CN1257262C - Pouring type biologic reactor system in vitro constructure of dual active skin - Google Patents
Pouring type biologic reactor system in vitro constructure of dual active skin Download PDFInfo
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- CN1257262C CN1257262C CNB2004100166516A CN200410016651A CN1257262C CN 1257262 C CN1257262 C CN 1257262C CN B2004100166516 A CNB2004100166516 A CN B2004100166516A CN 200410016651 A CN200410016651 A CN 200410016651A CN 1257262 C CN1257262 C CN 1257262C
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- liquid
- type bioreactor
- pouring type
- active skin
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- 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
- C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
- C12M29/10—Perfusion
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- 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
- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/58—Reaction vessels connected in series or in parallel
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- 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
- C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
- C12M41/26—Means for regulation, monitoring, measurement or control, e.g. flow regulation of pH
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- 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
- C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
- C12M41/30—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration
- C12M41/32—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration of substances in solution
Abstract
The present invention relates to a pouring type biological reactor system in vitro construction of dual active skin, which comprises a liquid feeding device, a pouring type biological reactor, a liquid collecting device, a liquid reflux device and an air supply device, wherein the liquid feeding device is connected with a liquid inlet of the pouring type biological reactor, the liquid collecting device is connected with a liquid outlet of the pouring type biological reactor and is used for collecting culture media cultivated in the reactor, the liquid reflux device is respectively communicated with the liquid feeding device and the liquid collecting device by pipe lines, the air supply device is communicated with a gas inlet of the pouring type biological reactor, and a support material or a cell material compound for skin construction is flatly arranged on a porous support board in the pouring type biological reactor. The reactor of the present invention has the advantages of simple system structure and easy amplification, the moist heat sterilization can be carried out, the sterile environment can be maintained for a long time, the growing environment in vivo can be well simulated, and a whole dual active skin construction process in vitro from inoculation to corium construction and to skin construction is realized.
Description
Technical field
The present invention relates to a kind of bio-reactor, relate in particular to the filling type bioreactor of external structure double-layer active skin in a kind of medical science and the organizational engineering.
Background technology
Skin is the tissue of human body maximum, the about 1.5-2.0m of its surface-area
2As one barrier of outermost of human body, skin also is the easiest tissue that sustains damage.Annual nearly 60000-80000 burn patient need carry out dermatoplasty.
The present auto-skin grafting that adopts, allogeneic skin graft, dermatoheteroplasty exist for problems such as district's deficiency, immunological rejection and pathophoresiss.New vitality has been injected in the dermatoplasty that appears as of organization engineering skin.
The research of organization engineering skin starts from nineteen seventies, has developed various Graftskins with clinical value so far, can be divided into epiderm substitute, dermal substitute and composite skin surrogate by forming.There are shortcomings such as thin and frangible, not wear-resisting, that contraction is bigger in epiderm substitute owing to lacking skin corium, and clinical application is restricted; There is not the dermal substitute of epidermal area to exist moisture easily to evaporate shortcomings such as easy infection.The ideal skin substitute products should be able to be repaired the corium and the epidermal area that are lacked simultaneously, because not only cutaneous function of these two kinds of compositions and profile, and can influence each other, promote differentiation each other.The Apligraf that U.S. Organogenesis company produces is first kind of organization engineering skin that contains epidermis and corium simultaneously.
The final purpose that organization engineering skin makes up is that to carry out dermatoplasty be that burn patient or some other skin injury (the acute skin corrupted that causes as wound or operation, chronic ulcer etc.) patient provides temporary transient or nonvolatil Graftskin.Yet a typical burn patient needs 3000-5000cm usually
2Graftskin, for can scale operation Graftskin to satisfy clinical demand, exploitation is applicable to that the bioreactor system of external structure artificial skin is organization engineering skin move towards application from the laboratory the only way which must be passed.
Apligraf (William HE, Eaglastein MD, and Vincent Falanga, MD.Tissue engineeringand the development of Apligraf, a human skin equivalent.Clinical Therapeutics, 1997,19 (5): production 894-905.) is to adopt traditional static cultivation, with inoblast be laid on culture dish not of uniform size after collagen gel mixes, cultivate and connect keratinocyte on the surface after 4-6 days, use liquid-gas interface after submerged culture 2 days instead and cultivate.This manual preparation mode complicated operation is difficult for amplifying, and can't simulate intravital growing environment, and organizes homogeneity, production repeatability and quality stability to be difficult to guarantee, the bio-reactor that therefore is used for the skin histology structure is more in recent years come out one after another.
