CN104673875A - Osteochondral cell-based method for rapidly screening orthopaedic drugs - Google Patents
Osteochondral cell-based method for rapidly screening orthopaedic drugs Download PDFInfo
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Abstract
The invention provides an osteochondral cell-based method for rapidly screening orthopaedic drugs to relatively well simulate an environment in a human body and rapidly screen drugs. The target of rapidly screening the drugs is realized by the methods of obtaining a plurality of cells by spinner bottle culture, preparing a three-dimensional matrix, crosslinking the three-dimensional matrix, carrying out three-dimensional culture by adding drugs, and detecting and screening various drugs. The cell growth states at different drug concentrations can be obtained by three-dimensional cell culture; a plurality of experimental data are provided for late-stage clinical trials; the availability and toxicity of the drugs are relatively rapidly judged; the pathogen contamination can be relatively easily prevented and treated in the drug screening process; the cell growth sate and the cell reaction can be observed for a long period of time; and data support is provided for side effects in late drug treatment.
Description
Technical field
The present invention relates to a kind of orthopaedics medicine rapid screening method based on bone chondrocyte, be specifically related to the application of the method in orthopaedics medicine primary dcreening operation, belong to technical field of pharmaceutical biotechnology.
Background technology
Current drug screening all adopts high flux screening, result due to high flux screening acquisition is the Activity Results of molecular level or cell levels, in most cases can only reflect the information of the mechanism of action of sample, can not prove to have preventive and therapeutic effect to certain disease completely, this is the important difference of itself and conventional screening methods.Traditional pharmaceutical research is generally first study its object effect, and then research mechanism of action.High-throughput screening method is adopted to be then that this process is also known as making reverse pharmacology from molecular level.High-throughput screening method discovery and developing drugs is adopted generally to have following several step.
Primary dcreening operation and multiple sieve are at molecule or cell-based screening sample, prove that a certain sample has pharmacologically active (or avidity) to this target spot.After primary dcreening operation, select the activated compound of tool, adopt series concentration, carry out the multiple sieve of same model, illustrate its action character to this target spot, action intensity and dose-effect relationship, find active compound (sample) thus.
Deeply screening is on the basis of primary dcreening operation and multiple sieve, by the sample obtained, adopts the molecule different but relevant from primary dcreening operation, cell model does further to screen, comprise selectivity, the cytotoxicity of proof sample, and other character.Through deeply screening, for the pharmaceutical use evaluating active compound more all sidedly provides experimental data more fully.According to these data, and the chemical structure of binding activities compound, character feature, comprehensively analyze, determine the compound in structure and effect with novelty and Development volue, as lead compound.Simultaneously also can conjunctive tissue organ or whole animal model, prove its pharmacological action, for sample provides experimental basis more fully.After obtaining lead compound, composition optimizes (also can directly develop as drug candidate compound according to data) is carried out according to practical situation, namely the transformation of compound structure is carried out, to obtain the active substance that activity is higher, shortcoming is less.
The lead compound that confirmation screening obtains deep screening or the best compound of the activity that is selected of Dai Huahou carry out wider and deeper study, comprise many-sided screenings such as pharmacological action, drug metabolism processes, general toxicity, to determine its DEVELOPMENT PROSPECT.Satisfactory sample is defined as drug candidate compound, enters development research program, i.e. preclinical study, for clinical study prepares necessary data.
Therefore, high-throughput screening method has the features such as range of choice is wide, screening cost is lower, reliable results as the means of new drug discovery, is the important means of new drug research.By high-flux medicaments sifting method, can find effective, clinical value is high, has the original new drug of independent intellectual property rights.But high-flux cell horizontal screen choosing method can not simulation tissue environment, all cells reaction is in same level, can not reflect at human body change procedure.
To be the Introduction To Cn Patent of CN101626781 a kind of prepares the method with anti-tumor immune response cell mass for publication number, comprise and tumour and mononuclearcell are placed in three dimentional cell cultivation equipment co-cultivation, and from the culture obtained, be separated the cell mass that amplification has immune response.The invention also discloses the cell mass with anti-tumor immune response adopting present method to obtain and the test kit containing described cell mass.
