CN102618442A - Artificial skin culture device and using method thereof - Google Patents
Artificial skin culture device and using method thereof Download PDFInfo
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- CN102618442A CN102618442A CN201210099969XA CN201210099969A CN102618442A CN 102618442 A CN102618442 A CN 102618442A CN 201210099969X A CN201210099969X A CN 201210099969XA CN 201210099969 A CN201210099969 A CN 201210099969A CN 102618442 A CN102618442 A CN 102618442A
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- C12M21/00—Bioreactors or fermenters specially adapted for specific uses
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- C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
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Abstract
The invention provides an artificial skin culture device and a using method thereof. The device consists of a control unit and a culture unit, wherein a gas mixing chamber and a liquid storage chamber are respectively connected with a culture bottom case through a control valve via pipelines; a plurality of sensors in the gas mixing chamber and the liquid storage chamber are connected with a control panel through circuits by virtue of a gas control module and a liquid control module, so that circulation of gas and culture fluid in the culturing process can be dynamically monitored; and setting, monitoring and adjustment for the culturing process can be realized by virtue of the control panel. Compared with the prior art, according to the artificial skin culture device and the using method thereof, static culture of artificial skin is replaced by dynamic culture, single culture is replaced by integral culture, the culture fluid is automatically replaced by virtue of dynamic monitoring and adjustment, and the stability of a culture system is increased; the risk of microorganism invaded contamination is remarkably reduced by adopting a closed culture system; and the uniformity of product shapes and the stability of product quality are improved, so that industrialization is easily realized. The artificial skin culture device and the using method are applicable to culture of various artificial skins.
Description
Technical field
The invention belongs to the preparation facilities technical field of organizational engineering biomaterial for medical purpose, be specifically related to the improvement of artificial skin culture apparatus and method of use thereof.
Background technology
Skin is to cover and the vital tissue organ of protecting body surface.Owing to reasons such as inflammation, ulcer, wound, burn, iatrogenic and congenital abnormality often cause the damaged and unusual of skin.In recent years, along with the development of tissue engineering technique, be widely used in the transplantation treatment of skin injury at the organization engineering skin of external structure.Organization engineering skin is to adopt organizational engineering principle and the artificial skin surrogate of method in external preparation, is after the human fibroblasts is increased with epidermic cell in a large number, again epidermic cell, dermal fibroblast is compound in collagen scaffold and recombinates and form.
Because tissue engineering technique also is in the starting stage, in the production of artificial skin, does not still have sophisticated specific equipment and produce utensil.Chinese patent 200910024283.2 discloses a kind of cultivation box that organization engineering skin is produced that is used for, and is changed end box, lid and the cultivation holder of liquid bath by band and forms; It is cultivated holder and in end box, puts for the single flat lay.There is following problem in this cultivation box in application: the application mode that box is cultivated in (1) is a static cultivation; Its culture system lacks dynamic monitoring; Only control adjustment culturing process (as changing nutrient solution) by rule of thumb; Be difficult to realize stdn and normalized operational administrative for the scale prodn process, cause quality instability, the effect of product undesirable; (2) cultivating the holder sidewall has the vertical groove that is uniformly distributed with, and can make in the artificial skin culturing process that the liquid nutrient oozes out in the holder, is prone to cause the skin edge of cultivating to form irregular projection; Especially when compound cells, can cause oozing out of cell mixture, cause cell to run off, cause the inhomogeneous of the cell quantity in the skin cultivated; (3) stand bar structure of cultivation holder outer bottom makes to cultivate to hold in the palm in end box, to be subjected to displacement easily, makes the position fixing, especially in the automatic operation to cultivation holder or skin surface, causes being difficult to accurate location; (4) change the liquid bath position end of at the box side, strengthened the horizontal volume of cultivating box, need greater room in intensive cultivation with when depositing; (5) seamless between end box and lid, be unfavorable for gaseous interchange, influence the effect that skin is cultivated.
Summary of the invention
For solving above-mentioned existing in prior technology defective, the purpose of this invention is to provide a kind of artificial skin culture apparatus and method of use thereof, its robotization that is more conducive to artificial skin and standardization are produced.