Halberstadt (Halberstadt CR, Hardin R, Bezverkov K, et al.The in vitro growthof a three-dimensional human dermal replacement using a single-pass perfusion system, Biotechnol Bioeng, 1994,43:740-746.) wait the people to design and develop a kind of unidirectional perfusion culture system of sealing, be used to produce the dermal tissue surrogate.This system adopts the dynamically mode of inoculation, but the culture systems complex structure, and does not realize the structure of epidermal area.Prenosil and Villeneuve (PrenosilJE, Villeneuve PE.Automated production of cultured epidermal autograpts and sub-confluentepiderlmal autografts in a computer controlled bioreactor.Biotechnol Bioeng, 1998,59:679-683.) having developed the cover epiderm substitute reactor assembly of control automatically, this system still can't realize the production of composite skin.
Kermer (Michael Kremer, Ekkehard Lang, Alfred Berger.Organotypical engineeringof differentiated composited-skin equivalents of human keratinocytes in a collagen-GAGmatrix (INTEGRA Artificial Skin) in a perfusion culture system.Langenbeck ' s Arch Surg, 2001,386:357-363.) etc. the filling system of human MINUCELLS and MINUTISSUE exploitation be used for the external structure of composite skin, yet they directly with the Integra artificial skin as skin corium, omit corium and made up this step, and this device for casting liquid-gas interface of structurally failing to support, therefore can't really finish the external structure of composite skin.People such as Cao Yilin are at Chinese utility model patent (application number: the new-type bioreactor that has disclosed a kind of organization engineering skin external structure 02266979.5), this reactor is complex structure not only, and little for the improvement effect of timbering material inside cell micro-environment.
Summary of the invention
The technical issues that need to address of the present invention are the pouring type bioreactor systems that disclose a kind of double-layer active skin external structure, with overcome complex structure that prior art exists, can't analogue body in growing environment, be difficult for the shortcoming of amplifying, satisfy the demand of clinical application.
Technical conceive of the present invention is such:
By regulating the liquid level of substratum, in same reactor, carry out submerged culture or the liquid-gas interface cultivation, to satisfy preparation technology's requirement of double-layer active skin; By monitoring to culturing process, determine nutrition wear rate and metabolism generating rate, in time reflect cell growing state in bio-reactor, to determine suitable irrigation rate and to carry out perfusion culture, thereby the optimization culture environment satisfies normal growth of cell and secretion extracellular protein; Realize the multiple spot inoculation by liquid distributor in the cell inoculation process, cell is more evenly distributed in biomaterial, the artificial skin of preparing is more near the histological structure and the physiological function of natural skin; Bio-reactor of the present invention has the amplification characteristic of simple, not only can amplify the size of single reactor, increase the area of the artificial skin of preparation, also can be by a plurality of reactor parallel connections, realize the increase of product amount, therefore, can be from truly realizing external scale operation artificial skin.
The pouring type bioreactor system of double-layer active skin external structure of the present invention comprises at least: liquid-adding device, filling type bioreactor, receipts liquid device, liquid reflux device and air feeder;
Said filling type bioreactor comprises: the closed container that is provided with loam cake; Be arranged on the porous support plate that in the closed container closed container is divided into last chamber and following chamber; Be arranged on the fit sealing spare of porous support plate top near the closed container inwall; The timbering material or the cell material mixture that are used for the skin structure lie in porous support plate, and fix by fit sealing spare, and porous support plate can provide liquid-gas interface when epidermal area made up; Last chamber is provided with liquid inlet and liquid exit, and following chamber is provided with liquid inlet and liquid exit, on be covered with liquid inlet, gas inlet and pneumatic outlet, loam cake below is provided with liquid distributor, is used to the substratum that distributes and enter from the liquid inlet of loam cake 1.
