CN101050418A - Bioreactor of artificial liver - Google Patents
Bioreactor of artificial liver Download PDFInfo
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- CN101050418A CN101050418A CN 200710020669 CN200710020669A CN101050418A CN 101050418 A CN101050418 A CN 101050418A CN 200710020669 CN200710020669 CN 200710020669 CN 200710020669 A CN200710020669 A CN 200710020669A CN 101050418 A CN101050418 A CN 101050418A
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Abstract
This invention relates to an artificial live bioreactor with nanofiber scaffold material as the culture medium for liver cells. A plasma inlet and an oxygen inlet are set at one end of the bioreactor hull, while a plasma outlet and an oxygen outlet are set at the other end. A nutrient solution inlet and an outlet are set at both ends of one side of the bioreactor hull, respectively. A culture medium composed of nanofiber scaffold and hollow fibers is set in the bioreactor hull. The nanofiber scaffold and the hollow fibers form coiled sandwich structure, with the nanofiber scaffold coiled into spiral drum and the hollow fibers encapsulated in the interlayer. The liver cell aggregates are adhered to the hollow fibers. An oxygen passage is set in the bioreactor to realize real-time oxygen supplement.
Description
Technical field
The present invention relates to a kind ofly, belong to the technical field of AISS with the bio-reactor that is used for the bioartificial liver of nano fiber scaffold material as liver cell culture matrix.
Background technology
Artificial liver is as effective assisting therapy technology widespread use in clinical of hepatic insufficiency, liver failure or relative disease, and the artificial liver of using clinically mainly comprises three classes now: abiotic type artificial liver, Biotype artificial liver, hybrid artificial liver.Wherein Biotype artificial liver curative effect in the clinical application of treatment hepatopathy patients is the most remarkable, and the liver cell of the general special finger artificial culture of Biotype artificial liver is the external biological reactive system of basic component.It not only has the specificity function of detoxification of liver, and has higher usefulness, as participating in energy metabolism, has the biosynthesizing transformation function, secretion hepatocyte growth-promoting active substance etc.The Biotype artificial liver has level of automation height, simple to operate, safe and reliable characteristics.The clinical effectiveness of its treatment hepatitis gravis shows that obvious effective rate is 36.7%, and efficient is 46.7%, and total effective rate is 83.3%.Be applied to clinical Biotype artificial liver at present and aspect cell cultures, have a lot of problems, such as low excessively 2～3 * 10 of the needed by human body that do not reach of the liver cell density of cultivating in the bio-reactor
10Cells/m
2Thereby, liver cell easily from culture medium come off cause liver cell to flow away with the round-robin nutrient solution and in bio-reactor skewness cause the artificial liver function reduction, and the liver cell death that oxygen causes for quantity not sufficient in the nutrient solution or the like.Following Biotype artificial liver is mainly being improved (Jung-Keug Park, Doo-Hoon Lee.Bioartificial LiverSystems:Current Status and Future Perspective.Journal ofBioscience and Bioengineering 2005.99 (4): 311-319.) aspect hepatocellular cultivation and the hepatocellular optimization.
Nano fiber scaffold is a kind of cell culture substrate of rising in recent years, Fibre diameter with huge specific surface area (porosity＞85%) and 10nm～10 μ m, the appearance structure of its weaving shape is similar to the intravital cell growth substrate-thiozell of vertebrates, helps sticking, grow and breeding of cell.There is data to show that this nano fiber scaffold helps the formation and the bioactive maintenance of spherule of liver cell spherule, its synthetic ability from albumen, urea of the liver cell of cultivation on nano fiber scaffold is all greater than the liver cell (Kian-Ngiap Chua, Wei-Seng Lima.Stable immobilization of rat hepatocytespheroidson galactosylated nanofiber scaffold.Biomaterials 2005.26:2537-2547) that utilizes traditional mode to cultivate.
