CN101724557B - Polysaccharide composite stent perfusion type liver cell reactor system for use in medicament-induced hepatotoxicity evaluation - Google Patents
Polysaccharide composite stent perfusion type liver cell reactor system for use in medicament-induced hepatotoxicity evaluation Download PDFInfo
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- CN101724557B CN101724557B CN2009102291091A CN200910229109A CN101724557B CN 101724557 B CN101724557 B CN 101724557B CN 2009102291091 A CN2009102291091 A CN 2009102291091A CN 200910229109 A CN200910229109 A CN 200910229109A CN 101724557 B CN101724557 B CN 101724557B
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- 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
- C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
- C12M25/16—Particles; Beads; Granular material; Encapsulation
Abstract
The invention discloses a polysaccharide composite stent perfusion type liver cell reactor system for use in medicament-induced hepatotoxicity evaluation. The structure of the reactor system comprises a polysaccharide composite stent immobilized bed reactor, wherein the upper port of the reactor is connected with a defoamer which is synchronously connected with perfusion culture solution and a cell inoculator through accesses; and the lower port of the reactor is connected to a liquid tank through a pipe and a peristaltic pump, and the liquid tank is connected to defoamer through a pipe and a regulating valve to form perfusion culture solution circulating circuit. In the invention, the small-sized perfusion type cell reactor is designed and manufactured by using three-dimensional porous cell culture tents prepared by compounding pectin and alginate, the interaction between the cell and the matrix and the interaction between cells are improved, the defoamer is used to dilute the inoculated cells to uniformly disperse the inoculated cells in the three-dimensional porous tents in the reactor, so simple three-dimensional dynamic inoculation of cells in the reactor is realized; and the physiological functions and the medicament-induced hepatotoxicity expression level of cells cultured in vitro are improved obviously.
Description
[technical field]: the present invention relates to a kind of is the small hepatocytes reactor assembly of carrier with three-dimensional pectin/Lalgine complex stephanoporate bracket, is used to realize external liver cell high-density culture and promotes to cultivate hepatocellular use in medicament-induced hepatotoxicity express.
[background technology]: the cell biological reactor assembly can be biologically active substances such as cultured cells, tissue the Three-Dimensional Dynamic microenvironment is provided; Through signal factors such as mechanics, biology and chemistry in the external regulation and control culture environment; Improve physiological condition in exchange of substance, the analogue body, promote to realize the 3 d function cultivation of cell.In view of liver is realization material and an energy exchange organ maximum in the human body; It has complicated cell and extracellular matrix is formed; And complicated blood vessel, gallbladder tube structure, the liver cell bio-reactor has become external three-dimensional high-density and has cultivated the important means that liver cell, inducing function hepatic tissue are built again.Study novel liver cell reactor system and develop its applied research and become current research focus in the liver tissue engineering field
[1,2]
In recent years, the reactor drum of following several kinds of forms has successively appearred in the liver tissue engineering research field.1. rolling bottle formula reactor drum, it rotates through glass in the culture apparatus or magnetic agitation and exchange of substance in the improvement system, and equipment is simple, easy to operate, but nutrient solution is prone to produce eddy current and influences the cell growth
[3]2. rotating and culturing tube wall formula reactor drum; Can the cultured tissue cell be placed in the culture tank of filling nutrient solution, drive culture tank and rotate along transverse axis, rotating speed can make cell tissue move the centripetal force and the gravitational equilibrium of generation when suitable; Reach the microgravity culture effect; It reduces, and destructive stress produces, the promotion exchange of substance, but equipment and aseptic technique are complicated, still locate fundamental research
[4]3. tubular fibre cell reactor; Select the semi-permeable membranes tubular fibre of perviousness to constitute by having, exocoel is respectively cell and nutrient solution in the fiber, has increased cell cultures area, volume; Improve cell culture density; The exchange of substance of improvement system also is the principal reaction device form of current bioartificial livers, but because the accumulation of the machinery of lumen inner cell; Lack the interaction between good cell-cell, cell-extracellular matrix, the long-term BA of cell is kept and is still waiting further raising
