CN1428418A - Culture method of external tissue and its fatiscent carrier - Google Patents
Culture method of external tissue and its fatiscent carrier Download PDFInfo
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- CN1428418A CN1428418A CN 01144738 CN01144738A CN1428418A CN 1428418 A CN1428418 A CN 1428418A CN 01144738 CN01144738 CN 01144738 CN 01144738 A CN01144738 A CN 01144738A CN 1428418 A CN1428418 A CN 1428418A
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Abstract
The present invention relates to a culture method for external tissue and its polycellular carrier. It is mainly characterized by that after the tissue block is partially dissolved by ferment, it can be placed in the hollow cavity of the polycellular carrier with hollow cavity to make the tissue grow toward the pores of peripheral polycellular carrier from interior in the mode of three-dimensional space and directly proliferate new tissue in the form of tissue. Said invention has the function of that uses smallest volume tissue quantity to culture required tissue in vitro in shortest time.
Description
Technical field
The invention relates to a kind of cultural method and multi-pore carrier thereof of vitro tissue, it is the tissue block of acquisition to be inserted hollow porous crack decompose in the carrier, and the supply nutrient makes tissue with the hole growth of porous crack base material towards periphery of three-dimensional space mode, to obtain newborn tissue.
Background technology
As everyone knows, when tissue or organ can't be repaired because of factors such as unexpected injury or disease are aging wreck, the life that tends to cause the disabled of sufferer limbs or threaten sufferer further caused great burden and the loss to family and society.Therefore seek a kind of suitable repairing and substitute tissue or organ, be the continuous target of making great efforts of investigator institute at present.
In recent years, along with the progress of biotechnology, biological, medical material and histiocytic culture technique mutually combine, develop gradually this new research field of organizational project.Wish future, impaired tissue or organ can be via the reconstruction techniques of organizational project, in external repairing or rebuild a new tissue or organ, substitute impaired position, to recover the life of the healthy of sufferer or continuity sufferer.
Content according to present tissue engineering technique, the tissue that needs acquisition sufferer or contributor's sub-fraction health, behind external a large amount of cultured tissue cells, implant the porous crack base material of a decomposability, utilize its three-dimensional space framework, allow histocyte attach and growth thereon, when histocyte gradually grow up become tridimensional tissue block after, replant go back to the position of the required repairing of sufferer.From histological viewpoint, one block organization is except containing its specific composition extracellular, and (extracellulamatrix ECM) coats by tridimensional extracellular matrix for cell and iuntercellular, matter also can show the specific function of this tissue except keeping its cell framework around here.
Cultivation with cartilaginous tissue is an example, cultivate at present external cartilage three-dimensional space, confirmed the three-dimensional space cultivation of chondrocyte in agar (agarosegel), can keep its original cellular form and function in tissue, and cultivate the chondrocyte that variation transforms through space twice, in the time of also can cultivating, reply original chondrocyte's form in the agar three-dimensional space.Aspect the framework of analog cell outer room matter, developed multiple differing materials at present as Collagenase (collagen) or poly(lactic acid) glycolic acid (poly (glycolicco-Lactic) acid (PLGA)) and different structure, as fibrous web-like or mushy artificial base material.Facing maximum problem at present is:
Cell when 1, histocyte is cultivated in bidimensional culture dish in a large number, because arranging, the three-dimensional space in the tissue transfers to when grow up in the space twice, cell will dedifferente the phenomenon of (dedifferentiation), thereby form and the function of lost cell in original tissue.
2, in addition, the implantation of cell (seeding) also is a problem that is difficult for overcoming, because provide cell enough to develop into the space of tissue, the aperture of porous crack base material needs the chi footpath greater than cell, when cell and substratum mixed solution implantation porous crack base material, cell tends to flow out from carrier, is difficult for remaining in the base material, for overcoming this problem, present way has two kinds:
A kind of is static implantation, earlier institute's cultured cells concentration is adjusted to the high density more than the 106cells/ml, utilize the aquosity of porous crack base material itself then, the cell of being implanted is contained in the base material, after treating that cell is attached at base material, adding a large amount of substratum again cultivates, though this method can know that many a spot of cells implant in the base materials, and the cell of implantable high density enters in the material, yet, the factor that the distribution of cell still can be subjected to gravity influences, the cell distribution inequality that causes levels in addition, is overflowed for preventing cell, can be quite limited with the volume of the substratum of cytomixis, therefore effect and the survival rate that attaches also need be considered.
