CN102719391A - Diphasic porous three-dimensional cell culture scaffold - Google Patents
Diphasic porous three-dimensional cell culture scaffold Download PDFInfo
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- CN102719391A CN102719391A CN2012101860491A CN201210186049A CN102719391A CN 102719391 A CN102719391 A CN 102719391A CN 2012101860491 A CN2012101860491 A CN 2012101860491A CN 201210186049 A CN201210186049 A CN 201210186049A CN 102719391 A CN102719391 A CN 102719391A
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- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
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- C12M25/14—Scaffolds; Matrices
Abstract
The invention relates to a diphasic porous three-dimensional cell culture scaffold which is formed by a coarse fiber phase and a fine fiber phase which are entirely different in diameter. The coarse fiber phase is larger than cultured cells in size while the fine fiber phase is smaller than the cultured cells in size, the coarse fiber phase comprises multilayer coarse fiber structures, each two adjacent coarse fiber structures are arranged according to a certain angle, and the fine fiber phase is individually combined on one side or multiple sides of the coarse fiber phase and is averagely or intensively distributed in hole structures of the three-dimensional cell culture scaffold formed by the coarse fiber phase. In the diphasic porous three-dimensional cell culture scaffold, the fine fiber phase is much smaller than the cells in diameter, so that the cells can attach to nanofibers quite easily, and differentiation of stem cells on the nanofibers can be effectively promoted. Therefore, by means of adding the fine fibers on the three-dimensional cell culture scaffold, cell vaccination efficiency can be improved, and the cells grown on the fine fibers, particularly growth and differentiation of the stem cells, can be promoted and regulated.
Description
Technical field
The present invention relates to a kind of two-phase porous three-dimensional cell culturing bracket, belong to cell, field of tissue culture.
Background technology
In the prior art, the conventional main cell cultures mode of using comprises following two kinds:
1) with individual layer two-dimensional approach culturing cell
Cell cultures be a kind of in drug development, cytobiology, toxicology, biotechnology and field of tissue engineering technology the very useful and technology that is widely used.Conventional cell cultures is to carry out in like 2,4,6,24,96 porocyte culture plates at Tissue Culture Plate, and above-mentioned Tissue Culture Plate comprises that by the polymkeric substance of nondegradation PS processes.These Tissue Culture Plates often with its surface of plasma treatment improving its surperficial wetting ability, thereby make cultured cells can adhere to the two-dimensional surface of said culture plate better.In the cell culture experiments that typical employing PS cell cultures flat board carries out, institute's cultured cells is with a kind of two-dimensional approach monolayer growth in cell culture medium.
2) with the three dimensional constitution culturing cell
The two dimension cell cultures is that a kind of preparation, observation and research cell and they and medicine, biotic factor and biomaterial of being used for is in external interactional method easily.But this and said cell growth in vivo mode are far apart.Really in vivo, cell normally three dimensional growth and make up and form three-dimensional living tissue or organ.More and more evidences shows that external three-dimensional cell culture system can deepen the structure-emic understanding to the tissue of normal and pathology.In order to study this functional and morphologic mutual relationship; Some investigators have explored and have used three-dimensional gel matrix; Comprise collagen gel [Douglas WHJ; Moorman GW and Teel RW, The formation of histotypic structures from monodisperse fetal rat lung cells cultured on a threedimensional substrate. In Vitro 1976; 12:373-381], gelatin, fibrinogen, agarose and alginate [Gruber HE; Fisher EC Jr, Desai B, Stasky AA; Hoelscher G; Hanley EN, Human intervertebral disc cells from the annulus:Three dimensional culture in agarose or alginate and responsiveness to TGF-b1. Exp. Cell Res. 1997,235:13-21; Gruber HE, Stasky AA, Hanley EN Jr, Characterization and phenotypic stability of human disc cells in vitro. Matrix Biol. 1997; 16:285-288].In these gelling systems, cell cultures is grown with three-dimensional mode in gel matrix.Recent research shows; Compare with the cell of monolayer growth, the human disc cell of cultivating in three-dimensional alginate or sepharose system (human annulus disc cells) demonstrates different forms, has increased the synthetic of proteoglycan; And form have be deposited on cell peripheral and between multicellular colony [the Gruber HE of extracellular matrix; Fisher EC Jr, Desai B, Stasky AA; Hoelscher G; Hanley EN, Human intervertebral disc cells from the annulus:Three dimensional culture in agarose or alginate and responsiveness to TGF-b1. Exp. Cell Res. 1997,235:13-21; Gruber HE, Stasky AA, Hanley EN Jr, Characterization and phenotypic stability of human disc cells in vitro. Matrix Biol. 1997; 16:285-288].In addition; The human disc cell of in said three-dimensional alginate jelly system, cultivating confirms to have produced I type and II Collagen Type VI, and this I type and II Collagen Type VI do not have discovery [Gruber HE and Hanley EN, Jr when monolayer is cultivated; Human disc cells in monolayer vs 3D culture:cell shape; Division and matrix formation, BMC Musculoskeletal Disorders, 2000; 1:1].The three dimensional growth of external zooblast promotes the polarization and differentiation [the Roskelley CD of normal epithelium cell; Bissell MJ; Dynamic reciprocity revisited:a continuous; Bidirectional flow of information between cells and the extracellular matrix regulates mammary epithelial cell function, Biochem Cell Biol 1995; 73 (7-8): 391-7].Compare with living tissue cells; The cell of dimensional culture moves and divides sooner and have a kind of distinctive asymmetric profile [Cukierman E, Pankov R, Stevens DR; Yamada KM; Taking cell matrix adhesions to the third dimension, Science, 2001; 294 (5547): 1708-12].
