CN1928077A - Cell three-dimensional culture method for tissue engineering - Google Patents
Cell three-dimensional culture method for tissue engineering Download PDFInfo
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- CN1928077A CN1928077A CNA200610016042XA CN200610016042A CN1928077A CN 1928077 A CN1928077 A CN 1928077A CN A200610016042X A CNA200610016042X A CN A200610016042XA CN 200610016042 A CN200610016042 A CN 200610016042A CN 1928077 A CN1928077 A CN 1928077A
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Abstract
The present invention discloses one kind of 3D cell culture method for tissue engineering, and belongs to the field of tissue engineering technology. Into various kinds of biological rack material, thrombin in working concentration of 100-500 IU/ml and calcium chloride and other catalyst in working concentration of 20-60 mmol are added. Various kinds of cell are suspended in the culture medium with fibrinogen in working concentration of 10-50 mg/ml and XIII factor in working concentration of 10-50 U/ml, and inoculated in various kinds of rack material homogeneously based on hematopexis principle, so as to obtain 3D cell-rack material-fibrin gel culturing system. By means of the said method, excellent 3D cultured cell may be obtained for constituting various kinds of engineering tissue.
Description
Technical field
The present invention relates to field of tissue engineering technology, more particularly, relate to a kind of cell three-dimensional culture method that is applied to organizational project.
Background technology
Cell is distributed in three dimensions in tissue, makes up engineering tissue, and it is very important to set up a kind of cell three-dimensional culture method.The cell suspension direct inoculation is carried out cell three-dimensional cultivate on timbering material, because action of gravity, cell settlement is difficult to uniform distribution in frame bottom, has a large amount of cells to fail to be attached on the timbering material in operating process and has wasted a large amount of cells; Utilize liquid collagen, ECM glue etc. to carry out cell three-dimensional and cultivate, operate cumbersome price and also compare costliness, also be difficult to reach cell uniform distribution in support; Directly utilize zymoplasm-scleroproein, in operating process, also be difficult to make the cell uniform distribution.
Summary of the invention
Technical problem to be solved by this invention is, overcomes the deficiencies in the prior art, and a kind of cell three-dimensional culture method that is applied to organizational project is provided.
The present invention is applied to the cell three-dimensional culture method of organizational project, is achieved by following technical proposals:
(1) at first the zymoplasm of 100-500IU/mL working concentration and the calcium chloride of 20-60mmol working concentration are added in the timbering material,
(2) then the Parenogen of 10-50mg/mL working concentration and the XIII factor of 10-50U/mL working concentration are dissolved in the liquid nutrient medium of mammalian cell cultivation usefulness,
(3) then with 1 * 10
7-2 * 10
8The cell of/mL concentration is added in the aforesaid liquid substratum and abundant mixing cell,
(4) again with above-mentioned cell suspension inoculation in the timbering material of above-mentioned steps (1), cell suspension solidifies in second at 5-20, forms the three-dimensional complex body of cell-scaffold-fibrin gel,
After (5) 37 ℃ placement was solidified in 1 hour fully down, add the aforesaid liquid substratum and in the incubator of 5% carbonic acid gas, cultivate this complex body, change liquid nutrient medium one time, and cultivated, and in every 1-2 days? promptly get the dimensional culture complex body of cell behind (scope) sky.
Timbering material of the present invention is the various timbering materials that are applied to organizational project, for example use chitosan, collagen, gelatin, chitin natural polymer and derivative thereof and poly(lactic acid), polyglycolic acid synthetic high molecular polymer, for example take off the Deproteinated biologically-derived timbering material of cell, bioceramic scaffold material, porous hydroxyapatite again.
Zymoplasm of the present invention and Fibrinogen and the XIII factor are zymoplasm, Fibrinogen and the XIII factors with people and other Mammalss source.
Cell of the present invention is for deriving from people and other mammiferous cells, for example myocardial cell, scleroblast, chondrocyte, inoblast, stem cell.
Liquid nutrient medium is DMEM substratum or RPMI1640 substratum in the above-mentioned steps of the present invention (2).
The present invention compares with ECMge1 glue with collagen gel, and the method that this invention provides saves more money than the above two and makes things convenient for.
1. can be solidified as gel (5-10 second) very soon by the dosage that improves catalyzer, this like cell has little time also in suspension that avaling removes photoresist has just solidified, and cell is very even in support, and collagen solidifies slow (about 60 seconds), complicated operation will be with also more expensive with price in the alkali.