Another kind is dynamic cell implantation, the general at present stirring reactor (spinnerflask) that adopts, bring cell into base material inside from substratum in the mode that current stir, this method can obtain preferable cell attaching rate and compare the more uniform cell distribution of static implantation mode, yet, the cell count of using is higher, the actual cell count that is attached to material internal also is difficult for knowing, and cell is to implant from outside to inside, peripheral cell count can inner cell density be high still, when tissue culture, the higher one deck that will form of peripheral cell growth rate hinders, stop the nutrient exchange of inner cell, thereby cause the inside and outside cell growth of material significantly uneven.
Summary of the invention
At the shortcoming and the drawback of above-mentioned prior art, the inventor creates technical scheme of the present invention through studying chronically and testing.
Main purpose of the present invention provides a kind of cultural method of vitro tissue, comprises the following step at least: a multi-pore carrier with cavity is provided; Tissue block is partly dissolved through ferment; To and dissolve the cavity that the histocyte that places aforementioned multi-pore carrier through the partly soluble tissue block of ferment; And include tissue block and histiocytic multi-pore carrier places substratum to cultivate with aforementioned.Overcome to be organized in when external space plane is twice cultivated and dedifferente phenomenon, or the histocyte unequal shortcoming of growing inside and outside the base material, reach the purpose of cultivating vitro tissue effectively.
Another object of the present invention provides a kind of employed multi-pore carrier of cultural method of vitro tissue, develop a kind of multi-pore carrier, it mainly comprises main body, at least one is positioned at body interior and has the hollow cavity of an opening and the loam cake of covering hollow cavity opening, loam cake is to be made by the mushy material identical with main body, the particle diameter of tissue block is greater than the aperture of the peripheral base material of carrier cavity, reaches the purpose of avoiding the tissue block overflow to go out.
The object of the present invention is achieved like this: a kind of cultural method of vitro tissue is characterized in that: it comprises the following step at least:
(1) provides a multi-pore carrier with cavity;
(2) tissue block is partly dissolved through ferment;
(3) will and dissolve the cavity that the histocyte that places this multi-pore carrier through the partly soluble tissue block of ferment;
(4) this is included tissue block and histiocytic multi-pore carrier places substratum to cultivate.
Has the hollow cavity that at least one places tissue block in this multi-pore carrier.The particle diameter of this tissue block is the aperture greater than the peripheral base material of this carrier cavity.The particle diameter of this tissue block is 500 to 1000 μ m.Being shaped as of this multi-pore carrier is cylindrical, cubic type or required arbitrary shape.The pore diameter range of this multi-pore carrier is that 50 to 500 these multi-pore carriers of μ m are the macromolecular material of Bioabsorbable, and this macromolecular material is selected from following at least a: polylactic acid, the acid of poly(lactic acid) lactide, poly(lactic acid) glycolic acid, polyanhydride, polycaprolactone, poly-two ketone esters or polyorthoesters.This multi-pore carrier is the poly(lactic acid) glycolic acid.This tissue block is an animal tissues.This animal tissues is a cartilaginous tissue.This ferment is soluble protein enzyme or ferment that tissue block cytolysis is come out.This nutrient solution is the nutrient solution that contains foetal calf serum.
The present invention also provides a kind of cultural method of vitro tissue employed multi-pore carrier, it is characterized in that: it comprises a main body, at least more than one the loam cake that is positioned at this body interior and has the hollow cavity of an opening and cover this hollow cavity opening.
This main body is cylindrical, cubic type or required arbitrary shape.The pore diameter range of this multi-pore carrier is to be 50 to 500 μ m.This loam cake is by the mushy material identical with main body.
Major advantage of the present invention is: tissue mass that can minimal volumes, in the shortest time in the required tissue of vitro culture.
Further specify below in conjunction with preferred embodiment and accompanying drawing.
