 | 2012-05-30 | 上海交通大学医学院附属第九人民医院 | Method for in-vitro fusion of stem cells and porous biological material |
| CN103263440A (en) * | 2013-02-08 | 2013-08-28 | 周胜利 | Method for extracting and preparing homology mesenchymal stem cell injection from placenta and umbilical cord |
| CN101574543B (en) * | 2009-06-09 | 2014-10-29 | 广州迈普再生医学科技有限公司 | Artificial articular cartilage based on autologous cells and preparation method thereof |
| CN107028981A (en) * | 2007-09-19 | 2017-08-11 | 普拉里斯坦有限公司 | Adherent cell from fat or placenta tissue and its purposes in the treatment |
| CN107206126A (en) * | 2015-01-23 | 2017-09-26 | 纽约市哥伦比亚大学理事会 | It is engineered mechanical functional human cartilage and its preparation method |
| CN111067676A (en) * | 2019-12-11 | 2020-04-28 | 中国人民解放军军事科学院军事医学研究院 | A prosthetic finger tactile sensor based on force-sensitive resistor and polyvinylidene fluoride |
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2004
- 2004-09-09 CN CNA2004100095328A patent/CN1746295A/en active Pending
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|---|---|---|---|---|
| CN107028981A (en) * | 2007-09-19 | 2017-08-11 | 普拉里斯坦有限公司 | Adherent cell from fat or placenta tissue and its purposes in the treatment |
| CN107028981B (en) * | 2007-09-19 | 2021-04-20 | 普拉里斯坦有限公司 | Adherent cells from adipose or placental tissue and their use in therapy |
| CN101574543B (en) * | 2009-06-09 | 2014-10-29 | 广州迈普再生医学科技有限公司 | Artificial articular cartilage based on autologous cells and preparation method thereof |
| CN102477419A (en) * | 2010-11-26 | 2012-05-30 | 上海交通大学医学院附属第九人民医院 | Method for in-vitro fusion of stem cells and porous biological material |
| CN102477419B (en) * | 2010-11-26 | 2014-04-23 | 上海交通大学医学院附属第九人民医院 | Method for in-vitro fusion of stem cells and porous biomaterial |
| CN103263440A (en) * | 2013-02-08 | 2013-08-28 | 周胜利 | Method for extracting and preparing homology mesenchymal stem cell injection from placenta and umbilical cord |
| CN107206126A (en) * | 2015-01-23 | 2017-09-26 | 纽约市哥伦比亚大学理事会 | It is engineered mechanical functional human cartilage and its preparation method |
| US11179498B2 (en) | 2015-01-23 | 2021-11-23 | The Trustees Of Columbia University In The City Of New York | Engineering mechanically functional human cartilage and method of making same |
| US12048779B2 (en) | 2015-01-23 | 2024-07-30 | The Trustees Of Columbia University In The City Of New York | Engineering mechanically functional human cartilage and method of making same |
| CN111067676A (en) * | 2019-12-11 | 2020-04-28 | 中国人民解放军军事科学院军事医学研究院 | A prosthetic finger tactile sensor based on force-sensitive resistor and polyvinylidene fluoride |
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