2. when making up the dimensional culture system, than on timbering material, saving cell to the cell direct inoculation, almost cell has very all directly suffered at three-dimensional rack, and the cell of direct inoculation on timbering material, a lot of cell detachments are arranged in operating process, and the final cell adherence rate is 50% to the maximum, and (cell inoculation density is 1 * 10
7/ ml)
3. price is than cheap with ECMge1 (Sigma), and the former 1ml is 1000 yuan, and about 500 yuan of the latter 1ml operate also simply, and normal temperature is operated down or must be kept low temperature.
The present invention utilizes the final stage reaction of blood coagulation process: i.e. the α β of zymoplasm broken fiber proteinogen and AB chain and discharge Acid polypeptide make Fibrinogen be converted into fibrin monomer, the spontaneous continuous polymer of no bridged bond that is gathered into of each monomer; The while thrombin activation XIII factor, the activated XIII factor is at Ca
2+Effect under between each fibrin monomer of catalysis the glutamine residue of two γ chains and the epsilon-amino acid of lysine residue form covalent linkage, clump and form stable netted fibrin polymer.Utilize this principle, catalyzer such as zymoplasm, calcium chloride are added on the timbering material, with the substratum that contains the Parenogen and the XIII factor various cells that suspend, then with this cell suspension inoculation on this timbering material, form the three-dimensional complex body of cell-scaffold-fibrin gel soon, because setting time is very short, but cell uniform distribution in three-dimensional rack.Utilize this method, can easily make up cell three-dimensional fast and cultivate complex body, timbering material is any shape, and it is exactly that the cell three-dimensional of formation is cultivated complex body for what shape.
Embodiment
Below in conjunction with embodiment the present invention is described further.
Embodiment 1: myocardial cell's dimensional culture
1. under aseptic condition, the collagen sponge scaffold material is immersed in the PBS solution that contains the 400IU/ml thrombin of beef, soak after 2 hours and blot standby (also can in preparation collagen sponge process, directly add zymoplasm, obtain containing the collagen sponge of zymoplasm) with filter paper;
2. under aseptic condition, get neonatal cardiac myocytes, the centrifugal collecting cell precipitation, with the resuspended myocardial cell of DMEM high glucose medium (containing 15% foetal calf serum) who contains 25mg/ml ox blood Fibrinogen, 20U/mlXIII, 40mmolCaCl2, cell concn is 1 * 10
8/ ml;
3. cell suspension is dripped in the collagen sponge scaffold material of preparation in 1, cell suspension has just solidified after several seconds, forms the three-dimensional complex body of myocardial cell-collagen-fibrin gel;
4.37 ℃ place down and to add DMEM high glucose medium substratum after 1 hour and in 37 ℃, the incubator of 5% carbonic acid gas, cultivate this complex body, change a subculture every day, used the substratum twice that contains 10mmolBrdU instead, and still used original substratum later on, around the cultivation in the 3rd day to the 5th day.
The result: the myocardial cell in the three-dimensional complex body of myocardial cell-collagen-fibrin gel began to beat after the 4th day, and this complex body begins consistent beating after two weeks.
Embodiment 2: osteoblastic dimensional culture
1. under aseptic condition, the collagen sponge scaffold material is immersed in the PBS solution of the zymoplasm that contains 100IU/mL and 20mmol calcium chloride, blots standby after 2 hours with filter paper.
2. under aseptic condition, get the neonate rat scleroblast, after the subculture in vitro separately amplification cultivation, the centrifugal collecting cell precipitation, with the resuspended scleroblast of DMEM/F12 substratum (containing 10% foetal calf serum) of the XIII factor of the Parenogen that contains the 50mg/mL working concentration and 10U/mL working concentration, making cell concn is 2 * 10
7/ mL.
3. cell suspension is dripped in the collagen sponge scaffold material of preparation in 1, cell suspension has just solidified after about 15 seconds, being formed into the three-dimensional complex body of osteocyte-collagen-fibrin gel, is control group with common collagen sponge scaffold material (not adding zymoplasm and calcium chloride) and the three-dimensional complex body of direct scleroblast with common DMEM/F12 substratum (not adding ox blood Fibrinogen and XIII) suspension cell-collagen.
4.37 place under ℃ in the incubator that adds 37 ℃ in DMEM/F12 culture medium culturing base, 5% carbonic acid gas after 20 minutes and cultivate this complex body, changed liquid nutrient medium one time in per 2 days, promptly get osteoblastic dimensional culture complex body.
The result: inverted microscope finds that down cell is evenly distributed in the timbering material, have in the control group a large amount of cells from support come off and cell distribution inhomogeneous; Vitro culture is carried out paraffin section and histochemical stain and is observed after January, find that this complex body alkaline phosphatase is a strong positive, has a large amount of calcification tubercles to form, control group neutral and alkali phosphatase activity a little less than, the calcification tubercle is also seldom.