Artificial skin culture apparatus proposed by the invention comprises and cultivates end box, lid and cultivate holder; It is characterized in that described artificial skin culture apparatus is made up of control unit and cultivation unit, described control unit comprises computer-controlled gas control module, liquid control module, transmitter, gas mixing chamber, liquid storage room and control panel; Described cultivation unit comprises the pipeline of cultivating end box, lid box with the cultivation holder and at the bottom of being connected some cultivations; Wherein gas mixing chamber is connected with cultivation end box respectively by pipeline through valve with liquid storage room; Some transmitters in the gas mixing chamber are connected with control panel through the gas control module through circuit, can realize the dynamic monitoring to gas circulation in the mixing section in the culturing process; The indoor some transmitters of fluid storage are connected with control panel through the liquid control module through circuit, can realize nutrient solution round-robin dynamic monitoring in the storing chamber in the culturing process; Operation through control panel can realize setting, monitoring and adjustment to culturing process.
The inner bottom surface of box is provided with prominent shape reference column at the bottom of the cultivation according to the invention, is used for the position that holder is cultivated in grappling; The size of cultivating end box adapts with the size of cultivating holder, and cultivating holder is 1～4 row's discharging in end box, and every discharging is put 4～8 and cultivated holder; Box outer bottom of the said end is provided with four supporting legs, high 4mm～10mm, can prevent outside liquid immersion, be convenient to the carrying; The box both sides are respectively equipped with the pipe interface of gas, liquid turnover the end of at, through pipeline with some cultivation boxes serial connections or and connect, make gas, liquid in the box form flow cycle respectively, so that carry out the dynamic monitoring and the replacing of gas and nutrient solution; Fasten sealing between end box and lid.There is equally distributed through hole the bottom surface of cultivation holder according to the invention, and the aperture is 1mm～2mm, to guarantee the infiltration of liquid in the skin culturing process; The height of cultivating in the holder is 4mm～10mm, is to confirm the complete imperforation of sidewall according to the thickness of cultivation skin; Cultivate the holder outer bottom and evenly be provided with at least three legs that turn up; Central authorities have pilot hole at the cross break position of the leg that turns up; The pilot hole reference column interior with cultivating end box cooperates to be inlayed, and makes to cultivate and holds in the palm and can accurately be anchored in the end box, is convenient to the accurate location of automated operation; The total height of cultivating holder is lower than cultivates more than the end box inner height 4mm, to guarantee the supply of nutrient solution.
Characteristic of the present invention also is, other is provided with and cultivates the circle of setting up that holder can be sealed and matched, and inserting to cultivate increases the sidewall height in the holder, set up the high 2mm～10mm of circle, is used for the loss that the artificial skin culturing process is avoided cell suspension.
The method of use of the artificial skin culture apparatus of the present invention is being cultivated the nylon membrane of backing portion laying with its identical size, celliferous gelating soln is injected cultivate holder, and its sidewall can effectively be avoided the loss of solution before curing; To cultivate holder again with pilot hole on the leg and the embedding grappling mutually of cultivation end box reference column; Sealing fastens lid, will cultivate box usefulness gas, liquid line connects respectively, and is communicated with gas mixing chamber and nutrient solution storing chamber respectively;, and start to cultivate culturing process and condition setting at control panel.Between during cultivation, cell stretches the height and the area that cause culture gradually and shrinks, and culture is broken away from cultivate the holder inwall.Open and cultivate the box lid, adopt automatic punch device to the punching of culture surface after, cover inserts and sets up circle in cultivating holder, injects cell suspension, sealing fastens the lid continued and cultivates, and cultivates completion to artificial skin.
Artificial skin culture apparatus of the present invention compared with prior art has the following advantages: 1) training method of artificial skin changes dynamic cultivation into from static cultivation, can be to the gas (O in the culturing process
2, CO
2) and the component condition of nutrient solution (glucose, temperature, pH value, oxyty) carry out dynamic monitoring and adjustment respectively; Realize the automatic replacing of nutrient solution; Increased the stability of culture system, reduced the blindness and the workload of changing liquid, made that culture condition more stablize by science, reasonable reaching; Owing to adopt closed system to cultivate, can significantly reduce external microbe and invade and the risk of polluting.2) training method changes whole the cultivation into from single cultivation, have consistent more culture condition, and the homogeneity between having improved batch has increased the stability of quality product, is easy to realize industrialization.3) cultivate holder accurately grappling of quilt in end box, the cultivation holder can be subjected to displacement in operation, guaranteed the accurate location of automated operation.4) accessory structures such as need not to change liquid bath is cultivated in the robotization of sealing, the space increase that makes intensive cultivation and deposit; 5) cultivate holder sidewall imperforation, avoided the seepage of liquid protein, improved the profile and the stability of artificial skin; Set up coil structures when compound cells, can effectively avoid overflowing of enchylema, be beneficial to the uniform distribution of cell, can significantly improve the quality of artificial skin.