Publication number a kind of Three-dimensional cell culture that has been the Introduction To Cn Patent of CN101448932, comprises and being incorporated in three dimensional support matrix by PECTORAL LIMB SKELETON, and allow that described cell vitro differentiation in described upholder matrix is adipocyte.Described matrix can be collagen stroma.Present method may be used for the growth studying stem cell, or for studying adipocyte to the response stimulated.Process provides such system, have thus and can grow in vitro with the adipocyte of those the similar biological characteristics in body.
Publication number a kind of Three-dimensional cell culture plate that has been the Introduction To Cn Patent of CN203429183U, it comprises Growth of Cells plate and liquid storage plate, and described Growth of Cells plate is installed on the top of described liquid storage plate; The lower surface of described Growth of Cells plate is provided with growth hole, and the upper surface of described liquid storage plate is provided with liquid storage hole, and described growth hole and described liquid storage hole cooperatively interact; Be provided with sample holes and ring-type isolation channel in described growth hole, described sample holes is positioned at the center in described growth hole and runs through wherein, and described ring-type isolation channel is positioned at the periphery of described sample holes.Three-dimensional cell culture plate of the present invention, has with low cost, simple operation and other advantages, can be conveniently used in being inverted in culture method carrying out Three-dimensional cell culture.
Publication number a kind of full-automatic three-dimensional cell culture system that has been the Introduction To Cn Patent of 203559059U, comprises pedestal, power supply adaptor, culture dish; Described pedestal is built with rotation motor; Described power supply adaptor is provided with tachometer gage; Described culture dish is culture plate or cultivates tubing string, and culture plate or cultivation tubing string dorsal part are equipped with silica gel gas-exchange membrane; Described culture dish is contained on pedestal; Described pedestal is connected with power supply adaptor.Cell or tissue in the present invention is suspend with the state of freely falling body in process of growth, there is no the destructive pressure such as agitator, bubble, therefore be organized in nutrient solution and be able to free-falling, upset fully mixing with nutrient solution, in its container, the strength of all directions reaches balance, so cell or tissue can not be subject to the strength impact of single direction, can towards any direction homoepitaxial, increase cell proliferation rate, reduce the system of necrocytosis and effectively increase cellular products secretion, solve the restriction that the interior life (ingrowth) of Three-dimensional cell culture is not enough.
Publication number a kind of method and device preparing bone marrow mesenchymal stem cells-tube scaffold compound that be the Introduction To Cn Patent of CN102188752A, comprise: (1) cell is inoculated: after supplying the pipe support of cell stereoscopic culture sterile-processed, draw the 3rd generation mesenchymal stem cells MSCs cell suspension, evenly drop to pipe support outside surface, to be absorbed completely after pipe support is placed in syringe, negative pressure-pumping 2 ~ 5 times; (2) static cultivation: postvaccinal pipe support is placed in cell culture apparatus, static cultivation 20 ~ 30 hours in interpolation stem cell medium; (3) perfusion is cultivated: pipe support is placed in perfusion culture tank, described perfusion culture tank internal diameter and described pipe support external diameter match, shaft core position is provided with the solid cylinder matched with described pipe support centre gangway, pipe support is positioned in the cavity between cylinder and outer wall, and cavity bottom has the Porous Base for nutrient solution circulation; In perfusion culture tank, pass into stem cell medium, under 0.1 ~ 0.3mL/min flow velocity, perfusion is cultivated 10 ~ 15 days, prepares described bone marrow mesenchymal stem cells-tube scaffold compound.This inventive method improves the inoculation efficiency of mesenchymal stem cells MSCs in PCL pipe support, and improve cell being uniformly distributed and amplification ability in pipe support outer wall, profit can obtain mesenchymal stem cells MSCs evenly and the compound pipeline complex pipeline support of high-density distribution in this way, is expected to be applied to the reparation of pipeline tissue injury clinically.