Three-dimensional cell is cultivated and also is used to study the interaction between cell and growth factor and cell and the medicine.The three-dimensional cell of cancer cells is cultivated and can be used for studying many basic problems relevant with cancer biology; Because compare with the two-dimentional tissue culture plate of standard; The phraseology of the acceptor of the tumor development growth factor of cancer cells when three-dimensional cell is cultivated is different [Wang F; Weaver VM, Petersen OW, Larabell CA; Dedhar S; Briand P, Lupu R, Bissell MJ. Reciprocal interactions between beta1-integrin and epidermal growth factor receptor in three dimensional basement membrane breast cultures:a different perspective in epithelial biology. Proc Natl Acad Sci USA 1998; 95 (25): 14821-6; Jacks T, Weinberg RA. Taking the study of cancer cell survival to a new dimension. Cell, 2002; 111 (7): 923-5].For mammary cancer; The three-dimensional cell culture system provides a kind of model system [Bissell MJ that cancer cell multiplication is regulated and control and is used to estimate different cancer therapy drugs that is used to understand; Rizki A; Mian IS, Tissue architecture:the ultimate regulator of breast epithelial function. Curr Opin Cell Biolm, 2003; 15 (6): 753-62; Padron JM, van der Wilt CL, Smid K; Smitskamp-Wilms E; Backus HH, Pizao PE, Giaccone G; Peters GJ. The multilayered postconfluent cell culture as a model for drug screening. Crit Rev Oncol Hematol, 2000; 36 (2-3): 141-57].A large amount of evidences show, and at individual layer or disperse cells in culture to compare, the cell pair cell toxic agent of growing in the dimensional culture has higher tolerance.Many researchs disclose; Compare with monolayer, the spheroid cell cultures has higher resistance [Hoffman RM. Three-dimensional histoculture:origins and applications in cancer research. Cancer Cells 1991; 3 (3): 86-92].Originally; Investigators with cell spheroid to the tolerance of medicine owing to the bad diffusion of medicine to the inner cell of spheroid, but verified now, resistance is cultivated by three-dimensional cell and is caused; Rather than only can't touch nutritive substance [Lawler EM; Miller FR, Heppner GH, Significance of three dimensional growth patterns of mammary tissues in collagen gels. In Vitro 1983; 19 (8): 600-10; Miller BE, Miller FR, Heppner GH, Factors affecting growth and drug sensitivity of mouse mammary tumor lines in collagen gel cultures. Cancer Res, 1985; 45 (9): 4200-5].Further research confirms; It is a kind of better model [Harpreet K. Dhiman that is used to estimate the vitro cytotoxicity of cancer therapy drug that three-dimensional cell is cultivated; Alok R Ray, Amulya K Panda, Three-dimensional chitosan scaffoldbased MCF-7 cell culture for the determination of the cytotoxicity of tamoxifen; Biomaterials, 2005; 26 979-986].
Ever-increasing evidence shows; Three-dimensional environment also can disclose the fundamental mechanism of cell function; And external dimensional culture system can promote structure-emic understanding [Abbott A. Cell culture:biology's new dimension. Nature, 2003 in normal and pathological conditions; 424 (6951): 870-2; Hutmacher DW. Scaffold design and fabrication technologies for engineering tissues-state of the art and future perspectives. J Biomater Sci Polym Ed, 2001; 12:107 – 24; Schmeichel KL, Bissell MJ. Modeling tissue-specific signaling and organ function in three dimensions. J Cell Sci, 2003; 116 (Pt12): 2377-88; Zahir N, Weaver VM, Death in the third dimension:apoptosis regulation and tissue architecture. Curr Opin Genet Dev 2004; 14:71 – 80; Martin I, Wendt D, Heberer M, The role of bioreactors in tissue engineering, Trends Biotechnol, 2004; 22:80 – 6].Receivedly at present be, the behavior of cell in three-dimensional with two-dimentional environment in bone and cartilage source is different, and; The outer culture system of above-mentioned said three-dimensional body can closer be simulated intravital situation [Kale S than two-dimentional culture system; Biermann S, Edwards C, Tarnowski C; Morris M; Long MW, Three-dimensional cellular development is essential for ex vivo formation of human bone. Nat Biotechnol, 2000; 18:954 – 8; Ferrera D, Poggi S, Biassoni C, Dickson GR; Astigiano S, Barbieri O, Favre A; Franzi AT, Strangio A, Federici A; Manduca P, Three-dimensional cultures of normal human osteoblasts:proliferation and differentiation potential in vitro and upon ectopic implantation in nude mice, Bone 2002; 30:718 – 25; Tallheden T; Karlsson C, Brunner A, Van Der Lee J; Hagg R; Tommasini R, Lindahl A. Gene expression during redifferentiation of human articular chondrocytes. Osteoarthritis Cartilage, 2004; 12:525 – 35; ].In recent research, in a kind of inside of Vltra tears hydrogel matrix, dimensional culture three-type-person's scleroblast system and normal human osteoblast cell.Verified, osteosarcoma cell is bred with the mode of group's spheroid, and described human osteoblast cell's colony at least 3 weeks of survival.The mineralising test and the gene expression analysis of scleroblast mark and cytokine show that the cell that in above-mentioned hydrogel matrix, carries out dimensional culture shows a kind of more sophisticated differentiation situation [Trojani C, Weiss P than the cell of monolayer culture in plastics cell cultures flat board; Michiels JF, Vinatier C, Guicheux J; Daculsi G, Gaudray P, Carle GF; Rochet N.; Three-dimensional culture and differentiation of human osteogenic cells in an injectable hydroxypropylmethylcellulose hydrogel, Biomaterials, 2005; 26 (27): 5509-17].Evidence up to now clearly illustrates that culturing cell has the incomparable great advantages of two-dimentional cell cultures under three-dimensional environment.Present three-dimensional cell cultured product mainly contains gelling system and three-dimensional cell is cultivated support.