Said liquid-adding device is connected with the liquid-inlet of filling type bioreactor, be used for fresh culture is imported filling type bioreactor, said receipts liquid device is connected with the liquid exit of filling type bioreactor, be used for collecting the reacted substratum of reactor, said liquid reflux device is connected with liquid-adding device and receipts liquid device respectively by pipeline, and said air feeder is connected with the gas inlet of filling type bioreactor by pipeline;
System of the present invention uses like this:
The timbering material or the cell material mixture that will be used for the skin structure lie in porous support plate in the filling type bioreactor, and fix by fit sealing spare, fresh culture is sent into filling type bioreactor by liquid-adding device, realize the inoculation of cell multiple spot by liquid distributor, in reactor, carry out submerged culture or the liquid-gas interface cultivation, to satisfy preparation technology's requirement of double-layer active skin, enter the receipts liquid device by the filling type bioreactor liquid exit then, the air air feeder is sent into filling type bioreactor, to satisfy the needed oxygen of reaction and to regulate pH, liquid reflux device can be sent the reacted substratum of receiving the liquid device collection back to liquid-adding device, to reuse.
Reactor assembly of the present invention is simple in structure, be easy to amplify, can carry out moist heat sterilization, can keep gnotobasis for a long time, growing environment in the analogue body is realized being building up to the whole double-layer active skin external structure process that epidermis makes up from being inoculated into corium well.
Description of drawings
Fig. 1 is that the pouring type bioreactor system one-piece construction of double-layer active skin external structure is formed synoptic diagram.
Fig. 2 is the structural representation of filling type bioreactor main body.
Embodiment
Referring to Fig. 1 and Fig. 2, the pouring type bioreactor system of double-layer active skin external structure of the present invention comprises at least: liquid-adding device, filling type bioreactor, receipts liquid device, liquid reflux device and air feeder;
Said filling type bioreactor comprises: the closed container 2 that is provided with loam cake 1; Be arranged on the porous support plate 5 that in the closed container 2 closed container 2 is divided into last chamber 3 and following chamber 4, be arranged on the fit sealing spare 6 of porous support plate 5 tops near closed container 2 inwalls; The timbering material or the cell material mixture 7 that are used for the skin structure lie in porous support plate 5, and fixing by fit sealing spare 6, and porous support plate 5 can provide liquid-gas interface when epidermal area made up; Last chamber 3 is provided with liquid inlet 8 and liquid exit 9, following chamber is provided with liquid inlet 8 and liquid exit 9, loam cake 1 is provided with liquid inlet 8, gas inlet 10 and pneumatic outlet 11, and loam cake 1 below is provided with liquid distributor 101, is used to the substratum that distributes and enter from the liquid inlet 8 of loam cake 1.
Said liquid-adding device is connected with the liquid inlet 8 of filling type bioreactor, said receipts liquid device is connected with the liquid exit 9 of filling type bioreactor, said liquid reflux device is connected with liquid-adding device and receipts liquid device respectively by pipeline, and said air feeder is connected with the gas inlet 10 of filling type bioreactor by pipeline;
As seen from Figure 1:
Said liquid-adding device comprises liquid feeding peristaltic pump 12 and coupled liquid feeding liquid storage bottle 13, liquid feeding liquid storage bottle 13 is connected with the liquid inlet 8 of filling type bioreactor by pipeline, because said bio-reactor has three liquid inlets 8, select the liquid inlet 8 of loam cake 1 during inoculation for use, perfusion culture may be selected the liquid inlet 8 of avris, therefore be preferably between liquid feeding peristaltic pump 12 and the pipeline that each liquid inlet 8 of filling type bioreactor links to each other by-pass valve control 26 is set, entering the mouth from which with controlled liq 8 enters;
Said receipts liquid device comprises: receive liquid peristaltic pump 14, receive liquid liquid storage bottle 15, receive hydraulic control valve 16 and receive liquid bottle 17, the import of receiving liquid peristaltic pump 14 is connected with the liquid exit 9 of filling type bioreactor, the outlet of receiving liquid peristaltic pump 14 is connected with the inlet of receiving liquid liquid storage bottle 15, the outlet of receiving liquid liquid storage bottle 15 is connected with receipts liquid bottle 17 by liquid storage peristaltic pump 25, receiving hydraulic control valve 16 is arranged on the pipeline of receiving between liquid liquid storage bottle 15 and the liquid storage peristaltic pump 25, be used for control and receive liquid measure, be provided with by-pass valve control 26 between each outlet of bio-reactor and the receipts liquid peristaltic pump 14.