Publication number a kind of Three-dimensional cell culture support that has been the Introduction To Cn Patent of CN102935246A and its preparation method and application, comprising: solid-phase calcium phosphate content is the preparation of the slip of 50 ~ 55% (weightmeasurement ratios); The preparation of poly (methyl methacrylate) micro-sphere; The preparation of porous calcium phosphate support; The sodium alginate hydrochloric acid soln that mass percentage is 4 ~ 7% put into by porous calcium phosphate support step 3 prepared, and vacuum soaks 1-2h, with ultrapure water repetitive scrubbing three times, to obtain final product.It is simple that this support has preparation, the advantage such as convenient operation and transport.The present invention also provides the preparation method and application of this dimensional culture support.
The publication number one that has been the Introduction To Cn Patent of CN102517211A can dissociative type three-dimensional support for cell culture and preparation method fast.For raw material with carboxymethyl modified chitosan, in the phosphoric acid salt being dissolved in pH=5 ~ 8 or 2-(N-morpholino) ethanesulfonic acid buffer, then the dithio diprotic acid linking agent of carbodiimide activation is added, form the crosslinked hydrogel of three dimensional chemical by the amidate action in amino in cm-chitosan and linking agent between carboxyl, obtain three-dimensional porous carrier finally by lyophilize.Disulfide linkage in this solid support material by the rapid reduction fracture of its reductive agent, can realize porous support and dissociates fast and dissolve, and can meet multiple external Three-dimensional cell culture and cultivate the specific demand terminating rear needs cell-vector material and be effectively separated.Chitosan-based hydrogel three-dimensional cell culture vector of the present invention has that raw material is easy to get, technique is simple, easy bioactive modification, and the advantage such as carrier physico-chemical property, mechanical property and degradation rate be controlled, have a good application prospect in various Three-dimensional cell culture.
The preparation method of the publication number a kind of halfcystine of the L for Allergic skin test/golden nanometer particle composite membrane cell sensor that has been the Introduction To Cn Patent of CN103018298A and application thereof, modify on gold electrode by galvanic deposit and self-assembling method by nanometer gold and Cys material; Adopt P815 mastocyte as sensor information, utilize mouse tail type i collagen to build collagen/cell 3D training mode, and be inoculated into the structure that modified electrode completes cell sensor; Finally be applied to the detection of sensitinogen standard substance C48/80, be good linear relationship in concentration range in 0.5 ~ 3 μ g/mL, detect and be limited to 0.13 μ g/mL.The inventive method achieves identification, the conduction to allergen reference material precisely, efficiently, rapidly and detects, and overcome the defect of traditional detection method, gained sensor production is simple, and cost is low, in detection anaphylactogen, have good application prospect.
The method of publication number a kind of highly efficient gas permeable devices that has been the Introduction To Cn Patent of CN101611132 and culturing cell, comprises and uses ventilative culturing room, to reduce the use in space, maintain uniform culture condition simultaneously, and is more suitable for automated fluid operation.They comprise gas permeable material are integrated into traditional multilayer form to solve the uneven problem of culture condition.They comprise culture apparatus, and it uses the surface be made up of gas permeable material, is filled with the silicone of plasma body, and integrated traditional attaching surface, those surfaces be such as made up of traditional polystyrene through tissue culture treated.They comprise the culture apparatus being integrated with ventilative transparent liquid film.The multiple advantage of such generation, the culture condition more optimized during comprising amplification, and to storage space, incubator space and the process more effective utilization in space.Further, workload and Pollution risk can be reduced.
The method of publication number a kind of high-performance device that has been the Introduction To Cn Patent of CN101611133 and culturing cell comprises and uses ventilative culturing room, to reduce the use in space, maintains uniform culture condition simultaneously, and is more suitable for automated fluid operation.They comprise gas permeable material are integrated into traditional multilayer form to solve the uneven problem of culture condition.They comprise culture apparatus, and it uses the surface be made up of gas permeable material, is filled with the silicone of plasma body, and integrated traditional attaching surface, those surfaces be such as made up of traditional polystyrene through tissue culture treated.They comprise the culture apparatus being integrated with ventilative transparent liquid film.The multiple advantage of such generation, the culture condition more optimized during comprising amplification, and to storage space, incubator space and the process more effective utilization in space, workload and Pollution risk can also be reduced.