The liver cell density of existing Biotype artificial liver is generally 5～8 * 10
8Cells/m
2Between, with the liver cell density 2～3 * 10 of people liver
10Cells/m
2Still there is a big difference, and therefore, existing artificial liver can not satisfy an adult's blood purification demand.And the liver cell spherule of existing Biotype artificial liver causes the minimizing of the even liver cell quantity of hepatocellular skewness along with flowing of nutrient solution easily comes off from cell matrix.Liver cell is in great demand to oxygen, but the prior biological artificial liver all has oxygen that insufficient shortcoming is provided, and causes hepatocellular biological activity to reduce even death.Above reason has a strong impact on the clinical usefulness of artificial liver.Also do not retrieve at present about utilizing nano fiber scaffold that bioreactor of artificial liver is carried out improved report or patent.
Summary of the invention
Technical problem: the object of the invention is to provide a kind of being beneficial to improve liver cell concentration and the hepatocellular bioreactor of artificial liver of immobilization.This bio-reactor has been realized hepatocellular tactophily in the bio-reactor, the cell losing issue that not only can avoid the liver cell suspension culture of traditional artificial liver to be brought, and the structure of the class organism thiozell of nano fiber non-woven fabric can promote hepatocellular breeding and growth, thereby can improve density and the hepatocellular biological activity of liver cell in bio-reactor to a great extent, the ability of synthetic albumin of enhance hepatocyte and secretion chromosin.
Technical scheme: the present invention is applied to bioreactor of artificial liver with nano fiber scaffold and by optimizing internal structure bio-reactor is improved, with the liver cell density that improves the Biotype artificial liver with increase the oxygen level of nutrient solution and improve liver cell distribution situation in bio-reactor, thereby improve the clinical usefulness of Biotype artificial liver.In improvement, introduce nano fiber scaffold at above problem the present invention to bioreactor of artificial liver, utilize nano fiber scaffold to be beneficial to the characteristics of cell adhesion, growth, can reach fixing liver cell and then improve the purpose of the liver cell density of Biotype artificial liver, and the formation that helps the circulation of cell culture fluid and promote hepatocellular breeding and spherule.Internal structure to bio-reactor is optimized design in addition, increases oxygen cathete, to improve hepatocellular surviving rate and biological activity.And then improve the ability that liver cell purifies the blood.
The structure of bioreactor of artificial liver of the present invention is: the end at the bio-reactor shell is provided with blood plasma import, oxygen inlet, the other end at the bio-reactor shell is provided with blood plasma outlet, oxygen outlet, be respectively equipped with nutritive medium import, nutritive medium outlet at two laterally of bio-reactor shell, be provided with the matrix of the cell cultures of forming by nano fiber scaffold, tubular fibre in the inside of bio-reactor shell; Form " sandwich " structure of curling between nano fiber scaffold and the tubular fibre, promptly nano fiber scaffold is curled into the drum of spiral, is provided with tubular fibre in the interlayer that curls, and the liver cell aggregate is attached to by the tubular fibre; Oxygen channel is set in bio-reactor, can realizes in good time oxygenation function.
The material of described nano fiber scaffold is one or more the mixture in alginates, poly-lysine, chitosan, agarose, the polyvinyl alcohol, and nonwoven fabric construct is rectangle.The Fibre diameter of described nano fiber scaffold between 30nm～400nm, porosity＞85%.5～10 at the every interval of the tubular fibre of arranging in the nano fiber scaffold is chosen one, according to said method with the tubular fibre selected passage, with the tubular fibre of remainder passage as blood plasma as oxygen.
Beneficial effect: this bio-reactor is introduced nano fiber scaffold first, realized hepatocellular tactophily in the bio-reactor, the cell losing issue that not only can avoid the liver cell suspension culture of traditional artificial liver to be brought, and the structure of the class organism thiozell of nano fiber non-woven fabric can promote hepatocellular breeding and growth, thereby can improve density and the hepatocellular biological activity of liver cell in bio-reactor to a great extent, the ability of synthetic albumin of enhance hepatocyte and secretion chromosin.At the big characteristics of hepatocellular cultivation oxygen requirement, in the design of bio-reactor, added logical oxygen facility, can realize the function of oxygen supply in reactor in good time.The modern design of this bio-reactor, being introduced in of nano fiber scaffold still belongs to the first both at home and abroad, and the preparation method all can realize technically.Can believe that this novel bio-reactor has broad application prospects at artificial liver and other field of cell culture.