[5]4. immobilization bed perfusion type reactor drum; All nutrient solution will slowly pass through three-dimensional stent material; Can promote the exchange of substance between system inner cell and nutrient solution; And can suitably adjust the hydromeehanics token stimulus through regulating flow velocity, can't evenly pour into, cause the difficulty that cellular form and function can't long term maintenance but this type of reactor drum of present majority all faces cell because of three-dimensional stent material lacks the liver cell pathoklisis
[6]
Particularly in recent years, in the medicament research and development process, follow the understanding to the chemical structure of potential drug target spot, endogenic ligand and substrate, the researchist can screen precursor for new drug provides in a large number.Traditional animal vivo test labor manpower financial capacity, experimental period is long, and the vitro culture liver cell has begun to be used for drug screening
[7,8]The small hepatocytes bio-reactor has can reduce liver cell and drug dose to be detected, and realizes characteristics such as high flux screening, and the application in medicament-induced hepatotoxicity evaluation also more and more causes different field investigator's concern.The small hepatocytes bio-reactor mainly concentrates on the scale down of traditional biological reactor drum at present
[9,10]
Make a general survey of the development of liver cell bio-reactor, various liver cell bio-reactors are all attempted to be beneficial to external hepatic tissue structural remodeling with proximate environment in one different of form constructions and the body, realize the structure of external functionalization hepatic tissue and substituting of liver function.Present result of study shows that all the three-dimensional uniform distribution of liver cell bio-reactor ubiquity reactor drum inner cell, hepatic tissue structure induce bottleneck problems such as reconstruction and cell culture recycling also not to be well solved.
[summary of the invention]: the present invention seeks to overcome the above-mentioned deficiency that prior art exists, provide a kind of medicament-induced hepatotoxicity evaluation with polysaccharide composite stent perfusion type liver cell reactor system.
The present invention is directed to shortcomings such as current liver cell reactor system cytoactive is poor, complicated operation, experimental cost height; Utilize the three-dimensional porous rack of pectin and the compound preparation of Lalgine to design and produce small-sized perfusion type cell reactor system; Improved cell-matrix and cell-intercellular interaction; And, realized reactor drum inner cell Three-Dimensional Dynamic inoculation easy and simple to handle through froth breaker dilution inoculating cell; Hepatocellular physiological function of vitro culture and use in medicament-induced hepatotoxicity expression level have been significantly improved.
Medicament-induced hepatotoxicity evaluation provided by the invention comprises with the formation of polysaccharide composite stent perfusion type liver cell reactor system: polysaccharide composite stent immobilization bed bioreactor; The last interface of reactor drum connects froth breaker, and froth breaker connects perfusion nutrient solution and cell inoculation device simultaneously through access port; The lower interface of reactor drum inserts liquid storage tank through pipeline and peristaltic pump, and liquid storage tank inserts froth breaker through pipeline and variable valve again, forms perfusion nutrient solution circulation loop.
Curing bed in the said reactor drum is processed by three-dimensional porous cell culturing bracket, and natural polysaccharide pectin and sodium-alginate that three-dimensional porous cell culturing bracket selects for use main chain to contain the semi-lactosi group are timbering material, through lyophilize with polyvalent cation is crosslinked processes.The natural polysaccharide pectin wherein and the mass ratio of sodium-alginate are 1: (0.5～5).
The natural polysaccharide pectin in the described three-dimensional porous cell culturing bracket and the gross weight concentration expressed in percentage by volume of sodium-alginate are 1～3%.
Described polyvalent cation can be calcium, copper, barium, magnesium, iron, mn ion.
Described polysaccharide composite stent immobilization bed bioreactor base adopts commercial membrane filter base, and itself and inner and outer sealing ring have been realized the fixing and nutrient solution uniform distribution of three-dimensional porous rack in the reactor drum.