Publication number a kind of bioartificial liver's reactor based on double-deck nitrocellulose filter perfusion type that has been the Introduction To Cn Patent of CN102166380A, comprise casing and be arranged on the cytoskeleton in cabinets cavity, wherein, casing is arranged fluid inlet, liquid outlet, inlet mouth, venting port; Described cytoskeleton is double-deck nitrocellulose filter, two long limits of this double-deck nitrocellulose filter are close on the front side of casing and the inwall of rear side, its two ends are fixed on the inwall of the arranged on left and right sides of casing, cabinets cavity is separated into upper and lower two separate space, nutrient solution flows and carries out exchange of substance in upper and lower two separate space; The intermembranous clearance space of described double-deck nitrocellulose is as hepatocellular three dimensional growth space, cell grows in clearance space, when can avoid liquid-flow while carrying out sufficient mass transfer, shearing force is to the injury of cell, also reaches the effect that immunity deadens simultaneously.
The publication number three-dimensional space cell culture system that to be the Introduction To Cn Patent of CN101007999 a kind of can see thoroughly and the application in histocyte and newborn organ culture thereof, comprise: container, containing cell culture medium and Three-dimensional cell culture unit in container, described Three-dimensional cell culture unit contains implantable cell and is beneficial to cell adhesion and long term growth, differentiation, ripe cavity.Described three-dimensional space is cultivated unit and can thoroughly be seen, the sticking of dynamic observation cell, expands stretch, divide a word with a hyphen at the end of a line, hyperplasia, differentiation, maturation, aging, death and develop formative tissue or organ.System of the present invention can be used for long-term cultured and produces a large amount of cell as the seed cell of organizational project, also can form regenerating tissues and micro-organs in a short time for transplantation treatment.In addition, present system is that the in vitro study of tumour provides unique environment, can help diagnosing tumor, observe neoplasm invasiveness and transfer, screening antineoplastic drugs.Also for the various cell of isolated culture, and the gene program for studying cell inherence changes and the impact etc. of extracellular environment change on cell.
The construction process of publication number a kind of human amniotic membrane epithelial cell that has been the Introduction To Cn Patent of CN103045535A and silk fibroin bracket complex body, it comprises the steps: S1: the separation and Culture of amniotic epithelial cells: the amnion getting hepatitis B surface antigen and the equal negative cesarian section Post operation acquisition of HIV antibody detection, size is 15 × 15cm about, be separated, remove remaining chorion, PBS rinses repeatedly, until wash most courageous and upright liquid, amnion is shredded into diameter and be about the little broken of about 1-2mm, pour in cover glass ware, add 0.25% pancreatin and each 15ml of EDTA respectively, 37 DEG C of digestion 7-8 minute, filter through 200 mesh filter screens after diluting with PBS liquid 100ml, add in filtrate simultaneously and stop digestion containing 5%FBS nutrient solution, the centrifugal 6min collecting cell of 1000rpm, be seeded in the culture dish containing the RPMI1640 of 10%FBS, in 37 DEG C containing 5%CO
2incubator in cultivate.S2: the purifying of primary cell: carry out purifying to after primary cell separation and Culture to 7 day, (1) adds Ara-C in nutrient solution, and activity is 10-6mol/L, and action time is 36h.(2) change fresh culture to continue to cultivate 1d.(3) again in culture dish, add Ara-C, activity is 10-7mol/L, and action time is 12h.(4) change fresh culture to continue to cultivate 1d.(5) abandon substratum, D-Hank cleans cell 2 times, and 0.25% trysinization 5min, FCS stop digestion, the centrifugal 5min of 800R/min, and after abandoning supernatant, full substratum continues to cultivate.S3: the qualification of primary cell: after getting the continuation of the cell after above-mentioned purifying cultivation 3d, with trysinization, PBS liquid adjustment cell concn is 1 × 106/ml, add following mouse anti-human monoclonal's antibody respectively: CD29-FITC, CD34-FITC, CD44-PE, CD45-PE, CD73-PE, CD90PE, CD105-FITC, CD106-PE, CD166-FITC, HLA-DR-PE, put 4 DEG C and hatch 30min, PBS washes 2 times, directly carries out flow cytometry analysis.S4: the structure of amnion cell silk fibroin bracket complex body: the amniotic epithelial cells adjustment concentration by above-mentioned vitro culture and after purifying is 1 × 106/ml, cell suspension is dropped in silk fibroin bracket that sterilising treatment crosses, put into that 24 well culture plates are unsettled hatches 4 hours, fully be adsorbed on after on support until cell and add the full substratum of 10%FBS+RPMI1640 again, make the complete submergence of cell, 2 to 3 day half amount changes liquid, carries out the adherent and growing state of observation of cell under inverted microscope every day.S5: scanning electron microscopic observation: ten day taking-up scaffold complex sample is put into 2.5% glutaraldehyde and is fixed, after PBS rinses, and conventional dehydration, the growing state of critical point drying metal spraying observation of cell under scanning electron microscope.