Description of drawings
Fig. 1 is a bio-reactor square section synoptic diagram,
Fig. 2 is a bio-reactor profile synoptic diagram,
Have among the above figure: nutritive medium import 1, blood plasma import 2, oxygen inlet 3, nutritive medium outlet 4, blood plasma outlet 5, oxygen outlet 6, bio-reactor shell 7, nano fiber scaffold 8, tubular fibre 9, liver cell aggregate 10.
Embodiment
Bioreactor of artificial liver of the present invention is provided with blood plasma import 2, oxygen inlet 3 at an end of bio-reactor shell 7, the other end at bio-reactor shell 7 is provided with blood plasma outlet 5, oxygen outlet 6, be respectively equipped with nutritive medium import 1, nutritive medium outlet 4 at two laterally of bio-reactor shell 7, be provided with the matrix of the cell cultures of forming by nano fiber scaffold 8, tubular fibre 9 in the inside of bio-reactor shell 7; Form " sandwich " structure of curling between nano fiber scaffold 8 and the tubular fibre 9, promptly nano fiber scaffold 8 is curled into the drum of spiral, is provided with tubular fibre 9 in the interlayer that curls, and it is other that liver cell aggregate 10 is attached to tubular fibre 9; Oxygen channel is set in bio-reactor, can realizes in good time oxygenation function.
The subject matter that wherein faces is the problem of the selection and the design of bio-reactor internal structure of timbering material.The nano fiber scaffold material should have excellent biological compatibility and certain mechanical strength.For this reason, the material of preparation nano fiber scaffold selects to help high molecular polymers such as liver cell breeding, the alginates of growing, poly-lysine, chitosan, agarose, polyvinyl alcohol.The design of internal structure should be beneficial to the in good time oxygenation of cell nutrient solution and reduce operation easier in the concrete preparation process.
Utilize method of electrostatic spinning that above high molecular polymer is prepared into to have 30nm～the rectangle non-woven fabrics support of 400nm diameter fibers, the tubular fibre rule is arranged on the non-woven fabrics support, wherein the part tubular fibre as oxygen channel and other tubular fibres as the blood plasma passage, (see accompanying drawing) with being filled in the bio-reactor behind the curling parcel of the nano fiber scaffold tubular fibre, promptly make this new-type bioreactor.This bio-reactor is characterised in that introduces the matrix of nano fiber scaffold as cell cultures, these nano fiber scaffold starting material are alginates or high molecular polymers such as poly-lysine or chitosan or agarose or polyvinyl alcohol, Fibre diameter with 30nm～400nm, nonwoven fabric construct is rectangle; " sandwich " structure of curling that is rule between nano fiber scaffold and tubular fibre; Hollow fiber bundle is divided into two bundles, and is wherein a branch of as oxygen channel, in good time oxygenation in cell culture fluid; On the shell of reactor, leave each two of the import and export of blood plasma, oxygen, nutritive medium.
The present invention is implemented by the following method.Utilize electrostatic spinning technique with polymkeric substance (alginates, poly-lysine, chitosan, agarose, polyvinyl alcohol etc.) be prepared into the rectangle non-woven fabrics support of certain area, (spacing is at 0.5mm～2mm) with being arranged on the nano fiber non-woven fabric of polysulfone hollow fibre rule then, every 5～15 passages of selecting a tubular fibre as oxygen, the nano fiber scaffold that curls wraps up polysulfone hollow fibre, after be filled in the outer casing stand, on outer casing stand, leave oxygen channel, the blood plasma passage, each two in nutrient solution passage is respectively as import and outlet.This method comprises following process:
1. the preparation of nano fiber scaffold: select different solvents that polymkeric substance (alginates, poly-lysine, chitosan, agarose, polyvinyl alcohol etc.) dissolving is prepared into electrostatic spinning solution.Formulate different electrostatic spinning condition (voltage, acceptance distance, injection speed) according to the different properties of polymers soln, carry out spinning then, in spinning process, the shape and the thickness of nano fiber scaffold are regulated by the position of mobile susceptor and the time of control spinning.The nano fiber scaffold of differing materials is carried out corresponding modification and sterilization, be prepared into the nano fiber scaffold that can be positioned in the cell culture fluid as cell growth substrate.