Said liquid reflux device comprises by reflux pipeline 19 and is arranged on the recycle control valve of receiving between liquid liquid storage bottle 15 and the liquid feeding liquid storage bottle 13 18;
Said air feeder comprises interconnective successively pneumatic pump 20, air flowmeter 21 and air filter 22, and air filter 22 is connected with filling type bioreactor;
Said proofing unit comprises and is inserted in liquid feeding liquid storage bottle 13, receives the dissolved oxygen electrode 23 in the liquid liquid storage bottle 15 and receives pH electrode 24 in the liquid liquid storage bottle 15, dissolved oxygen electrode 23 is connected with the pH determinator with dissolved-oxygen content analyser respectively with pH electrode 24, to detect and to control the oxyty and the pH of substratum in liquid feeding liquid storage bottle 13, the receipts liquid liquid storage bottle 15.
As seen from Figure 1, a plurality of said filling type bioreactors can be connected in parallel, to satisfy the needs that enlarge turnout.
Claims (9)
1. the pouring type bioreactor system of a double-layer active skin external structure is characterized in that, comprises at least: liquid-adding device, filling type bioreactor, receipts liquid device, liquid reflux device and air feeder;
Said filling type bioreactor comprises: the closed container (2) that is provided with loam cake (1), be arranged on the porous support plate (5) that in the closed container (2) closed container (2) is divided into last chamber (3) and following chamber (4), be arranged on the fit sealing spare (6) of porous support plate (5) top near closed container (2) inwall, last chamber (3) is provided with liquid inlet (8) and liquid exit (9), following chamber is provided with liquid inlet (8) and liquid exit (9), loam cake (1) is provided with liquid inlet (8), gas inlet (10) and pneumatic outlet (11), loam cake (1) below is provided with liquid distributor (101);
Said liquid-adding device is connected with the liquid inlet (8) of filling type bioreactor, said receipts liquid device is connected with the liquid exit (9) of filling type bioreactor, said liquid reflux device is connected with liquid-adding device and receipts liquid device respectively by pipeline, and said air feeder is connected with the gas inlet (10) of filling type bioreactor by pipeline;
Said receipts liquid device comprises: receive liquid peristaltic pump (14), receive liquid liquid storage bottle (15), receive hydraulic control valve (16) and receive liquid bottle (17), the import of receiving liquid peristaltic pump (14) is connected with the liquid exit (9) of filling type bioreactor, the outlet of receiving liquid peristaltic pump (14) is connected with the inlet of receiving liquid liquid storage bottle (15), the outlet of receiving liquid liquid storage bottle (15) is connected with receipts liquid bottles (17) by liquid storage peristaltic pump (25), receive hydraulic control valve (16) and be arranged on the pipeline of receiving between liquid liquid storage bottle (15) and the liquid storage peristaltic pump (25), be used for control and receive liquid measure.
2. the pouring type bioreactor system of double-layer active skin external structure according to claim 1, it is characterized in that, said liquid-adding device comprises liquid feeding peristaltic pump (12) and coupled liquid feeding liquid storage bottle (13), and liquid feeding liquid storage bottle (13) is connected with the liquid inlet (8) of filling type bioreactor by pipeline.
3. the pouring type bioreactor system of double-layer active skin external structure according to claim 1, it is characterized in that said liquid reflux device comprises by reflux pipeline (19) and is arranged on the recycle control valve of receiving between liquid liquid storage bottle (15) and the liquid feeding liquid storage bottle (13) (18).
4. the pouring type bioreactor system of double-layer active skin external structure according to claim 1, it is characterized in that, said air feeder comprises interconnective successively pneumatic pump (20), air flowmeter (21) and air filter (22), and air filter (22) is connected with filling type bioreactor.
5. the pouring type bioreactor system of double-layer active skin external structure according to claim 1, it is characterized in that, also comprise proofing unit, said proofing unit comprises and is inserted in liquid feeding liquid storage bottle (13), receives dissolved oxygen electrode (23) in the liquid liquid storage bottles (15) and the pH electrode (24) in the receipts liquid liquid storage bottles (15) that dissolved oxygen electrode (23) is connected with the pH determinator with dissolved-oxygen content analyser respectively with pH electrode (24).