Publication number a kind of cells in vitro stereoscopic culture system that has been the Introduction To Cn Patent of CN103589637A, comprise plane culture dish, it is characterized in that: described culture dish internal surface is coated with bio-matrix, the inoculation of bio-matrix upper strata has sustenticular cell, and the inoculation of sustenticular cell upper strata has object cell.The invention solves the solid space problem of modelling of cell injuring model, object cell can be grown being similar in the environment in its body, maintain the fidelity of its Morphology and structure, thus be conducive to the reliability of its external various test.
Publication number a kind of electrical stimulation module cell culture apparatus that has been the Introduction To Cn Patent of CN103849565A, described device comprises: electrostatic spinning electrode 1, culture dish substrate 2, Electrical excitability cell 3, electrical stimulation module 4, electrode connecting line 5.Structure of the present invention is simple, can add electricity irritation while based on skeleton stereoscopic culture, and improving can the training quality of Electrical excitability cell.
Publication number a kind of artificial liver fiber mat superposed type biological reactor that has been the Introduction To Cn Patent of CN201418905, comprise shell of reactor, the tubular fibre mesh sheet of some longitudinally superpositions is arranged in shell of reactor, tubular fibre mesh sheet is formed by woven hollow fiber, the tubular fibre of some vertical layouts passes the net center of a lattice of said hollow fiber mat, and described tubular fibre is polysulfone hollow fibre; Cell suspension entrance and liquid inlet are established in the bottom of shell of reactor, and top establishes cell suspension to export and liquid exit, and sidewall subtend arranges gas inlet and pneumatic outlet.The present invention possess 3 D stereo cultivate structure, have oxygenate concurrently and immune barriers function, cytoactive and function are good, be easy to amplify.
A kind of 3 D stereo of the Introduction To Cn Patent of CN1563364 to be cultivated and the method for inducing bone mesenchymal stem cell chondroblast, comprise the steps:: (1), by mesenchymal stem cells MSCs suspension culture in the suspension culture device presetting substratum and microcarrier, under agitation increase mesenchymal stem cells MSCs; Said substratum is the α-MEM containing 2% ~ 20% volume percent foetal calf serum; Said microcarrier is Cytodex3 microcarrier; (2) then chondroblast inducing culture is added in the culture apparatus of the Cytodex3 microcarrier containing results and the mesenchymal stem cells MSCs grown thereon, the induction of agglomerate chondroblast is carried out to mesenchymal stem cells MSCs; Chondroblast inducing culture, based on DMEM, adds Regular Insulin, the transforming growth factor-beta 3 of 1 ~ 20ng/ml, the dexamethasone of 10 ~ 150nmol/L, the ascorbic acid phosphoric acid esters of 10 ~ 100mg/ml, the proline(Pro) of 10 ~ 100mg/ml, the Sodium.alpha.-ketopropionate of 0.5 ~ 5mmol/L that concentration is 1 ~ 10 μ g/ml.The method of this invention can once obtain a large amount of chondrocytes at short notice, reduces pollution youngster and leads, decrease cell injury, be conducive to the transmission of iuntercellular nutritive substance.Directly this mixture can be implanted in body after having induced and carry out cartilage defect repair experiment or provide sufficient seed cell source for scientific research.