Description of drawings
Accompanying drawing 1 is the structural representation of the cultivation box of the artificial skin culture apparatus of the present invention, and 1 is lid among the figure, and 2 is sealring; 3 is end box, and 4 is the gas piping interface, and 5 is the liquid line interface; 6 is reference column, and 7 is supporting leg, and end box and lid adopt the buckle structure sealing.Accompanying drawing 2 by the present invention design cultivate holder and the structural representation of setting up circle, 8 for setting up circle among the figure, 9 be pilot hole, 10 be the leg that turns up, 11 is that (sidewall) held in the palm in cultivation.Accompanying drawing 3 is the structural representation of the artificial skin culture apparatus of the present invention, and (three) cultivate box and adopt series system to connect.Accompanying drawing 4 is the another kind of structural representation of accompanying drawing 3, and (three) cultivate box and adopt parallel way to connect.
Embodiment
Below in conjunction with accompanying drawing technical scheme of the present invention is done further explain.Cultivation box among the present invention, cultivation are held in the palm and are set up circle and can select medical grade macromolecular material or metallic substance preparation.
Shown in accompanying drawing 1, cultivate box and constitute by cultivating end box and lid.Box internal surface bossed reference column in the end is used for the position that holder is cultivated in grappling; The length and width of end box adapts with the size of cultivating holder, and cultivating holder is 2 row's dischargings in end box, and every row can place 7 and cultivate holder; End box outer bottom has four supporting legs, highly is 5mm.Respectively there are upper and lower two pipe interfaces the box both sides end of at, make in the culturing room to be interlinked to the outside; Wherein the top is used for gaseous interchange for the gas piping interface, and its height will surpass the liquid level of cultivating nutrient solution in the end box; The below is used for the circulation of nutrient solution for the nutrient solution pipe interface, and its height should be lower than the liquid level of nutrient solution, is beneficial to the mobile of nutrient solution; Through these pipe interfaces, can adopt silicone tube with a plurality of cultivation boxes serial connection or and meet (referring to Fig. 3,4), make gas and liquid in the system can form the loop of circulation respectively, so that carry out the dynamic monitoring and the replacing of gas and nutrient solution.The sealing means of end box and lid can adopt buckle structure, and box is provided with the buckle structure that matches with the outside of lid the end of at, can be provided with sealring between it, to guarantee to cultivate the tight closure of end box and lid, avoids gas leakage.
Shown in accompanying drawing 2, cultivating the backing face has equally distributed through hole, and aperture 1mm obtains sufficient nutrient solution to guarantee artificial skin when cultivating; Cultivating the holder height is 5mm, the complete imperforation of sidewall; Outer bottom has four legs that turn up, and the central authorities at its cross break position have pilot hole, matches with described reference column in its aperture, makes to cultivate to hold in the palm and can accurately be anchored in the end box, is convenient to the accurate location of automated operation.Set up circle and be circular ring, its external diameter is complementary with cultivation holder internal diameter, and height is 5mm, can avoid the loss of cell suspension from the artificial skin surface, helps the uniform distribution of cell, improves the artificial skin quality of stability.
Shown in accompanying drawing 3 and 4, the artificial skin culture apparatus of the present invention is made up of control unit and cultivation unit, and control unit comprises computer-controlled gas control module, liquid control module, transmitter, gas mixing chamber, liquid storage room and control panel.Wherein gas mixing chamber is connected with the cultivation box respectively by pipeline through valve with liquid storage room, and (the dotted line pipeline is the gas communication pipeline among the figure; The solid line pipeline is the liquid communication pipeline); Some transmitters in the gas mixing chamber are connected with control panel through the gas control module through circuit, can realize mixing indoor gas (O in the culturing process
2, CO
2Deng) the round-robin dynamic monitoring; The indoor some transmitters of fluid storage are connected with control panel through the liquid control module through circuit, can realize nutrient solution in the storing chamber in the culturing process (glucose, temperature, pH value, oxyty) round-robin dynamic monitoring; Operation through control panel can realize setting, monitoring and adjustment to culturing process, and when appearance is unusual, can correct, and the nutrient solution of liquid storage room can carry out regularly changing automatically according to monitored results.