Advantage of the present invention and positively effect:
One; The present invention has access port and is used to connect the cell inoculation device on froth breaker; Inoculating cell injects froth breaker; It at first through dilution of perfusion nutrient solution and the rapid reactor drum that gets into, has realized reactor drum inner cell Three-Dimensional Dynamic inoculation easy and simple to handle, and the result shows that cell can be uniformly distributed in the three-dimensional porous rack in the reactor drum.
Two; The present invention is through the specific receptors-ligand interaction of semi-lactosi group in the timbering material pectin and hepatic parenchymal cells surface asialoglycoprotein acceptor; Improve the inoculum density of support inner cell; And improve the cell-matrix interphase interaction, and and then promote cell aggregation to form cell aggregation, improve the hepatocyte functions such as urea synthesis, liver toxicity expression of cell; The compound mechanical property that significantly improves support of while sodium-alginate and pectin.Reported first of the present invention uses pectin/alginates compound rest to realize hepatocellular Three-Dimensional Dynamic cultivation.
Three; But related major parts is transparent autoclave sterilization material among the present invention; Easy handling and observation, reusable, greatly reduce experimental cost; Utilize liver cell reactor system of the present invention to investigate the drug toxicity of PARACETAMOL BP98, set up simple and easy medicament-induced hepatotoxicity evaluation and used the liver cell model.
[description of drawings]:
Fig. 1 is that medicament-induced hepatotoxicity evaluation is used the polysaccharide composite stent perfusion type liver cell reactor structural representation;
Fig. 2 is alginates (A) and the middle liver cell form of pectin/alginates compound rest (B) in the phase microscope observing response device;
Fig. 3 is the cell inoculation method is cultivated liver cell urea synthesis function to Three-Dimensional Dynamic influence;
Fig. 4 is that the hepatocellular urea synthesis function of the dynamic cultivation of reactor drum of the present invention is significantly higher than static cultivation;
Fig. 5 is that mtt assay detects the liver toxicity evaluation that reactor drum of the present invention is dynamically cultivated hepatocellular PARACETAMOL BP98.
Among Fig. 1,1 inoculator, 2 froth breakers, 3 reactor drums, 3-1 inner seal ring, the three-dimensional porous polysaccharide compound cells of 3-2 support, 3-3 exterior seal ring, 4 liquid storage tanks, 5 peristaltic pumps, 6 variable valve.
[embodiment]:
Embodiment 1:
As shown in Figure 1; Medicament-induced hepatotoxicity evaluation provided by the invention comprises with the formation of polysaccharide composite stent perfusion type liver cell reactor system: reactor drum 3; The last interface of reactor drum 3 connects froth breaker 2, and froth breaker connects perfusion nutrient solution and cell inoculation device 1 simultaneously through the access port of top, after inoculating cell injects froth breaker 2; At first, realized reactor drum inner cell Three-Dimensional Dynamic inoculation easy and simple to handle through dilution of perfusion nutrient solution and rapid the entering in the reactor drum 3; The lower interface of reactor drum 3 inserts liquid storage tank 4 through pipeline and peristaltic pump 5, and liquid storage tank 4 inserts froth breaker 2 through pipeline and variable valve 6 again, forms perfusion nutrient solution circulation loop.
Reactor base of the present invention adopts commercial membrane filter base, and it has realized that with the inner and outer sealing ring that increases the uniform distribution and the reactor drum of liquid in the reactor drum are airtight; The froth breaker that interface connects on the reactor drum is connected with the cell inoculation device with the perfusion nutrient solution simultaneously; But the total system building block is autoclave sterilization all, and connects into the recycle system through silicone tube, places CO2gas incubator to use.
Embodiment 2:
The concrete formation of reactor drum of the present invention such as embodiment 1; Curing bed in the said reactor drum 3 is made up of three-dimensional porous cell culturing bracket; Natural polysaccharide pectin and sodium-alginate that three-dimensional porous cell culturing bracket selects for use main chain to contain the semi-lactosi group are timbering material, through lyophilize with divalent cation is crosslinked processes.The present invention with three-dimensional porous cell culturing bracket serve as solidify bed designed and produced can parallel connection volume be 5 milliliters small-sized curing bed perfusion type liver cell reactor system (Fig. 1).