Description of drawings
Fig. 1 is the flow process block schematic diagram of the cultural method of vitro tissue of the present invention.
Fig. 2 is the structural representation with multi-pore carrier of hollow cavity of the present invention.
Fig. 3 is the growth view that is organized in the multi-pore carrier with hollow cavity of the present invention.
Fig. 4 is the schematic flow sheet of cultural method of the vitro tissue of experimental group of the present invention.
Fig. 5 is the schematic flow sheet of cultural method of the vitro tissue of control group of the present invention.
When Fig. 6 is the cartilaginous tissue cultivation fortnight of experimental group of the present invention, the photo of tissue around the hollow cavity carrier filling cartilaginous tissue and cell growth situation.
When Fig. 7 is the cartilaginous tissue cultivation fortnight of experimental group of the present invention, the tissue of the peripheral porous of carrier cavity crack base material and the photo of cell growth situation.
Fig. 8 is that the cartilaginous tissue of experimental group of the present invention is cultivated four stars during the phase, the tissue of around the hollow cavity carrier filling cartilaginous tissue and the peripheral porous of carrier cavity crack base material and the photo (amplifying 40 times) of cell growth situation.
Fig. 9 is the photo (amplifying 200 times) of Fig. 8 the same terms.
Figure 10 is that the cartilaginous tissue of the control group of the embodiment of the invention is cultivated four stars during the phase, the tissue of the peripheral porous of carrier cavity crack base material and the photo (amplifying 40 times) of cell growth situation.
Figure 11 is that the cartilaginous tissue of the control group of the embodiment of the invention is cultivated four stars during the phase, the tissue of the peripheral porous of carrier cavity crack base material and the photo (amplifying 200 times) of cell growth situation.
Figure 12 is grow into a cross-sectional area quantized result synoptic diagram in the PLGA multi-pore carrier of the cartilaginous tissue of experimental group more of the present invention and control group.
Embodiment
Consult shown in Figure 1ly, the cultural method of vitro tissue of the present invention mainly comprises the following step: a multi-pore carrier with cavity is provided; Tissue block is partly dissolved through ferment; To and dissolve the cavity that the histocyte that places aforementioned multi-pore carrier through the partly soluble tissue block of ferment; And include tissue block and histiocytic multi-pore carrier places substratum to cultivate with aforementioned.
Consulting shown in Figure 2ly, is to show multi-pore carrier 10 of the present invention, and it mainly comprises: a main body 1; At least more than one hollow cavity 2, it is to be positioned at aforementioned body 1 inside and to have an opening 5; And a loam cake 6, be as the usefulness that covers above-mentioned hollow cavity 2 openings 5.
Method of the present invention is that directly acquisition is organized, and shred and ferment dissolving superficial cell after, not in addition in external amplification culture in advance, but tissue block is inserted in the hollow cavity of a multi-pore carrier 10 2, the periphery then is mushy base material, afterwards, the perforate 5 that aforementioned loam cake 6 is covered hollow cavity 2 avoids tissue block to flow out again.The aforementioned fragment of tissue that shreds via its size of mode may command of sieving, makes its aperture greater than the hollow cavity 2 peripheral base materials of multi-pore carrier 10.When tissue block is received in the hollow cavity 2 of multi-pore carrier 10, because of the aperture of peripheral base material less than tissue block, therefore, it is inner and can overflow not go out that tissue block will be restricted to carrier 10.
Consult shown in Figure 3, multi-pore carrier 10 inside (being hollow cavity 2) will be contained through ferment dissolved high density cell mass and tissue block 4, because the cell concn of multi-pore carrier 10 inside is quite high, in culturing process, histocyte will be by interior to the lower peripheral porous crack base material climbing diffusion growth of density, from inside to outside along its tissue block structure originally, with the three-dimensional space mode to the periphery the hole 3 of porous crack base material grow newly-increased tissue 4 '.This cultural method is as external tissue culture, and be directly to cultivate new tissue with tissue morphology, the method of tissue culture outside conventional bulk can solve and cultivate the phenomenon of dedifferenting that causes in space twice, tissue mass that can also be minimum in the short period of time in the external tissue culture of carrying out.