The quick method of publication number a kind of separation and purification embryo and brain neural stem cell that has been the Introduction To Cn Patent of CN103031271A, use thermal conversion polymkeric substance NIPA and polyoxyethylene glycol to carry out 3 D stereo cultivation to neurocyte, utilize neural stem cell can breed in gel environment and the characteristic forming neural ball reaches the object of the separation and purification to neural stem cell.The neural ball warp immunofluorescence label formed measures, and shows that the cell of about 93% is that Nestin is positive, illustrates that this method separation and purification of nerve stem cell is simply effective.Embryo neural stem cells through this method separation and purification can be differentiated to form neurone, oligodendrocyte and neurogliocyte in vitro.
Publication number has been the Introduction To Cn Patent of CN103849566A, and a kind of Electrical excitability cell culture processes comprises: electrostatic spinning electrode 1, culture dish substrate 2, Electrical excitability cell 3, electrical stimulation module 4, electrode connecting line 5.Structure of the present invention is simple, can add electricity irritation while based on skeleton stereoscopic culture, and improving can the training quality of Electrical excitability cell.
Publication number a kind of perforated brick type filling support type reactor used in artificial liver that has been the Introduction To Cn Patent of CN201418901, comprises shell of reactor; Shell of reactor inside is divided into three cabins by the division board of two densely covered grids: from bottom to top for oxygenate cabin, filling bracket cabin, immunity intercept cabin.The present invention adopts unique porous brick fixed bed as inside reactor main body, miniature separate unit is become by elongated hole post not connected in fixed bed segmentation inside reactor, reactor inner chamber is broken the whole up into parts, reduces dead space, dead space, liquid flowing resistance, make inside reactor liquid stream even.Solve support rack type reactor and lack immunity isolation, oxygen for insufficient problem, make up again the defect that hollow fiber type reactor Growth of Cells insufficient space, cell adhesion are poor, lack 3 D stereo growing environment, easily block semi-permeable membranes.Have oxygenate concurrently, immunity intercepts, 3 D stereo cultivates function, over-all properties is powerful, and manufacture craft is simple, cost is low, be easy to amplification.
The method of publication number to be the Introduction To Cn Patent of CN101092606 a kind of neural stem cell 3 D stereo cultivates amplification in vitro, comprising: the porous microcarrier selecting to have three-dimensional environment; Pre-treatment is carried out to microcarrier, it is characterized in that: it is further comprising the steps of: the DMEM/F12 neural stem cell serum-free medium bag of the Prostatropin (bFGF) containing 40-60ng/ml, 40-60ng/ml epithelical cell growth factor (EGF) and B27 by above-mentioned microcarrier, makes the fissional Prostatropin of promotion and epithelical cell growth factor permeate equably in microcarrier by (1); (2) the above-mentioned microcarrier handled well is put into Tissue Culture Flask, add above-mentioned neural stem cell serum-free medium, then 1 × 105-1 × 106 neural stem cell is added at culturing bottle, after mixing, be filled with the carbon dioxide containing 5% concentration again, and cultivate under the constant temperature of about 37 DEG C, according to the proliferative conditions of cell, within every 5-7 days, change neural stem cell serum-free medium; (3) taken out from culturing bottle by the microcarrier covering with neural stem cell, put into containing 0.05% tryptic D-hank ' s damping fluid, at room temperature digest 10-30 minute, remove microcarrier, rinsing cell, obtains neural stem cell.Compared with traditional cultural method, porous microcarrier contributes to enlarged culturing area, Prostatropin and epithelical cell growth factor facilitate the multiple fission of cell, improve the microenvironment of cells survival, be conducive to cell proliferation of nerve cord division, reach the object of neural stem cell amplification in vitro.