When adopting gelling system, said cultured cells is embedded in gel matrix inside, because the diffusion of material in said gel receives certain restriction, so the exchange of the nutrition and metabolism product of this cell cultures is a problem.And,, reclaim after the cultivation or separate very difficulty of said cell because said cultured cells is embedded in gel inside.This with use two-dimentional cell cultures flat board different, two-dimentional cultured cells can use trypsinase simply with cell wash-out and separate through centrifugal from the culture plate.In addition; Culturing cell requires the required gelling system of preparation before each culturing cell in gel matrix; This not only can cause inconvenience for investigators when a large amount of the cultivation; And owing on the preparation method of gel between Different researchers and the laboratory, exist slight difference, thereby can between the preparing gel of different batches, cause the inconsistent of quality.
Three-dimensional porous rack is that another kind can be used for carrying out the system that three-dimensional cell is cultivated.Three-dimensional porous rack in use with cell inoculation in the vesicular structure of support, under suitable cell culture condition, cell can be cultivated in this vesicular structure and form engineering three-dimensional tissue structures.Common have porous calcium phosphate support, polylactic acid bracket, POLYACTIC ACID and glycolide copolymer support, collagen scaffold, a sodium-alginate support.Its common characteristic is that they have random void shape and size.Connective bad between the hole; Therefore cell is not easy to get into carriage center, even enter into the center of porous support, because the exchange of nutrition and metabolism product is not easy; Therefore cell is restricted in the center growth of these three-dimensional cells cultivation supports, even dead.Therefore big limitations adopt three-dimensional porous rack to advance the progress that three-dimensional cell is cultivated.
In sum, although three-dimensional cell is cultivated more advantage is arranged, because the existing variety of problems of three dimensional gel culture system of present use above-mentioned, two-dimentional cell cultures remains main cell culture processes.Therefore, a kind of dimensional culture system that has whole convenient parts of two-dimentional cell culture system concurrently, valuable for medicine, life science and bio-engineering research field.This ideal three-dimensional cell culture system need at first have can let the user as using the Tissue Culture Plate of plane easily observation of cell to cultivate the growing state in the support at three-dimensional cell.
For a long time, PS has become a kind of culture base material that is successfully used to two-dimentional cell cultures.Be widely used and have a multiple dimensions that can be purchased acquisition by the cell cultures flat board of PS manufacturing from many suppliers.Because the PS culture plate is quite familiar concerning the investigators that do cell or tissue culture; Therefore can imagine; A kind of advantage that the dimensional culture environment can not only be provided by the three-dimensional cell culture system of PS manufacturing; And many other advantages of PS two dimension cell culture system can be provided, have clear and definite surface properties and be easy to and use.Yet PS was but almost never explored as being applied in of three-dimensional cell culture system in the past.Recently, people such as Baker report its use electrostatic spinning technique and process a kind of three-dimensional porous styroflex matrix [Baker SC, Atkin N; Gunning PA, Granville N, Wilson K; Wilson D and Southgate J; Characterisation of electrospun polystyrene scaffolds for three-dimensional in vitro biological studies, Biomaterials, 2006; 27,3136-46].The three-dimensional styroflex matrix that they obtain is similar to a kind of non-woven pad, and it is exactly the porous space that the space between internal fiber is wherein arranged.They should cut into the size in the suitable hole that can be placed on 6 hole polystyrene culture plates by three-dimensional styroflex matrix.After handling, carry out cell cultures with these three-dimensional styroflex matrix inoculating cells and in 6 hole polystyrene cell cultures flat boards commonly used at argon gas ionic medium body.The result shows that these three-dimensional styroflex matrixes have the surface of good character that is suitable for the cell attaching.These data disclose, and these PS three-dimensional fiber property supports can be that a kind of of the dull and stereotyped system of two-dimentional PS cell cultures replenishes.Yet; The shortcoming of these fibering PS matrixes is: the aperture size of said matrix and the shape in hole are difficult for confirming and this fibrous matrix is soft under state of nature, produces difficulty thereby make said matrix under situation about not deforming, further operate.Patent of invention " three-dimensional cell is cultivated insert, its producing apparatus and kit utility " (200720187562.7) provides a kind of three-dimensional cell with regular pore space structure to cultivate support.This support is used for three-dimensional cell cultivate to be used, and comprises that with present two-dimentional tissue culturing system tissue culture plate uses.This support has 100% hole connectivity, and cell can be easy to be seeded in equably on the support and well-grown.Yet, when carrying out cell inoculation because the hole is 100% UNICOM, therefore have a spot of cell from then on three-dimensional cell cultivate on the support and drain on the Tissue Culture Plate, thereby have influence on the inoculation efficient of cell on three-dimensional rack.
Summary of the invention
The objective of the invention is to overcome above-mentioned deficiency, a kind of two-phase porous three-dimensional cell culturing bracket is provided, can improve cell inoculation efficient, and can play promotion and regulating and controlling effect the growth and the differentiation of the cell, particularly stem cell of growth above that.