The particle diameter of aforementioned tissue block 4 is to be 500 to 1000 μ m.
The shape of aforesaid multi-pore carrier 10 can change arbitrarily according to actual needs, for example: cylindrical, cubic type or arbitrary shape.
Aforesaid multi-pore carrier 10 is the macromolecular materials for absorbability, and this material is to be selected from following group: polylactic acid (polyglycolicacid; PGA), poly(lactic acid) lactide acid (polylacticacid; PLA), poly(lactic acid) glycolic acid (poly (glycolicco-Lactic) acid; PLGA), polyanhydride (polyanhydrides), polycaprolactone (polycapralactone), poly-two ketone esters (polydioxanone) and polyorthoesters (polyorthoester); Wherein preferable multi-pore carrier material is poly(lactic acid) glycolic acid (PLGA).
The pore diameter range of aforesaid multi-pore carrier 10 is to be 50 to 500 μ m.
Embodiment below by the cultivation of external cartilaginous tissue is described in more detail method of the present invention.
Embodiment
A kind of cultural method of external cartilaginous tissue:
(1) preparation has the Bioabsorbable multi-pore carrier 10 of hollow cavity 2:
Selected decomposability macromolecular material is the PLGA polymer (molecular weight is about 200,000) for preparing in the ring-opening polymerization mode in the present embodiment.Blocky PLGA macromolecular material is pulverized in pulverizer, and the particle of pulverizing can obtain the polymeric particles of particle diameter at 177-250 μ m after sieving by 60-80 hole purpose screen cloth; The water-soluble material of selecting for use that is added into creation pore space structure in the base material is the sodium chloride particle about particle diameter 250 μ m; The organic solvent of the dissolving polymeric particles of selecting for use is to be acetone.
PLGA polymeric particles and sodium chloride particle are complied with 10/90 weight ratio, with the mode uniform mixing that stirs; Mixed PLGA polymeric particles and sodium chloride particle are poured termination into, and air extractor, diameter are arranged is in the strainer of 7mm circle and compacting, pour into organic solvent-acetone in the composite grain and soak into this moment, open extraction valve and produce a negative pressure and take out unnecessary solvent downwards, and the polymeric particles that has dissolved on the surface is bondd mutually thereafter.After polymeric particles bonds mutually, pour a large amount of deionized waters in strainer top, also open extraction valve and bleed simultaneously, a large amount of water by in the material, is separated out and solidified polymeric particles, simultaneously that it is inner sodium chloride particle is washed out.Polymer after the curing takes out from strainer, place the large beaker that deionized water is housed, under room temperature, changed water once in per 6 hours, with the mode soaking and water washing that stirs 24 hours, solvent and salt grain that inside is residual wash out, and place 50 ℃ vacuum drying oven to heat the universe dry 24 hours again, can obtain a pore size distribution between 150 to 350 μ m, the about 90vol% of porosity, hole are the porous crack base material that is interconnected.
The multi-pore carrier with hollow cavity 2 10 that present embodiment is prepared, be that to cut diameter with scalpel be 7mm, it highly is the right cylinder of 9mm, digging a diameter in base material inside is the hollow cylindrical cavity of 6mm for the 3mm degree of depth, cutting a diameter again is 3mm, highly be the right cylinder stopper of 3mm (being loam cake 6 shown in Figure 2), can be when tissue block be inserted, to seal inner hollow cavity 2.After multi-pore carrier 10 preparation of hollow is finished, the alcohol-pickled sterilization with 75% 6 hours, and with a large amount of aseptic phosphate-buffered salts (phosphatebufferedsaline, PBS) the solution displacement includes disinfectant alcohol.