2. the structure of tubular fibre and nano fiber scaffold design: the nano fiber non-woven fabric support is smooth, order polysulfone hollow fibre (the spacing 0.5mm～2mm) that arranges on support, the nano fiber scaffold that curls then is wrapped in its inside with tubular fibre, promptly makes the core component of bio-reactor.The passage that a tubular fibre is selected at 5～10 at every interval as supply oxygen, the tubular fibre with two ends is divided into two bundles in this way, a branch of passage as blood plasma, a branch of passage as oxygen.
3. the encapsulation of core component: step 2 kind of the core component that obtains with the encapsulation of a polypropylene shell, is left each two of the manhole appendixs of blood plasma, oxygen, nutrient solution on shell.The two intrafascicular hollow fibers at two ends are put into the access opening of blood plasma and oxygen in order respectively, and, promptly make this new-type bioreactor with medical glue encapsulation.
Example one: make this new-type bioreactor as the starting material of nano fiber scaffold with alginates.
1. the preparation of nano fiber scaffold: the preparation of (1) electrostatic spinning solution: sodium alginate is dissolved in to be prepared into concentration in the deionized water be mass ratio W
Sodium alginate: W
Deionized water=4% solution.Polyoxyethylene (Mw=900kD) is dissolved in to be prepared into concentration in the deionized water be mass ratio W
Polyoxyethylene: W
Deionized water=4% solution.Then with two kinds of solution V by volume
Sodium alginate soln: V
Polyethylene oxide solutions=8: 2 ratio mixes, and stirring at normal temperature 100min obtains the mixing solutions of sodium alginate and polyoxyethylene to mixing.V by volume then
Tensio-active agent: V
Mixing solutions=0.5% ratio adds the tensio-active agent triton x-100, continues to stir 10min.V by volume afterwards
Solubility promoter: V
Mixing solutions=10% ratio adds the solubility promoter methyl-sulphoxide.At room temperature continue to stir 60min.Promptly obtain homogeneous, stable light yellow spinning solution.(2) electrostatic spinning process prepares nano fiber scaffold: use whizzer with the spinning solution centrifugal 5min under the rotating speed of 5000rpm that obtains in the step (1), be injected into afterwards in the syringe (diameter is 20mm), syringe is fixed on the micro-injection pump, apart from syringe needle 15cm place fixedly aluminium foil as receptor.Apply the high pressure of 18kv on syringe needle, the syringe pump flow velocity is 1.0mL/h.Continuous mobile receiver in spinning process promptly obtains the tunica fibrosa of certain area, and its Fibre diameter is at 50nm～500nm.(3) modification of sodium alginate nano fiber support is handled: the nano-fiber material of gained in the step (2) is immersed in 10min in the dehydrated alcohol, and this material being immersed in concentration afterwards is mass ratio W
CaCl2: W
H2O=2% CaCl
2In the solution and continue 120min, then with deionized water rinsing 10min to remove residual CaCl
2Promptly make the sodium alginate nano fiber support.
2. the structure of tubular fibre and nano fiber scaffold in the core component: alginate nano fabric nonwoven cloth support is smooth, the polysulfone hollow fibre its spacing of arranging in proper order on support is 1mm, the nano fiber scaffold that curls then is wrapped in its inside with tubular fibre, form " sandwich " structure, promptly make the core component of bio-reactor.The passage that a tubular fibre is selected at 5～10 at every then interval as supply oxygen, the tubular fibre with two ends is divided into two bundles in this way, a branch of passage as blood plasma, a branch of passage as oxygen.
3. the encapsulation of core component: step 2 kind of the core component that obtains with the encapsulation of a polypropylene shell, is left each two of the manhole appendixs of blood plasma, oxygen, nutrient solution on shell.The two intrafascicular hollow fibers at two ends are put into the access opening of blood plasma and oxygen in order respectively, and, promptly make this new-type bioreactor with medical glue encapsulation
Example two: make this new-type bioreactor as the starting material of nano fiber scaffold with chitosan.