The objective of the invention is to realize like this:
A kind of two-phase porous three-dimensional cell culturing bracket; Said two-phase porous three-dimensional cell culturing bracket by robust fibre mutually with fine-fibered mutually two kinds of distinct materials of diameter constitute; Wherein to compare the size of institute's cultured cells big for robust fibre, and the size that fine-fibered is compared institute's culturing cell is little, and robust fibre is divided into the multilayer coarse fiber structure mutually; Said adjacent two layers coarse fiber structure is pressed certain angle and is arranged, and fine-fibered is combined in one or more surfaces of robust fibre phase mutually separately; But fine-fibered mutually uniform distribution or concentrate is distributed in the three-dimensional cell that robust fibre constitutes mutually and cultivates in the pore space structure of support.
Two-phase three-dimensional cell of the present invention is cultivated support, and the three-dimensional cell that said robust fibre constitutes is cultivated support by the pillar of constant or different diameter and/or fibrous.
Two-phase three-dimensional cell of the present invention is cultivated support, and the porosity of said cell culturing bracket changes through pillar and/or the quantity and the size of fiber that changes in the said support; And/or said vesicular structure can change through the three-dimensional localization pattern that changes pillar and/or fiber.
Two-phase three-dimensional cell of the present invention is cultivated support, and robust fibre has circle, trilateral, square and/or orthogonal xsect.
Two-phase three-dimensional cell of the present invention is cultivated support and is made the size that is fit to cell cultures plate well, culturing room, culturing bottle and/or bio-reactor.
Two-phase three-dimensional cell of the present invention is cultivated support, and the material of robust fibre phase is a commaterial with fine-fibered material mutually.
Two-phase three-dimensional cell of the present invention is cultivated support, and said robust fibre and fine-fibered are processed by the material of no cytotoxicity.
Two-phase three-dimensional cell of the present invention is cultivated support, and robust fibre is the nondegradation material mutually with fine-fibered.
Two-phase three-dimensional cell of the present invention is cultivated support, and robust fibre is degradable materials mutually with fine-fibered.
Two-phase three-dimensional cell of the present invention is cultivated support, and wherein a phase material is non-degradable material, and another is degradation material mutually.
Two-phase three-dimensional cell of the present invention is cultivated support, and said cell culturing bracket adopts process for modifying surface to handle the said cytoskeletal cell attaching effect of improvement.
Two-phase three-dimensional cell of the present invention is cultivated support, and said process for modifying surface is that the physical chemistry mode comprises plasma treatment, photoglow processing, and/or chemical mode, uses H
2SO
4, HNO
3Strong acid treatment.
Two-phase three-dimensional cell of the present invention is cultivated support; Said process for modifying surface is a kind of surface coating technique, and through applying a kind of coated substance that is different from the polymkeric substance that is used to prepare the said construct of claim 1, said coated substance is naturally occurring polymkeric substance; And/or synthetic polymer; And/or inorganic substance, and/or the compound coating formed of two or more organic materialss, and/or a kind of inorganic/organic mixture.
Two-phase three-dimensional cell of the present invention is cultivated support, and said coated substance comprises albumen, peptide, TGSS C3, collagen, Fibronectin, Z 150PH, polyoxyethylene glycol, Vinylpyrrolidone polymer, polylysine, calcium phosphate, TiO
2, SiO
2, Al
2O
3, in gel and WOT Recovery Floc T, ROHM and polyoxyethylene glycol, Z 150PH and Vinylpyrrolidone polymer, calcium phosphate/collagen mixture, calcium phosphoric acid/polyoxyethylene glycol mixture, the calcium phosphate/extracellular matrix one or more.
Two-phase three-dimensional cell of the present invention is cultivated support, and described top coat material is to comprise that with chemical mode covalent linkage, hydrogen bond, ionic linkage or Van der Waals force are attached on the pillar and/or fiber of said cell culturing bracket.
Two-phase three-dimensional cell of the present invention is cultivated the making method of support, and said method comprises:
(A) adopt quick molding method to prepare the work in-process porous three-dimensional support that robust fibre constitutes mutually;
(B) adopt electrostatic spinning technique spraying fine-fibered at work in-process porous three-dimensional rack outer surface;
(C) continue on the face that has the fine-fibered phase, to adopt quick molding method to prepare the porous three-dimensional support, thereby make fine-fibered be fixed on the three-dimensional rack inside that robust fibre constitutes mutually mutually;
(D) utilize the cut mechanically method, two-phase porous three-dimensional cell culturing bracket is cut into desired size;
(E) use a kind of plasma surface treatment device, in argon gas atmosphere, said cell culturing bracket is carried out plasma treatment;
(F) pack this plasma treated cell culturing bracket respectively, and finally sterilize with the gamma-rays radiation of 20KGy dosage.
Two-phase three-dimensional cell of the present invention is cultivated the method for support culturing cell, and said method comprises:
(A) use dynamically inoculation or static inoculation method, with cell inoculation in said cell culturing bracket;
(B) after the inoculating cell, said cell culturing bracket is remained on the cell cultures upholder that is immersed among the growth medium, comprise in tissue culture plate, culturing room, culturing bottle and/or the bio-reactor and cultivating.