(2) processing of experimental group
Schema as shown in Figure 4, at first, cartilaginous tissue is the Thigh bone 12 that takes out about 2 to the 4 days big white mouse 11 of new life with microinstrument, remove the muscle and the periosteum on surface, be dipped in and do not add foetal calf serum (fetalcalfserum is among DMEM FCS), in the operator's console (laminarflow) of cell cultures, Thigh bone 12 is taken out, place the centrifuge tube 13 of 15ml, the phosphate buffered saline(PBS) that adds 10ml is cleaned, and repeats twice.After Thigh bone 12 cleaned, fall in the culture dish of 10cm, cut with the tissue of the bacterium of going out and joint cartilage is separated and shred, tissue block 4 chip size are controlled between the 400-800 μ m with 20-40 hole purpose screen cloth.The garbage collection that shreds is poured the phosphate buffered saline(PBS) flushing of 10ml into, triplicate again in the centrifuge tube of 15ml.Cartilaginous tissue piece 4 fragments after the flushing, phosphate buffered saline(PBS) is removed, added soluble protein enzyme (collagenase) 5ml (1mg/ml phosphate buffered saline(PBS)) again, place 37 ℃ incubator, left standstill 2 hours, the chondrocyte on surface is dissociated out.Afterwards,, place whizzer, with rotating speed 1500r.p.m. centrifugal 5 minutes, the soluble protein enzyme is separated with the cartilage piece with soluble protein enzyme separate dissolved cartilage bone piece later.After centrifugal, draw clarifying soluble protein enzyme supernatant liquor, remaining fragment and cell tissue is with phosphate-buffered salt flushing twice, and is centrifugal twice, to remove residual soluble protein enzyme.Cartilage piece and histocyte after cleaning are inserted it in hollow cavity 2 of multi-pore carrier 10 of volume 0.02ml, and cover the loam cake 6 of multi-pore carrier, run off to prevent the cartilage piece.
(3) processing of control group
Schema as shown in Figure 5, at first, control group is to adopt identical training method, take from the cartilage of above-mentioned Thigh bone 12 equally, and with same method cartilage is peeled off and shredded, cartilage is placed on culture dish 8 through shredding, and is soaked in soluble protein enzyme 5ml (1mg/ml phosphate buffered saline(PBS)) and the glass uronic acid (Hyaluronidase), in 37 ℃ incubators (figure does not show), left standstill 24 hours, all chondrocytes are dissociated out.Usually the cartilage of 1ml volume has 1.5x10 approximately
7Individual chondrocyte, with respect to multi-pore carrier 10 hollow cavities 2 volume 0.02ml, chondrocyte's amount that control group will add is 3x10
5Individual chondrocyte.The cell that adds is after whizzer is centrifugal, counted with the cell counting dish, cell behind the counting is added in multi-pore carrier 10 hollow cavities 2 (the cell solution volume that is added is 200 μ l) by the upper end, for preventing that cell from flowing out, cover loam cake 6 again and place culture dish 8, in 37 ℃ of incubators that contain saturated aqueous vapor, leave standstill the cell that made adding in 6 hours and attach.
(4) the common processing of experimental group and control group
After the histocyte of experimental group and control group is added into multi-pore carrier 10, will contains histiocytic carrier 10 and put into the rotary incubator (spinnerflask) 7 that contains cell culture fluid, as Fig. 4 and last step shown in Figure 5.Cell culture fluid is the DMEM nutrient solution that contains the 10%wt foetal calf serum, and the nutrient of each carrier is 3ml/ days, and according to the fresh nutrient solution of regular displacement of the time of cultivating, culture environment is the incubator that contains 37 ℃ of 5% carbonic acid gas.Cultivating the back takes out multi-pore carrier 10 in different incubation times from reactor, washed with phosphate buffered saline(PBS) earlier, be dipped in then in the phosphate buffered saline(PBS) that contains 4% formalin test piece is fixed, mode with the paraffin organization embedding is cut into slices, method with hematoxylin-Yihong dyes again, and observes the result that its cartilaginous tissue is grown up.
(5) experimental result
Cultivate result such as Fig. 6, shown in Figure 7 of fortnight with the method for experimental group, can find out that hole has newborn cartilaginous tissue 9 around the carrier hollow cavity, directly the cartilage piece on every side to the periphery hole growth of kenel from implanting to organize, as shown in Figure 6, the hole at other positions of carrier also can be observed by the center and overgrows with cell to external diffusion, as shown in Figure 7.