During the listed numerical value of the natural polysaccharide pectin wherein and the mass ratio of sodium-alginate and its total weight percent concentration such as table 1; Crosslinked through-20 ℃ of lyophilizes and divalent cation calcium; Make the support aperture all at the 150-220 mu m range; Porosity is more than 90%, and the mass ratio of pectin and sodium-alginate and total weight percent concentration are to made supporting structure and physicochemical property influence there are no significant difference; It is used for the liver cell dimensional culture, and the result shows that support inner cell adherence rate significantly improves, and the dynamic cellular rate of vaccination is higher than 75%, dynamically cultivates 24 h observation results in the reactor drum and shows that most of liver cell has formed cell aggregation (Fig. 2) in the support.
Table 1 is the composite three-dimensional porous support preparation condition of polysaccharide
| |
1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 |
| Pectin and sodium- |
2∶1 | 1∶1 | 1∶3 | 1∶5 | 2∶1 | 1∶1 | 1∶3 | 1∶5 | 2∶1 | 1∶1 | 1∶3 | 1∶5 |
| Pectin sodium-alginate bulking |
1 | 1 | 1 | 1 | 2 | 2 | 2 | 2 | 3 | 3 | 3 | 3 |
Embodiment 3:
Three-dimensional porous cell culturing bracket with preparation among the embodiment 2 among the present invention serves as to solidify a cell reactor system (Fig. 1) that makes to be respectively applied for sound attitude cell inoculation subsequent dynamic culture experiment; Investigation result to the urea synthesis function of expressing one of index as the hepatocellular liver function of vitro culture shows: more static cell inoculation, dynamically the liver cell urea synthesis function of constant current speed (2ml/min) cell inoculation subsequent dynamic cultivation significantly raise (Fig. 3); And this reactor drum is dynamically cultivated hepatocellular urea synthesis function and is about 2.5 times (Fig. 4) that three-dimensional static is cultivated.
Embodiment 4:
The small hepatocytes reactor assembly that the present invention is designed and produced is used for the use in medicament-induced hepatotoxicity of acetyl aminophenol to be investigated, and promptly adds 0uM in the nutrient solution, 5uM, and the PARACETAMOL BP98 of 10uM concentration, perfusion was cultivated after 24 hours, detected hepatocellular activity with MTT.The result shows: compare with traditional two-dimensional collagen, three-dimensional rack static cultivation, the dynamic culture system of reactor drum of the present invention can significantly improve the susceptibility (Fig. 5) of liver cell to preceding drug toxicity PARACETAMOL BP98.
Reference
1.G.Catapano，J.C.Gerlach.Bioreactor?for?liver?tissue?engineering.Topics?in?TissueEngineering，2007，(3)：1-48.
2.Salman?R.K.，Sangeeta?N.B.Microscale?culture?of?human?liver?cells?for?drug?development.Nat.Biotechnol.2008，26(1)：120-126.
3.Yu?CB，Lv?GL，Pan?XP，Chen?YS，et?al.In?vitro?large-scale?cultivation?and?evaluation?ofmicroencapsulated?immortalized?human?hepatocytes(HepLL)in?roller?bottles.Int?J?ArtifOrgans.2009，32(5)：272-281.
4.Chang?TT，Hughes-Fulford?M.Monolayer?and?spheroid?culture?of?human?liverhepatocellular?carcinoma?cell?line?cells?demonstrate?distinct?global?gene?expression?patternsand?functional?phenotypes.Tissue?Eng?Part?A.2009，15(3)：559-567.
5.Park?JK，Lee?DH.Bioartificial?liver?systems：current?status?and?future?perspective.J?Biosciand?Bioeng，2005，99(4)：311-319.
6.Cheng-Bo?Yu，Xiao-Ping?Pan，Lan-Juan?Li.Progress?in?bioreactors?of?bioartificial?livers.Hepatobiliary?Pancreat?Dis?Int.2009，8(2)：134-140.