1. the preparation of nano fiber scaffold: the preparation of (1) electrostatic spinning solution: (Mw=190kD, Deacetylation=85%) being dissolved in mass ratio is that to be prepared into concentration in 5% the acetum be mass ratio W with chitosan
Chitosan: W
Acetum=2% solution.It is that to be prepared into concentration in 5% the acetum be mass ratio W that polyoxyethylene (Mw=900kD) is dissolved in mass ratio
Polyoxyethylene: W
Acetum=3% solution.Then with two kinds of solution V by volume
Chitosan solution: V
Polyethylene oxide solutions=9: 1 ratio mixes, and stirring at normal temperature 100min obtains the mixing solutions of chitosan and polyoxyethylene to mixing.V by volume then
Tensio-active agent: V
Mixing solutions=0.3% ratio adds the tensio-active agent triton x-100, continues to stir 10min.V by volume afterwards
Solubility promoter: V
Mixing solutions=10% ratio adds the solubility promoter dimethyl formamide.At room temperature continue to stir 60min.Promptly obtain homogeneous, stable spinning solution.(2) electrostatic spinning process prepares nano fiber scaffold: use whizzer with the spinning solution centrifugal 5min under the rotating speed of 5000rpm that obtains in the step (1), be injected into afterwards in the syringe (diameter is 20mm), syringe is fixed on the micro-injection pump, apart from syringe needle 18cm place fixedly aluminium foil as receptor.Apply the high pressure of 22kv on syringe needle, the syringe pump flow velocity is 1.5mL/h.Continuous mobile receiver in spinning process promptly obtains the tunica fibrosa of certain area, and its Fibre diameter is at 30nm～80nm.At room temperature make the nano fibrous membrane drying promptly make the sodium alginate nano fiber support.
2. the structure of tubular fibre and nano fiber scaffold in the core component: chitosan nano fiber non-woven fabrics support is smooth, the polysulfone hollow fibre its spacing of arranging in proper order on support is 1mm, the nano fiber scaffold that curls then is wrapped in its inside with tubular fibre, form " sandwich " structure, promptly make the core component of bio-reactor.The passage that a tubular fibre is selected at 5～10 at every then interval as supply oxygen, the tubular fibre with two ends is divided into two bundles in this way, a branch of passage as blood plasma, a branch of passage as oxygen.
3. the encapsulation of core component: step 2 kind of the core component that obtains with the encapsulation of a polypropylene shell, is left each two of the manhole appendixs of blood plasma, oxygen, nutrient solution on shell.The two intrafascicular hollow fibers at two ends are put into the access opening of blood plasma and oxygen in order respectively, and, promptly make this new-type bioreactor with medical glue encapsulation
Claims (4)
1. bioreactor of artificial liver, it is characterized in that: the end at bio-reactor shell (7) is provided with blood plasma import (2), oxygen inlet (3), the other end at bio-reactor shell (7) is provided with blood plasma outlet (5), oxygen outlet (6), be respectively equipped with nutritive medium import (1), nutritive medium outlet (4) at two laterally of bio-reactor shell (7), be provided with the matrix of the cell cultures of forming by nano fiber scaffold (8), tubular fibre (9) in the inside of bio-reactor shell (7); Form " sandwich " structure of curling between nano fiber scaffold (8) and the tubular fibre (9), it is the drum that nano fiber scaffold (8) is curled into spiral, be provided with tubular fibre (9) in the interlayer that curls, it is other that liver cell aggregate (10) is attached to tubular fibre (9); Oxygen channel is set in bio-reactor, can realizes in good time oxygenation function.
2. bioreactor of artificial liver according to claim 1, the material that it is characterized in that described nano fiber scaffold (8) is one or more the mixture in alginates, poly-lysine, chitosan, agarose, the polyvinyl alcohol, and nonwoven fabric construct is rectangle.
3. bioreactor of artificial liver according to claim 1 and 2, the Fibre diameter that it is characterized in that described nano fiber scaffold (8) between 30nm～400nm, porosity＞85%.