Fig. 8, Fig. 9 is for cultivating the experimental result of four stars after date, can find in the carrier except the hole of hollow cavity periphery, the hole at other positions also grows many newborn cartilaginous tissues 9, as shown in Figure 8, can be observed with higher multiplying power has many chondrocyte's lacunas (lacunae) in the cartilaginous tissue, as shown in Figure 9, chondrocyte in its lacuna has begun to split into microscler one-tenth isogenous group, show that formed cartilage is a quite active newborn cartilaginous tissue, and around the cartilaginous tissue also around many flat chondroblasts (chondroblast), this cartilaginous tissue is grown up sustainable expansion.
Figure 10 and Figure 11 show the result who is cultivated with the control group method, in cultivating four stars during the phase, in the porous lyriform pore hole of PLGA hollow cavity carrier to overgrow with cell, as shown in figure 10, observe and to find with higher multiplying power, newborn cell kenel still is fusoid fibrocartilage cells, does not form cartilaginous tissue as shown in figure 11.
Figure 12 is with in microstructure image software analytical calculation experimental group and the control group, the shared area of formed cartilaginous tissue in the peripheral hole of multi-pore carrier.Result by Figure 12 can learn:
The cartilaginous tissue of being cultivated with method of the present invention can be filled about 26% hole area in four stars during the phase, compared to the result of control group evident difference is arranged, and showing can be at short notice in the external newborn cartilage that grows volume with cultural method of the present invention.
Effect of the present invention:
The cultural method of vitro tissue of the present invention, mainly be tissue block to be inserted one have in the cavity of multi-pore carrier of hollow cavity, make the hole growth of the multi-pore carrier that tissue encloses from inside to outside in the three-dimensional space mode, directly the tissue that makes new advances with the form hyperplasia of tissue.This method can minimal volumes tissue mass, in the shortest time, cultivate required tissue in external connecing.The breakthrough of this technology will make the vitro tissue cultivation utilize culturing cell to come formative tissue by the past, change entering directly again and will come cultured tissue with tissue, to providing a quicker and effective culture technique with the organizational project industry that forms future.
Claims (18)
1, a kind of cultural method of vitro tissue is characterized in that: it comprises the following step at least:
(1) provides a multi-pore carrier with cavity;
(2) tissue block is partly dissolved through ferment;
(3) will and dissolve the cavity that the histocyte that places this multi-pore carrier through the partly soluble tissue block of ferment;
(4) this is included tissue block and histiocytic multi-pore carrier places substratum to cultivate.
2, the cultural method of vitro tissue according to claim 1 is characterized in that: have the hollow cavity that at least one places tissue block in this multi-pore carrier.
3, the cultural method of vitro tissue according to claim 1 is characterized in that: the particle diameter of this tissue block is the aperture greater than the peripheral base material of this carrier cavity.
4, the cultural method of vitro tissue according to claim 1 is characterized in that: the particle diameter of this tissue block is 500 to 1000 μ m.
5, the cultural method of vitro tissue according to claim 1 is characterized in that: being shaped as of this multi-pore carrier is cylindrical, cubic type or required arbitrary shape.
6, the cultural method of vitro tissue according to claim 1 is characterized in that: the pore diameter range of this multi-pore carrier is 50 to 500 μ m.
7, the cultural method of vitro tissue according to claim 1, it is characterized in that: this multi-pore carrier is the macromolecular material of Bioabsorbable, and this macromolecular material is selected from following at least a: polylactic acid, the acid of poly(lactic acid) lactide, poly(lactic acid) glycolic acid, polyanhydride, polycaprolactone, poly-two ketone esters or polyorthoesters.
8, the cultural method of vitro tissue according to claim 7 is characterized in that: this multi-pore carrier is the poly(lactic acid) glycolic acid.
9, the cultural method of vitro tissue according to claim 1 is characterized in that: this tissue block is an animal tissues.
10, the cultural method of vitro tissue according to claim 9 is characterized in that: this animal tissues is cartilaginous tissue or os osseum tissue.
11, the cultural method of vitro tissue according to claim 1 is characterized in that: this ferment is soluble protein enzyme or ferment that tissue block cytolysis is come out.