Wherein, the accessory of control unit can be by following type selecting, and control panel selects GP4000M series man-machine interface touch-screen, or 3.5 and 4 inches liquid crystal displays backlight and film key; Programmable logic controller (PLC) or 80c51 micro-chip that control module selects Omron, Mitsubishi or siemens etc. to produce; Device has SFC4000 (Sensirion) mass-flow gas meter/flow director in the gas mixing chamber, and the liquid control module connects the WX-10B miniature peristaltic pump.The transmitter of realizing dynamic monitoring has O
2Concentration inductor block (Japan produces the KE-25 oxygen concentration sensor), CO
2Concentration inductor block (GT03-1112-BZ); The XLFX-SBA-40C transmitter is selected in the glucose concn monitoring; Oxyty is selected QFOA-DH type series oxygen analyser transmitter; Liquid flow sensor selects LG1626 OEM (Sensirion), and pressure transmitter selects SDP1000 (Sensirion), and the pH transmitter selects 5510GST three combined electrodes; The potential of hydrogen indicator gauge selects the BPH-200A indicator gauge, and TP selects PT100 transmitter or assembled TP or sheet type TP (Switzerland Steiner Technik AG).
The method of use of the artificial skin culture apparatus of the present invention is: cultivating the nylon membrane of backing portion laying with its equal size; Again inoblast and collagen gel solution are mixed back injection cultivation holder, build-in is fixed mutually with reference column with pilot hole, makes to cultivate to hold in the palm accurately to be anchored in the cultivation end box; Sealing fastens lid; To cultivate box usefulness gas, liquid line connects respectively, can and connect or be connected in series, and be communicated with gas mixing chamber and nutrient solution storing chamber respectively;, and start to cultivate culturing process and condition setting at control panel.Between during cultivation, inoblast stretches the height and the area that cause culture gradually and shrinks, and culture is broken away from cultivate the holder inwall.Open the lid after accomplishing cultivation, adopt automatic punch device to the punching of culture surface, circle is set up in insertion in cultivating holder; Its bottom is closely contacted with artificial skin, inject epidermal cell suspension, avoid the loss of epidermic cell; Sealing fastens the lid continued and cultivates, and cultivates to artificial skin and accomplishes.In culturing process, the gas of cultivation box internal recycle and the component condition of nutrient solution (glucose, temperature, pH value, oxyty etc.) are carried out dynamic monitoring and adjustment.
Claims (5)
1. an artificial skin culture apparatus comprises and cultivates end box, lid and cultivate holder; It is characterized in that described artificial skin culture apparatus is made up of control unit and cultivation unit, described control unit comprises computer-controlled gas control module, liquid control module, transmitter, gas mixing chamber, liquid storage room and control panel; Described cultivation unit comprises the pipeline of cultivating end box, lid box with the cultivation holder and at the bottom of being connected some cultivations; Wherein gas mixing chamber is connected with cultivation end box respectively by pipeline through valve with liquid storage room; Some transmitters in the gas mixing chamber are connected with control panel through the gas control module through circuit, realize the dynamic monitoring to gas circulation in the mixing section in the culturing process; The indoor some transmitters of fluid storage are connected with control panel through the liquid control module through circuit, realize nutrient solution round-robin dynamic monitoring in the storing chamber in the culturing process; Operation through control panel can realize setting, monitoring and adjustment to culturing process.
2. artificial skin culture apparatus according to claim 1 is characterized in that, the inner bottom surface of box is provided with prominent shape reference column at the bottom of the described cultivation, is used for the position that holder is cultivated in grappling; The size of cultivating end box adapts with the size of cultivating holder, and cultivating holder is 1～4 row's discharging in end box, and every discharging is put 4～8 and cultivated holder; Box outer bottom of the said end is provided with four supporting legs, high 4mm～10mm; The box both sides are respectively equipped with the pipe interface of gas, liquid turnover the end of at, through pipeline with some cultivation boxes serial connections or and connect, make gas, liquid in the box form flow cycle respectively; Fasten sealing between end box and lid.