6. the pouring type bioreactor system of double-layer active skin external structure according to claim 1, it is characterized in that, the timbering material or the cell material mixture (7) that are used for the skin structure lie in porous support plate (5), and fixing by fit sealing spare (6).
7. the pouring type bioreactor system of double-layer active skin external structure according to claim 2, it is characterized in that, between liquid feeding peristaltic pump (12) and pipeline that each liquid inlet (8) of filling type bioreactor links to each other, by-pass valve control (26) is set.
8. the pouring type bioreactor system of double-layer active skin external structure according to claim 2 is characterized in that, is provided with by-pass valve control (26) between each liquid exit of bio-reactor (9) and the receipts liquid peristaltic pumps (14).
9. according to the pouring type bioreactor system of each described double-layer active skin external structure of claim 1～8, it is characterized in that, comprise a plurality of filling type bioreactors that are connected in parallel.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2004100166516A CN1257262C (en) | 2004-03-01 | 2004-03-01 | Pouring type biologic reactor system in vitro constructure of dual active skin |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2004100166516A CN1257262C (en) | 2004-03-01 | 2004-03-01 | Pouring type biologic reactor system in vitro constructure of dual active skin |
Publications (2)
| Publication Number | Publication Date |
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| CN1560225A CN1560225A (en) | 2005-01-05 |
| CN1257262C true CN1257262C (en) | 2006-05-24 |
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| CNB2004100166516A Expired - Fee Related CN1257262C (en) | 2004-03-01 | 2004-03-01 | Pouring type biologic reactor system in vitro constructure of dual active skin |
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Families Citing this family (15)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101402917B (en) * | 2008-11-25 | 2011-07-06 | 中国人民解放军第三军医大学第三附属医院 | Bioreactor for producing tissue engineering products |
| CN102071137B (en) * | 2009-11-19 | 2014-10-22 | 上海坤巨科技发展有限公司 | Device and method for preparing stem cells through continuous perfusion bioreactor/tank (bag) system |
| CN102199536A (en) * | 2010-03-23 | 2011-09-28 | 上海坤巨科技发展有限公司 | Apparatus and method for monitoring cell quantity or cell proportion during dynamic stem cell differentiation |
| CN102199535A (en) * | 2010-03-23 | 2011-09-28 | 上海坤巨科技发展有限公司 | Device and method for noninvasive continuous monitoring of quantity or concentration of dynamic cells |
| CN102296029B (en) * | 2010-06-28 | 2013-01-30 | 裴国献 | Perfusion bioreactor system |
| CN102618442A (en) * | 2012-04-06 | 2012-08-01 | 中国人民解放军第四军医大学 | Artificial skin culture device and using method thereof |
| CN103266182B (en) * | 2013-05-30 | 2015-04-15 | 中山大学 | Method and device for regulating tower plate type perfusion bioreactor |
| CN103290145B (en) * | 2013-06-03 | 2015-09-23 | 中山大学 | A kind of filling type bioreactor experimental technique and using appts thereof |
| CN105683351A (en) * | 2013-10-24 | 2016-06-15 | 株式会社日立制作所 | Automated culture device |
| WO2015176653A1 (en) * | 2014-05-22 | 2015-11-26 | 第三军医大学第三附属医院 | Bioreactor for three-dimensional tissue perfusion culture |
| CN103966095B (en) * | 2014-05-22 | 2016-01-20 | 中国人民解放军第三军医大学第三附属医院 | A kind of bio-reactor being applicable to three-dimensional tissue's Cell infusion and cultivating |
| CN105176815B (en) * | 2015-10-20 | 2017-03-22 | 哈尔滨工业大学 | Gas-liquid interface perfusion bioreactor for artificial skin tissue culture and preparation method and application method of gas-liquid interface perfusion bioreactor |
| CN105524832B (en) | 2016-02-26 | 2018-03-30 | 广州洁特生物过滤股份有限公司 | Cell culture apparatus and method |
| CN111132595A (en) * | 2017-09-11 | 2020-05-08 | 三维生物科技有限公司 | Large-scale bioreactor |
| CN112933298B (en) * | 2021-04-01 | 2023-11-17 | 领博生物科技(杭州)有限公司 | Biological material preparation device and biological material preparation system |
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2004
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