4. bioreactor of artificial liver according to claim 1, it is characterized in that 5～10 at the every interval of tubular fibre (9) of arranging in the nano fiber scaffold (8) is chosen one, according to said method with the tubular fibre (9) selected passage, with the tubular fibre (9) of remainder passage as blood plasma as oxygen.
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Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102188759A (en) * | 2011-03-11 | 2011-09-21 | 西安交通大学 | Liver tissue engineering scaffold for cell composite culture and preparation method thereof |
| CN102225222A (en) * | 2011-05-13 | 2011-10-26 | 浙江大学 | U-shaped hollow fiber pipe artificial liver bioreactor |
| CN101549181B (en) * | 2009-05-07 | 2011-11-02 | 浙江大学 | Nanometer fiber net sheet superposed type biological reactor |
| CN101559246B (en) * | 2009-05-26 | 2011-11-02 | 浙江大学 | Hybrid artificial liver support system equipped with nano fibre sheet stacked reactor |
| CN102258817A (en) * | 2011-05-13 | 2011-11-30 | 浙江大学 | Helical hollow fiber tube artificial liver bioreactor |
| CN102409020A (en) * | 2010-09-26 | 2012-04-11 | 上海泰因生物技术有限公司 | Non woven/polyester fiber carriers for culturing cells and use method thereof |
| WO2013007224A1 (en) | 2011-07-14 | 2013-01-17 | Primecell A.S. | Cell culture substrate and a method for producing thereof |
| US8530237B2 (en) | 2009-01-08 | 2013-09-10 | Hitachi, Ltd. | Method for culturing animal hepatocyte |
| CN101428154B (en) * | 2008-12-04 | 2013-10-30 | 浙江大学 | Implantation type artificial hepar |
| CN106222086A (en) * | 2016-07-29 | 2016-12-14 | 武汉仝干医疗科技股份有限公司 | Lint wire type bioartificial liver's reactor |
| CN109266549A (en) * | 2018-10-25 | 2019-01-25 | 江苏久腾医学科技有限公司 | A kind of immunocyte culture bracket |
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2007
- 2007-03-20 CN CN 200710020669 patent/CN101050418A/en active Pending
Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101428154B (en) * | 2008-12-04 | 2013-10-30 | 浙江大学 | Implantation type artificial hepar |
| US8530237B2 (en) | 2009-01-08 | 2013-09-10 | Hitachi, Ltd. | Method for culturing animal hepatocyte |
| CN102272288B (en) * | 2009-01-08 | 2015-03-11 | 株式会社日立制作所 | Method for culture of animal hepatocyte |
| CN101549181B (en) * | 2009-05-07 | 2011-11-02 | 浙江大学 | Nanometer fiber net sheet superposed type biological reactor |
| CN101559246B (en) * | 2009-05-26 | 2011-11-02 | 浙江大学 | Hybrid artificial liver support system equipped with nano fibre sheet stacked reactor |
| CN102409020A (en) * | 2010-09-26 | 2012-04-11 | 上海泰因生物技术有限公司 | Non woven/polyester fiber carriers for culturing cells and use method thereof |
| CN102188759A (en) * | 2011-03-11 | 2011-09-21 | 西安交通大学 | Liver tissue engineering scaffold for cell composite culture and preparation method thereof |
| CN102225222A (en) * | 2011-05-13 | 2011-10-26 | 浙江大学 | U-shaped hollow fiber pipe artificial liver bioreactor |
| CN102258817A (en) * | 2011-05-13 | 2011-11-30 | 浙江大学 | Helical hollow fiber tube artificial liver bioreactor |
| WO2013007224A1 (en) | 2011-07-14 | 2013-01-17 | Primecell A.S. | Cell culture substrate and a method for producing thereof |
| CN106222086A (en) * | 2016-07-29 | 2016-12-14 | 武汉仝干医疗科技股份有限公司 | Lint wire type bioartificial liver's reactor |
| CN109266549A (en) * | 2018-10-25 | 2019-01-25 | 江苏久腾医学科技有限公司 | A kind of immunocyte culture bracket |
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