Embodiment 3: chondrocyte's dimensional culture
1. under aseptic condition, the chitosan sponges bracket material is immersed in the PBS solution of calcium chloride of the zymoplasm that contains 200IU/mL and 60mmol, blots standby after 2 hours with filter paper.
2. under aseptic condition, separate tame rabbit cartilage cell, after the subculture in vitro separately amplification cultivation, the centrifugal collecting cell precipitation, with the Parenogen that contains the 10mg/mL working concentration and the resuspended chondrocyte of XIII factor D MEM/F12 substratum (containing 10% foetal calf serum) of 50U/mL working concentration, making cell concn is 1 * 10
7/ mL.
3. cell suspension is dripped in the chitosan sponges bracket material of preparation in 1, cell suspension has just solidified after about 10 seconds, form forming the three-dimensional complex body of chondrocyte-collagen-fibrin gel, is control group with common chitosan sponges bracket material (not adding zymoplasm and calcium chloride) and direct chondrocyte-chitosan three-dimensional complex body with common DMEM/F12 substratum (not adding ox blood Fibrinogen and XIII) suspension cell.
4.37 ℃ place down and to add the DMEM/F12 substratum after 20 minutes and in 37 ℃, the incubator of 5% carbonic acid gas, cultivate this complex body, changed liquid nutrient medium one time in per 2 days, promptly get chondrocyte's dimensional culture complex body.
The result: inverted microscope finds that down cell is evenly distributed in the timbering material, have in the control group a large amount of cells from support come off and cell distribution inhomogeneous; This complex body of vitro culture carries out paraffin section after January and histochemical stain is observed, this complex body II Collagen Type VI immunostaining is a strong positive, glycosaminoglycan content than the high 1-2 of control group doubly, Toluidine blue staining is also dark than control group, in the control group II Collagen Type VI immunostaining positive than experimental group a little less than.
Claims (5)
1. a cell three-dimensional culture method that is applied to organizational project is characterized in that, comprises the steps:
(1) at first the zymoplasm of 100-500IU/mL working concentration and the calcium chloride of 20-60mmol working concentration are added in the timbering material,
(2) then the Parenogen of 10-50mg/mL working concentration and the XIII factor of 10-50U/mL working concentration are dissolved in the liquid nutrient medium of mammalian cell cultivation usefulness,
(3) then with 1 * 10
7-2 * 10
8The cell of/mL concentration is added in the aforesaid liquid substratum and abundant mixing cell,
(4) again with above-mentioned cell suspension inoculation in the timbering material of above-mentioned steps (1), cell suspension solidifies in second at 5-20, forms the three-dimensional complex body of cell-scaffold-fibrin gel,
After (5) 37 ℃ placement was solidified in 1 hour fully down, add the aforesaid liquid substratum and in the incubator of 5% carbonic acid gas, cultivate this complex body, changed liquid nutrient medium one time in every 1-2 days, promptly get the dimensional culture complex body of cell.
2. the cell three-dimensional culture method that is applied to organizational project according to claim 1, it is characterized in that, described timbering material is the various timbering materials that are applied to organizational project, for example use chitosan, collagen, gelatin, chitin natural polymer and derivative thereof and poly(lactic acid), polyglycolic acid synthetic high molecular polymer, for example take off the Deproteinated biologically-derived timbering material of cell, bioceramic scaffold material, porous hydroxyapatite again.
3. the cell three-dimensional culture method that is applied to organizational project according to claim 1 is characterized in that, described zymoplasm and Fibrinogen and the XIII factor are zymoplasm, Fibrinogen and the XIII factors with people and other Mammalss source.
4. the cell three-dimensional culture method that is applied to organizational project according to claim 1 is characterized in that, described cell is for deriving from people and other mammiferous cells, for example myocardial cell, scleroblast, chondrocyte, inoblast, stem cell.
5. the cell three-dimensional culture method that is applied to organizational project according to claim 1 is characterized in that, liquid nutrient medium is DMEM substratum or RPMI1640 substratum in the described step (2).