7.Gómez-Lechón?MJ，Castell?JV，Donato?MT，et?al.Hepatocytes-the?choice?to?investigate?drugmetabolism?and?toxicity?in?man：in?vitro?variability?as?a?reflection?of?in?vivo.Chem?BiolInteract，2007，168(1)：30-50.
8.Guillouzo?A，Guguen-Guillouzo?C.Evolving?concepts?in?liver?tissue?modeling?andimplications?for?in?vitro?toxicology.Expert?Opin?Drug?Metab?Toxicol，2008，4(10)：1279-1294.
9.Stephanie?Schmitmeier，Angelika?Langsch，Wolfgang?Schmidt-Heck，et?al.Improvement?ofmetabolic?performance?of?primary?hepatocytes?in?hyperoxic?cultures?by?vitamin?C?in?a?novelsmall-scale?bioreactor.Journal?of?Membrane?Science，2007，298：30-40.
10.Shvartsman?I，Dvir?T，Harel-Adar?T，et?al.Perfusion?cell?seeding?and?cultivation?inducethe?assembly?of?thick?and?functional?hepatocellular?tissue-like?construct.Tissue?Eng?Part?A，2009，15(4)：751-760.
Claims (3)
1. a medicament-induced hepatotoxicity evaluation is used polysaccharide composite stent perfusion type liver cell reactor system; The formation of this perfusion type liver cell reactor system comprises: polysaccharide composite stent immobilization bed bioreactor; The lower interface of reactor drum inserts liquid storage tank through pipeline and peristaltic pump; Liquid storage tank inserts froth breaker through pipeline and variable valve again, it is characterized in that, the last interface of reactor drum connects froth breaker; Froth breaker connects perfusion nutrient solution and cell inoculation device simultaneously through access port, forms perfusion nutrient solution circulation loop;
Curing bed in the described reactor drum is propped up by three-dimensional porous liver cell pathoklisis and is configured to; Natural polysaccharide pectin and sodium-alginate that described three-dimensional porous liver cell pathoklisis support selects for use main chain to contain the semi-lactosi group are timbering material, through lyophilize with polyvalent cation is crosslinked processes;
The natural polysaccharide pectin in the described three-dimensional porous liver cell pathoklisis support and the mass ratio of sodium-alginate are 1: (0.5～5);
The natural polysaccharide pectin in the described three-dimensional porous liver cell pathoklisis support and the gross weight concentration expressed in percentage by volume of sodium-alginate are 1～3%.
2. medicament-induced hepatotoxicity evaluation according to claim 1 is characterized in that with polysaccharide composite stent perfusion type liver cell reactor system described polyvalent cation is: calcium, copper, barium, magnesium, iron or mn ion.
3. medicament-induced hepatotoxicity evaluation according to claim 1 is with polysaccharide composite stent perfusion type liver cell reactor system; It is characterized in that reactor base adopts commercial membrane filter base, itself and inner and outer sealing ring have been realized the fixing and nutrient solution uniform distribution of three-dimensional porous liver cell pathoklisis support in the reactor drum.
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| CN102174397B (en) * | 2011-03-07 | 2013-08-07 | 四川大学 | Bionic three-dimensional fluid shear stress cell culture device and shear stress loading method thereof |
| CN106680053A (en) * | 2017-01-04 | 2017-05-17 | 浙江大学 | Tissue treatment fluid circulation system |
| CN108588006A (en) * | 2018-05-10 | 2018-09-28 | 华东理工大学 | A kind of biological support and its preparation method and application for liver cell dimensional culture |
| CN109337863A (en) * | 2018-10-25 | 2019-02-15 | 南京鼓楼医院 | A kind of external model construction method that predictive compound is viral to fatty liver |
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| CN2761164Y (en) * | 2004-12-30 | 2006-03-01 | 中国科学院电工研究所 | Biological reactor |
| CN2817763Y (en) * | 2005-03-18 | 2006-09-20 | 中国人民解放军第三军医大学第一附属医院 | Artificial hepar tissue biological reactor |
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