Compared with prior art, the invention has the beneficial effects as follows:
Two-phase porous three-dimensional cell culturing bracket of the present invention has further increased the fine-fibered that many littler than cell dia, comprises the fiber of nanometer diameter.Fine-fibered, particularly nanofiber can promote cell absorption.Nanofiber has the diameter that many littler than cell, and is similar with spandex fiber at the intravital collagen of structure aspects and Mammals, so cell can be easy to attach on the nanofiber.Nanofiber can also effectively promote stem cell differentiation above that.Therefore, add fine-fibered and to three-dimensional cell cultivation support, can improve cell inoculation efficient, and can play promotion and regulating and controlling effect the growth and the differentiation of the cell, particularly stem cell of growth above that.
Description of drawings
Fig. 1 is the structural representation of two-phase porous three-dimensional cell culturing bracket of the present invention.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention., these embodiment are not used in restriction scope of the present invention but only being used to the present invention is described.One of ordinary skill in the art should be appreciated that the experimental technique for unreceipted concrete experiment condition in the following example, usually according to normal condition, or the condition of advising according to manufacturer.
Referring to Fig. 1; Two-phase porous three-dimensional cell culturing bracket of the present invention is made up of two kinds of distinct materials of diameter, and wherein one is to be made up of mutually the robust fibre bigger than institute's cultured cells size mutually, and robust fibre is divided into four layers of coarse fiber structure mutually; Said adjacent two layers coarse fiber structure is pressed certain angle and is arranged; Be 90 degree between the adjacent two layers robust fibre and arrange, perhaps become 60 degree to arrange, perhaps become 45 degree to arrange; Robust fibre provides the vesicular structure of rule to supply cell to carry out three dimensional growth therein mutually; Another then is to be made up of mutually the fine-fibered littler than institute's culturing cell size mutually; Fine-fibered is combined in one or more surfaces of robust fibre phase mutually separately; Impel cell to be adsorbed on the fine-fibered, thereby stop cell from the porous three-dimensional cell culturing bracket that robust fibre constitutes mutually, to spill.Fine-fibered is joining in the porous three-dimensional cell culturing bracket that robust fibre constitutes mutually of being selected property also mutually.
Configuration
Two-phase porous three-dimensional cell culturing bracket of the present invention can be formed for realizing any size and the shape of concrete application purpose, and said application purpose is fit to the size and the shape of cell/tissue culture plate, flask and bio-reactor.
In one embodiment, the present invention provides a kind of three-dimensional cell that is made up of two kinds of different fibers of thickness to cultivate support.Wherein, robust fibre or pillar constitute the main skeleton construction that three-dimensional cell is cultivated support.Said pillar is in the same place according to a kind of mode or the combination of patterns of design in advance with fiber.Fine-fibered relies on robust fibre, the structure and growth that influences cell and the differentiation situation that provide cell to adsorb more easily.
Confirm the porosity and the aperture size of cell culturing bracket through the global design of said support; The design of said support comprises pillar or coarse-fibred size and geometry; Pillar or coarse-fibred number in the per unit volume, and the structure plan of robust fibre or pillar in this three-dimensional rack.Fine-fibered then joins in the vesicular structure of support with a kind of mode unordered or ordered arrangement.
In one embodiment, support Design of the present invention be a kind of by pillar and/or robust fibre in the junction vertical combination the and the three-dimensional cell cultivation support formed each other.Fine-fibered then joins in the vesicular structure of support with a kind of mode unordered or ordered arrangement.
In one embodiment, the present invention provides a kind of three-dimensional cell of being made up of pillar and/or robust fibre to cultivate support, and in this support, said pillar and/or fiber also not all are perpendicular to one another in the junction, but combine with different angles.Fine-fibered then joins in the vesicular structure of support with a kind of mode unordered or ordered arrangement.
In a kind of concrete embodiment, said two-phase porous three-dimensional cell culturing bracket is a kind of three-dimensional disc vesicular structure.In the concrete embodiment of another kind, said two-phase porous three-dimensional cell culturing bracket is a kind of three-dimensional cube shaped vesicular structure.
Size
Two-phase porous three-dimensional cell culturing bracket preprocess of the present invention becomes standard size, perhaps is customized to the size that is fit to concrete cell cultures plate well, case, flask, bio-reactor.In one embodiment, the present invention provides a kind of two-phase porous three-dimensional cell culturing bracket with a kind of size (diameter and height), and it is fit to a kind of circular port of commercially available tissue culture plate.In another embodiment, the present invention provides a kind of two-phase porous three-dimensional cell culturing bracket with a kind of cube size (length * wide * height), and it is fit to a kind of rectangular opening of tissue culture plate.In another embodiment, this two-phase porous three-dimensional cell culturing bracket has the size and dimension of the chamber that is fit to a kind of bio-reactor.In a kind of embodiment, the size of this two-phase porous three-dimensional cell culturing bracket is fit to a kind of cubic space of tissue culture flasks.
It is 50 μ m-1mm that this two-phase three-dimensional cell is cultivated stent strut/coarse-fibred diameter.
The mean pore size of cell culturing bracket is 50 μ m-2mm.
Three-dimensional cell that said robust fibre constitutes is mutually cultivated support by the pillar of constant or different diameter and/or fibrous; Preferably, the porosity of said cell culturing bracket changes through pillar and/or the quantity and the size of fiber that changes in the said support; And/or preferably, vesicular structure can change through the three-dimensional localization pattern that changes pillar and/or fiber.But fine-fibered mutually uniform distribution or concentrate is distributed in the three-dimensional cell that robust fibre constitutes mutually and cultivates in the pore space structure of support.