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| CNA200610016042XA CN1928077A (en) | 2006-09-28 | 2006-09-28 | Cell three-dimensional culture method for tissue engineering |
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Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101053679B (en) * | 2007-04-17 | 2010-05-26 | 浙江大学 | Method for preparing polymer multiporous holder filled with fiber protein gel |
| CN102614547A (en) * | 2012-03-13 | 2012-08-01 | 中山大学中山眼科中心 | Method for rapidly constructing multilayer cells |
| CN103160468A (en) * | 2011-12-19 | 2013-06-19 | 中国科学院大连化学物理研究所 | Human tumor invasion and metastasis histioid in vitro three-dimensional model and construction and evaluation thereof |
| CN103372233A (en) * | 2012-04-13 | 2013-10-30 | 杭州龙禧生物医药科技有限公司 | Preparation method and product of tissue-engineered cartilage graft |
| CN104004709A (en) * | 2014-05-05 | 2014-08-27 | 曾奕明 | Fibrous-protein-gel release system of multiple-growth-factor compound and for promoting proliferation and differentiation of human embryonic lung fibroblast |
| CN105154397A (en) * | 2015-07-09 | 2015-12-16 | 深圳爱生再生医学科技有限公司 | Preparation method of CIK (cytokine-induced killer) cells |
| CN108130314A (en) * | 2018-01-24 | 2018-06-08 | 郑州大学第附属医院 | A kind of monoclonal cell cultural method |
| CN105911096B (en) * | 2016-03-29 | 2018-07-10 | 南京艾尔普再生医学科技有限公司 | A kind of artificial heart system that can carry out drug pharmacological toxicology screening in vitro |
| CN109091704A (en) * | 2018-08-08 | 2018-12-28 | 青岛大学 | A kind of composite support of tissue engineering and preparation method thereof for bone repair of cartilage |
| CN111690596A (en) * | 2020-06-17 | 2020-09-22 | 广东源心再生医学有限公司 | Novel 3D culture medium for human iPS differentiated cardiac muscle cells and preparation method and application thereof |
| CN112210526A (en) * | 2020-10-13 | 2021-01-12 | 北京健为医学检验实验室有限公司 | Three-dimensional cell culture scaffold and preparation method and application thereof |
| CN113677788A (en) * | 2019-04-01 | 2021-11-19 | 凸版印刷株式会社 | Three-dimensional tissue body, method for producing same, and method for producing cell-containing composition |
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2006
- 2006-09-28 CN CNA200610016042XA patent/CN1928077A/en active Pending
Cited By (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101053679B (en) * | 2007-04-17 | 2010-05-26 | 浙江大学 | Method for preparing polymer multiporous holder filled with fiber protein gel |
| CN103160468A (en) * | 2011-12-19 | 2013-06-19 | 中国科学院大连化学物理研究所 | Human tumor invasion and metastasis histioid in vitro three-dimensional model and construction and evaluation thereof |
| CN102614547A (en) * | 2012-03-13 | 2012-08-01 | 中山大学中山眼科中心 | Method for rapidly constructing multilayer cells |
| CN102614547B (en) * | 2012-03-13 | 2015-04-15 | 中山大学中山眼科中心 | Method for rapidly constructing multilayer cells |
| CN103372233A (en) * | 2012-04-13 | 2013-10-30 | 杭州龙禧生物医药科技有限公司 | Preparation method and product of tissue-engineered cartilage graft |
| CN104004709A (en) * | 2014-05-05 | 2014-08-27 | 曾奕明 | Fibrous-protein-gel release system of multiple-growth-factor compound and for promoting proliferation and differentiation of human embryonic lung fibroblast |
| CN105154397A (en) * | 2015-07-09 | 2015-12-16 | 深圳爱生再生医学科技有限公司 | Preparation method of CIK (cytokine-induced killer) cells |
| CN105911096B (en) * | 2016-03-29 | 2018-07-10 | 南京艾尔普再生医学科技有限公司 | A kind of artificial heart system that can carry out drug pharmacological toxicology screening in vitro |
| CN108130314A (en) * | 2018-01-24 | 2018-06-08 | 郑州大学第附属医院 | A kind of monoclonal cell cultural method |
| CN108130314B (en) * | 2018-01-24 | 2022-05-17 | 郑州大学第一附属医院 | Monoclonal cell culture method |
| CN109091704A (en) * | 2018-08-08 | 2018-12-28 | 青岛大学 | A kind of composite support of tissue engineering and preparation method thereof for bone repair of cartilage |
| CN113677788A (en) * | 2019-04-01 | 2021-11-19 | 凸版印刷株式会社 | Three-dimensional tissue body, method for producing same, and method for producing cell-containing composition |
| CN111690596A (en) * | 2020-06-17 | 2020-09-22 | 广东源心再生医学有限公司 | Novel 3D culture medium for human iPS differentiated cardiac muscle cells and preparation method and application thereof |
| CN112210526A (en) * | 2020-10-13 | 2021-01-12 | 北京健为医学检验实验室有限公司 | Three-dimensional cell culture scaffold and preparation method and application thereof |
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Open date: 20070314 |