12, the cultural method of vitro tissue according to claim 1 is characterized in that: this nutrient solution is the nutrient solution that contains foetal calf serum.
13, the employed multi-pore carrier of cultural method of the described vitro tissue of a kind of claim 1 is characterized in that: it comprises a main body, at least more than one the loam cake that is positioned at this body interior and has the hollow cavity of an opening and cover this hollow cavity opening.
14, the employed multi-pore carrier of the cultural method of vitro tissue according to claim 13 is characterized in that: this main body is cylindrical, cubic type or required arbitrary shape.
15, the employed multi-pore carrier of the cultural method of vitro tissue according to claim 13 is characterized in that: the pore diameter range of this multi-pore carrier is to be 50 to 500 μ m.
16, the employed multi-pore carrier of the cultural method of vitro tissue according to claim 13, it is characterized in that: this multi-pore carrier is the macromolecular material of Bioabsorbable, and this macromolecular material is selected from following at least a: polylactic acid, the acid of poly(lactic acid) lactide, poly(lactic acid) glycolic acid, polyanhydride, polycaprolactone, poly-two ketone esters or polyorthoesters.
17, the employed multi-pore carrier of the cultural method of vitro tissue according to claim 16 is characterized in that: this multi-pore carrier is the poly(lactic acid) glycolic acid.
18, the employed multi-pore carrier of the cultural method of vitro tissue according to claim 13 is characterized in that: this loam cake is by the mushy material identical with main body.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200910118097 CN101491703B (en) | 2001-12-24 | 2001-12-24 | Vitro tissue culturing method and multi-pore carrier |
| CNB011447389A CN100532543C (en) | 2001-12-24 | 2001-12-24 | Culture method of external tissue and its porous carrier |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB011447389A CN100532543C (en) | 2001-12-24 | 2001-12-24 | Culture method of external tissue and its porous carrier |
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| CN 200910118097 Division CN101491703B (en) | 2001-12-24 | 2001-12-24 | Vitro tissue culturing method and multi-pore carrier |
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| CN1428418A true CN1428418A (en) | 2003-07-09 |
| CN100532543C CN100532543C (en) | 2009-08-26 |
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| CNB011447389A Expired - Lifetime CN100532543C (en) | 2001-12-24 | 2001-12-24 | Culture method of external tissue and its porous carrier |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102586106A (en) * | 2006-01-23 | 2012-07-18 | 杨炜 | Three-dimensional space cell culture system preparation method |
| CN103958670A (en) * | 2011-12-01 | 2014-07-30 | 富士软件株式会社 | Method for long-term storage of porous body bearing chondrocytes |
| CN105265353A (en) * | 2015-06-07 | 2016-01-27 | 肖义军 | Pearl nucleus having mantellum cell hydrogel layer at surface and preparation method therefor |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR100372751B1 (en) * | 1999-11-16 | 2003-02-17 | 한국과학기술원 | Fabrication Method of Porous Biodegradable Polymer Scaffolds for Tissue Engineering |
-
2001
- 2001-12-24 CN CN 200910118097 patent/CN101491703B/en not_active Expired - Lifetime
- 2001-12-24 CN CNB011447389A patent/CN100532543C/en not_active Expired - Lifetime
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102586106A (en) * | 2006-01-23 | 2012-07-18 | 杨炜 | Three-dimensional space cell culture system preparation method |
| CN102586106B (en) * | 2006-01-23 | 2014-02-05 | 杨炜 | Three-dimensional space cell culture system preparation method |
| CN103958670A (en) * | 2011-12-01 | 2014-07-30 | 富士软件株式会社 | Method for long-term storage of porous body bearing chondrocytes |
| CN105265353A (en) * | 2015-06-07 | 2016-01-27 | 肖义军 | Pearl nucleus having mantellum cell hydrogel layer at surface and preparation method therefor |
| CN105265353B (en) * | 2015-06-07 | 2018-07-10 | 肖义军 | There are pearl nucleus of the hydrogel layer containing external carbon dosage and preparation method thereof on a kind of surface |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101491703B (en) | 2013-02-20 |
| CN100532543C (en) | 2009-08-26 |
| CN101491703A (en) | 2009-07-29 |
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