Preferably, said robust fibre adopts material of the same race or not of the same race mutually with fine-fibered.
Preferably, fine-fibered is made up of differing materials.
It is made that said material is preferably the material of no cytotoxicity.Material is a polymkeric substance, ceramic, the mixture of metallic substance and above-mentioned materials.The preferred self-polystyrene of polymer materials, gather the multipolymer of racemic lactic acid (PDLLA), POLYACTIC ACID and NSC 403079, the polymer materials of polycarbonate, polymeric amide and SE is processed.Ceramic is preferably from tricalcium phosphate, Win 40350, silicate, aluminium sesquioxide.Metallic substance is preferably from titanium, titanium alloy, stainless steel, tantalum, magnesiumalloy.
Said two-phase porous three-dimensional cell culturing bracket is handled through process for modifying surface, thereby improves cell attaching effect.Preferably, said process for modifying surface is the physical chemistry mode, comprises plasma treatment, photoglow processing, and/or chemical mode, uses H
2SO
4, HNO
3Strong acid treatment.
Preferably, said process for modifying surface is an a kind of top coat technology, through applying the coated substance that one kind of multiple are different from the polymkeric substance that is used to prepare construct according to the invention.Preferably, said coated substance is a natural polymer, comprises albumen, peptide, TGSS C3, collagen, Fibronectin and/or synthetic polymer, comprises Z 150PH, polyoxyethylene glycol, Vinylpyrrolidone polymer, polylysine; And/or inorganic substance, comprise calcium phosphate, TiO
2, SiO
2, Al
2O
3And/or the compound coating of two or more organic materialss compositions; Comprise gel and WOT Recovery Floc T; ROHM and polyoxyethylene glycol; Z 150PH and Vinylpyrrolidone polymer and/or a kind of inorganic/organic mixture, comprise calcium phosphate/collagen mixture, calcium phosphoric acid/polyoxyethylene glycol mixture, calcium phosphate/extracellular matrix.
Preferably, the fine-fibered of said two-phase porous three-dimensional cell culturing bracket applies coating of the same race or not of the same race with support.
Preferably, said top coat material is to comprise that with chemical mode covalent linkage, hydrogen bond, ionic linkage or Van der Waals force are attached on the pillar and/or fiber of said two-phase porous three-dimensional cell culturing bracket.
Preferably, said two-phase porous three-dimensional cell culturing bracket is made the size that is fit to cell cultures plate well, case, flask and/or bio-reactor.
Preferably, said robust fibre is by the polymkeric substance pillar of constant or different diameter and/or fibrous mutually.
Preferably, the porosity of said robust fibre phase changes through changing the quantity and the size that constitute said stent strut and/or fiber.The vesicular structure of said robust fibre phase also can change through the three-dimensional localization pattern that changes pillar and/or fiber.
Preferably, the pillar of said robust fibre phase and/or fiber have circle, trilateral, square and/or orthogonal xsect.
Preferably, the fine-fibered that has of said two-phase porous three-dimensional cell culturing bracket covers the outside surface that three-dimensional cell that robust fibre constitutes is mutually cultivated support mutually.
Preferably, fine-fibered is positioned in the middle of the timbering material that robust fibre constitutes mutually mutually.
Preferably, fine-fibered is distributed in the porous support that robust fibre constitutes mutually.
Embodiment 1: the method for manufacture of two-phase porous three-dimensional cell culturing bracket
Method one
(A) use polystyrene material to make two-phase porous three-dimensional cell culturing bracket, adopt quick molding method to prepare the work in-process porous three-dimensional support that robust fibre constitutes mutually.
(B) adopt electrostatic spinning technique spraying fine-fibered at work in-process porous three-dimensional rack outer surface.
(C) continue to adopt quick molding method to prepare the porous three-dimensional support, thereby make fine-fibered be fixed on the three-dimensional rack inside that robust fibre constitutes mutually mutually on the surface that has the fine-fibered phase.
(D) utilize the cut mechanically method.Two-phase porous three-dimensional cell culturing bracket is cut into desired size.
(E) use a kind of plasma surface treatment device.In argon gas atmosphere, two-phase porous three-dimensional cell culturing bracket is carried out plasma treatment.
(F) pack this plasma treated two-phase porous three-dimensional cell culturing bracket respectively, and finally sterilize with the gamma-rays radiation of 20KGy dosage.
Method two
(A) use conventional polymer processing method, comprise that injection moulding, fiber weaving and adhering technique make the single lamella of said support.
(B),, be assembled into described cell culturing bracket with the described a plurality of porous lamella structure cuts of step (A) or without after the cutting according to structure design.The polymer fiber or the clip web member of non-cell toxicity preferably used in said assembling.
(C), can fine-fibered be sprayed on the three-dimensional rack outside surface that assembles through electrostatic spinning technique according to structure design.Also can with fiber coating on the single lamella of unassembled support, carry out the support assembling again.
Said surface-coated handle preferably with said coated substance chemically crosslinked, heat cross-linking, heating under vacuum is crosslinked and/or radiation crosslinking to said cytoskeleton.Preferably, said crosslinked be that chemistry carries out.Preferably, saidly crosslinkedly be to use heating to carry out.Preferably, the said crosslinked heating under vacuum that is to use is carried out.Preferably, saidly crosslinkedly be to use radiation to carry out.Further preferably, said radiation is electron beam (e-beam) radiation, gamma-radiation and/or ultraviolet radiation.