3. artificial skin culture apparatus according to claim 2 is characterized in that there is equally distributed through hole the bottom surface of described cultivation holder, and the aperture is 1mm～2mm; The height of cultivating in the holder is 4mm～10mm, the complete imperforation of sidewall; Cultivate the holder outer bottom and evenly be provided with at least three legs that turn up, central authorities have pilot hole at the cross break position of the leg that turns up, and the pilot hole reference column interior with cultivating end box cooperates to be inlayed; The total height of cultivating holder is lower than cultivates more than the end box inner height 4mm.
4. based on the described artificial skin culture apparatus of claim 3, it is characterized in that described cultivation is cultivated holder and is provided with it can be sealed and matched and sets up circle, inserting to cultivate increases the sidewall height in the holder, set up the high 2mm～10mm of circle.
5. by the method for use of the said artificial skin culture apparatus of claim 1 to 4, it is characterized in that, cultivating the nylon membrane of backing portion laying, celliferous gelating soln is injected cultivate holder with its identical size; To cultivate holder again with pilot hole on the leg and the embedding grappling mutually of cultivation end box reference column; Sealing fastens lid, will cultivate box usefulness gas, liquid line connects respectively, and is communicated with gas mixing chamber and nutrient solution storing chamber respectively;, and start to cultivate culturing process and condition setting at control panel; After stage cultivate to finish, open and cultivate the box lid, adopt automatic punch device to the punching of culture surface after, cover inserts and sets up circle in cultivating holder, injects cell suspension, sealing fastens the lid continued and cultivates, and cultivates completion to artificial skin.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103614295A (en) * | 2013-11-27 | 2014-03-05 | 绍兴振德医用敷料有限公司 | Epidermal cell enriching instrument and dynamic enriching method thereof |
| WO2015078137A1 (en) * | 2013-11-27 | 2015-06-04 | 鲁建国 | Epidermal cell collecting instrument and dynamic collecting method thereof |
| CN106591120A (en) * | 2015-10-19 | 2017-04-26 | 梁彦 | Incubation nourishing circulation system for easily necrotic tissues |
| CN111334430A (en) * | 2020-03-27 | 2020-06-26 | 济南磐升生物技术有限公司 | Double-layer artificial skin culture device and use method thereof |
| US11566215B2 (en) | 2016-08-27 | 2023-01-31 | 3D Biotek Llc | Bioreactor with scaffolds |
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| CN101691535A (en) * | 2009-10-14 | 2010-04-07 | 张光晟 | Culture box for producing tissue engineering skin |
| CN101985598A (en) * | 2010-11-08 | 2011-03-16 | 中国人民解放军第三军医大学第三附属医院 | Dynamic three-dimensional strain culture device for tissue-engineered skin |
| CN102277298A (en) * | 2011-07-04 | 2011-12-14 | 李胜蓝 | Culturing system of biological tissue engineering |
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| CN2565838Y (en) * | 2002-09-09 | 2003-08-13 | 上海组织工程研究与开发中心 | New type bioreactor of tissue engineering skin in vitro structure |
| CN1560225A (en) * | 2004-03-01 | 2005-01-05 | 华东理工大学 | Pouring type biologic reactor system in vitro constructure of dual active skin |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103614295A (en) * | 2013-11-27 | 2014-03-05 | 绍兴振德医用敷料有限公司 | Epidermal cell enriching instrument and dynamic enriching method thereof |
| CN103614295B (en) * | 2013-11-27 | 2014-07-16 | 绍兴振德医用敷料有限公司 | Epidermal cell enriching instrument and dynamic enriching method thereof |
| WO2015078137A1 (en) * | 2013-11-27 | 2015-06-04 | 鲁建国 | Epidermal cell collecting instrument and dynamic collecting method thereof |
| CN106591120A (en) * | 2015-10-19 | 2017-04-26 | 梁彦 | Incubation nourishing circulation system for easily necrotic tissues |
| CN106591120B (en) * | 2015-10-19 | 2018-10-02 | 梁彦 | A kind of incubation for easy slough nourishes the circulatory system |
| US11566215B2 (en) | 2016-08-27 | 2023-01-31 | 3D Biotek Llc | Bioreactor with scaffolds |
| US11926810B2 (en) | 2016-08-27 | 2024-03-12 | 3D Biotek, Llc | Bioreactor with scaffolds |
| CN111334430A (en) * | 2020-03-27 | 2020-06-26 | 济南磐升生物技术有限公司 | Double-layer artificial skin culture device and use method thereof |
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