Said coating is preferably crosslinked.Further preferably, saidly crosslinkedly be to use radiation to carry out, said radiation is electron beam irradiation, gamma-radiation and/or ultraviolet radiation preferably.Further preferably, said crosslinked be that chemistry carries out.Further preferably, said crosslinked heating and the vacuum of being to use carried out.
Embodiment 2: use two-phase porous three-dimensional cell culturing bracket to come culturing cell
The present invention also is provided at the method for using this two-phase porous three-dimensional cell culturing bracket cultivating living cells in tissue culturing polystyrene's flat board.That the two-phase porous three-dimensional cell culturing bracket that is used for this research has 10mm is wide * size that 10mm length * 0.3mm is thick, have the square hole of 200 μ m and the Fibre diameter of 400 μ m.The fine-fibered diameter is 1 μ m.
Use static inoculation method inoculation smooth muscle cell: with transfer pipet the upper surface of 500 μ l smooth muscle cell suspension-s (1 * 105 cells/ml) from said support added, make cell after attaching 2 hours under 37 ℃, pour into more many cells substratum again.After having inoculated cell, said cell culturing bracket is put into the porous flat plate that contains cell culture medium, and under 37 ℃, in a kind of air atmosphere that contains 5-10% CO2 of 90% humidity, in incubator, cultivate.Said growth medium by the improved Eagle's substratum of DulbeccoShi that comprises 5% (v/v) foetal calf serum (Dulbecco ' s Modified Eagle ' s Medium, DMEM) form.Under the situation of using dynamic inoculation method, inoculation is to carry out through said cell culturing bracket being immersed in the cell suspending liquid that is contained in the rotary flask, under 60rpm, stirring, and this rotary flask is placed down in moistening 5%CO at 37 ℃
2In the incubator.After the inoculation, said cell culturing bracket is placed in the hole of the tissue culture plate that has cell culture medium moistening 5%CO under 37 ℃
2Further cultivate in the incubator, and the periodic replacement substratum.
After accomplishing cell cultures, said cell culturing bracket is taken out from the cell cultures flat board, and carry out conventional analysis in a certain definite time point.And microscopically is observed at the different layers of said cell culturing bracket or locational cell and is attached and cytoactive.
Under the situation that said cell need reclaim, use trypsinase-EDTA solution (Sigma T4049) that said cell is carried out trysinization.After cell breaks away from from said cell culturing bracket, cell is suspended in once more in the fresh substratum that comprises serum of small volume, thereby makes the trypsinase inactivation.Then these cells are used for other purpose.
Embodiment 3: use with the polystyrene tissue culture flat board
The present invention also provides the method for using said cell culturing bracket cultivating living cells in a kind of polystyrene tissue culture flat board.This cell culturing bracket is a kind of disk or cubical shape, to be fit to the hole of tissue culture plate.Use dynamically inoculation or static inoculation method, with cell inoculation in said cell culturing bracket.
In one embodiment, use static inoculation method, the cell suspending liquid of certain volume is added with the upper surface of valinche from said cell culturing bracket, and let cell have the regular hour to attach on it, and then use the nutrient solution lavation.After the inoculating cell, said cell culturing bracket is put into the orifice plate that contains cell culture fluid, under 37 ℃, in incubator, cultivate in the atmosphere of relative humidity 90%, carbon dioxide content 5-10%.
In another embodiment, use dynamic inoculation method, said inoculation is through in a kind of rotary flask, with what carry out in the said cell culturing bracket immersion cell suspending liquid.After inoculation, cell culturing bracket is placed in the hole of the tissue culture plate that has substratum, under 37 ℃, in the incubator of 5% carbonic acid gas, further cultivate.Regularly replace cell culture medium.
After accomplishing cell cultures, said cell culturing bracket is taken out from this cell cultures flat board, and carry out conventional test at a certain definite time point.Cell culturing bracket is taken apart, so that different layers or the cell on the different positions examined under a microscope at said cell culturing bracket attach and cytoactive.
Under the situation that said cell need reclaim, use trypsinase-EDTA solution that said cell is carried out trysinization.After cell separates from said cell culturing bracket, cell is suspended in once more in the fresh substratum that comprises serum of small volume, thereby makes the trypsinase inactivation.Be used for other purpose after can these cells being cleaned recovery then.
Embodiment 4: use with bio-reactor
The present invention also provides the method for using said cell culturing bracket cultivating living cells in bio-reactor.This cell culturing bracket is a kind of disc or cube, and its size and dimension is fit to be positioned over said bio-reactor.
In using an embodiment of static inoculation method, the cell suspending liquid of certain volume is added with the upper surface of valinche from said cell culturing bracket, and before with the substratum lavation, allow the cell attaching regular hour.After using static inoculation method inoculation, these cell culture supports of having inoculated cell are put into the bio-reactor that is full of cell culture medium, under 37 ℃, in the atmosphere of 90% humidity, 5-10% carbonic acid gas, cultivate.In whole cell cultivation process, cell culture fluid flows the hole through cell culturing bracket constantly, and is regularly replaced.
After accomplishing cell cultures, said cell culturing bracket is taken out from this bio-reactor, and carry out conventional test at a certain definite time point.Cell culturing bracket is taken apart, so that the cell of examining under a microscope on said cytoskeletal different layers or different positions attaches and cytoactive.
Under the situation that said cell need reclaim, use trypsinase-EDTA solution that said cell is carried out trysinization.After cell separates from said cell culturing bracket, cell is suspended in once more in the fresh substratum that comprises serum of small volume, thereby makes the trypsinase inactivation.Be used for other purpose after can these cells being cleaned recovery then.
Claims (16)
1. two-phase porous three-dimensional cell culturing bracket; It is characterized in that, said two-phase porous three-dimensional cell culturing bracket by robust fibre mutually with fine-fibered mutually two kinds of distinct materials of diameter constitute, wherein to compare the size of institute's cultured cells big for robust fibre; The size that fine-fibered is compared institute's culturing cell is little; Robust fibre is divided into the multilayer coarse fiber structure mutually, and said adjacent two layers coarse fiber structure is pressed certain angle and arranged, and fine-fibered is combined in one or more surfaces of robust fibre phase mutually separately; Fine-fibered phase uniform distribution or concentrate is distributed in the three-dimensional cell that robust fibre constitutes mutually and cultivates in the pore space structure of support.
2. two-phase three-dimensional cell according to claim 1 is cultivated support, it is characterized in that, the three-dimensional cell that said robust fibre constitutes is cultivated support by the pillar of constant or different diameter and/or fibrous.
3. two-phase three-dimensional cell according to claim 1 is cultivated support, it is characterized in that, the porosity of said cell culturing bracket changes through pillar and/or the quantity and the size of fiber that changes in the said support; And/or said vesicular structure changes through the three-dimensional localization pattern that changes pillar and/or fiber.
4. two-phase three-dimensional cell according to claim 1 is cultivated support, it is characterized in that robust fibre has circle, trilateral, square and/or orthogonal xsect.
5. two-phase porous three-dimensional cell culturing bracket according to claim 1 is characterized in that, said two-phase porous three-dimensional cell culturing bracket is made the size that is fit to cell cultures plate well, culturing room, culturing bottle and/or bio-reactor.
6. cultivate support according to each described two-phase three-dimensional cell of claim 1-5, it is characterized in that the material of robust fibre phase is a commaterial with fine-fibered material mutually.
7. the described two-phase three-dimensional cell of claim 6 is cultivated support, it is characterized in that robust fibre is the nondegradation material mutually with fine-fibered.
8. two-phase three-dimensional cell according to claim 6 is cultivated support, it is characterized in that robust fibre is degradable materials mutually with fine-fibered.
9. two-phase three-dimensional cell according to claim 6 is cultivated support, it is characterized in that wherein a phase material is non-degradable material, and another is degradation material mutually.
10. two-phase three-dimensional cell according to claim 1 is cultivated support, it is characterized in that, said cell culturing bracket adopts process for modifying surface to handle the said cytoskeletal cell attaching effect of improvement.
11. two-phase three-dimensional cell according to claim 10 is cultivated support, it is characterized in that, said process for modifying surface is that the physical chemistry mode comprises plasma treatment, photoglow processing, and/or chemical mode, uses H
2SO
4, HNO
3Strong acid treatment.
12. two-phase three-dimensional cell according to claim 11 is cultivated support, it is characterized in that said process for modifying surface is a kind of surface coating technique; Through applying a kind of coated substance that is different from the polymkeric substance that is used to prepare the said construct of claim 1; Said coated substance is naturally occurring polymkeric substance, and/or synthetic polymer, and/or inorganic substance; And/or the compound coating formed of two or more organic materialss, and/or a kind of inorganic/organic mixture.
13. two-phase three-dimensional cell according to claim 12 is cultivated support; It is characterized in that said coated substance comprises albumen, peptide, TGSS C3, collagen, Fibronectin, Z 150PH, polyoxyethylene glycol, Vinylpyrrolidone polymer, polylysine, calcium phosphate, TiO
2, SiO
2, Al
2O
3, in gel and WOT Recovery Floc T, ROHM and polyoxyethylene glycol, Z 150PH and Vinylpyrrolidone polymer, calcium phosphate/collagen mixture, calcium phosphoric acid/polyoxyethylene glycol mixture, the calcium phosphate/extracellular matrix one or more.
14. two-phase three-dimensional cell according to claim 13 is cultivated support; It is characterized in that described top coat material is to comprise that with chemical mode covalent linkage, hydrogen bond, ionic linkage or Van der Waals force are attached on the pillar and/or fiber of said cell culturing bracket.
15. make the method that the described two-phase three-dimensional cell of claim 1 is cultivated support for one kind, said method comprises:
(A) adopt quick molding method to prepare the work in-process porous three-dimensional support that robust fibre constitutes mutually;
(B) adopt electrostatic spinning technique spraying fine-fibered at work in-process porous three-dimensional rack outer surface;
(C) continue on the face that has the fine-fibered phase, to adopt quick molding method to prepare the porous three-dimensional support, thereby make fine-fibered be fixed on the three-dimensional rack inside that robust fibre constitutes mutually mutually;
(D) utilize the cut mechanically method, two-phase porous three-dimensional cell culturing bracket is cut into desired size;
(E) use a kind of plasma surface treatment device, in argon gas atmosphere, said cell culturing bracket is carried out plasma treatment;
(F) pack this plasma treated cell culturing bracket respectively, and finally sterilize with the gamma-rays radiation of 20KGy dosage.
16. a method of using the described two-phase three-dimensional cell of claim 1 to cultivate the support culturing cell, said method comprises:
(A) use dynamically inoculation or static inoculation method, with cell inoculation in said cell culturing bracket;
(B) after the inoculating cell, said cell culturing bracket is remained on the cell cultures upholder that is immersed among the growth medium, comprise in tissue culture plate, culturing room, culturing bottle and/or the bio-reactor and cultivating.
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