Publication number a kind of perforated brick type filling support type reactor used in artificial liver that has been the Introduction To Cn Patent of CN101549179, comprises shell of reactor; Shell of reactor inside is divided into three cabins by the division board of two densely covered grids: from bottom to top for oxygenate cabin, filling bracket cabin, immunity intercept cabin.The present invention adopts unique porous brick fixed bed as inside reactor main body, miniature separate unit is become by elongated hole post not connected in fixed bed segmentation inside reactor, reactor inner chamber is broken the whole up into parts, reduce dead space, dead space, liquid flowing resistance, make inside reactor liquid stream even.Both solved support rack type reactor and lacked immunity isolation, oxygen for insufficient problem, make up again hollow fiber type reactor Growth of Cells insufficient space, cell adhesion is poor, lacks 3 D stereo growing environment, easily the defect of blocking semi-permeable membranes.Have oxygenate concurrently, immunity intercepts, 3 D stereo cultivates function, over-all properties is powerful, and manufacture craft is simple, cost is low, be easy to amplification.
From current documents and materials, at home and abroad main with mechanically resistant material as electrostatic spinning electrode, microcarrier etc. provide stereoscopic culture carrier, but human body environment is mainly formed with soft material, human internal environment can not be simulated, greatly slow down the speed of drug screening, also reduce the accuracy of drug screening.
Summary of the invention
In drug screening process, medicine directly mixes with cell, all cells reaction is consistent, but body metabolism process Chinese traditional medicine is unlikely in each cell peripheral concentration, each cell response is not identical yet, in order to better simulate human internal environment, observes medicine to the impact of people's cell, there is provided abundant experimental data for clinical, the invention provides a kind of orthopaedics medicine rapid screening method.
The technical solution used in the present invention comprises: get that spinner culture obtains a large amount of human marrow mesenchymal stem cell (hBMSCs), three-dimensional matrix preparation, three-dimensional matrix carry out being cross-linked, add medicine carries out three-dimensional cultivation, detects the various orthopaedics medicine of screening.
Therefore, the invention provides the orthopaedics medicine rapid screening method based on bone chondrocyte, its concrete steps comprise:
(1) spinner culture obtains a large amount of cell: adopt stem cell conventional culture methods to obtain the nutrient solution containing a large amount of human marrow mesenchymal stem cell;
(2) three-dimensional matrix is prepared and is cross-linked: the 1.2% thiolated hyaluronic acid aqueous solution getting 100mL puts into 300mL culturing bottle, add the nutrient solution 50mL that step (1) obtains, 2-4min is left standstill after mixing, add the 15% high-molecular weight polyethylene glycol diacrylate aqueous solution of 50mL again, pour into immediately after carrying out Homogeneous phase mixing in 6 orifice plates, 28-32 DEG C of standing 12-20min, obtains orthopaedics drug screening matrix;
(3) orthopaedics drug screening Establishing: the various medicines adding different concns in orthopaedics drug screening matrix, at the CO of 37 DEG C
2cultivate in incubator;
(4) microscopy observation of cell state: when cultivating with the various medicines of different concns, after 2h, directly the human marrow mesenchymal stem cell of cultivation is put into inverted microscope observe, determine various medicine cell growth state under different concns, the validity of the various medicine of rapid screening and toxicity.
The described 15% high-molecular weight polyethylene glycol diacrylate aqueous solution and the 1.2% thiolated hyaluronic acid aqueous solution, refer to mass volume ratio, the 15% high-molecular weight polyethylene glycol diacrylate aqueous solution is obtained with 15g high-molecular weight polyethylene glycol diacrylate 100mLPBS solubilize, 2.4g thiolated hyaluronic acid 200mLPBS solubilize is obtained the 1.2% thiolated hyaluronic acid aqueous solution, and above-mentioned various solution is through the degerming acquisition sterile solution of membrane filtration.
Described PBS solution refers to sodium chloride nacl 0.9g, potassium primary phosphate KH
2pO
40.024g, Sodium phosphate dibasic Na
2hPO
40.164g, sodium bicarbonate NaHCO
30.3g water is made into 100mL.
Described medicine refers to the water-soluble or fat-soluble medicines such as small organic molecule, microbiotic, polypeptide protein class medicine.
Accompanying drawing illustrates:
Fig. 1. the shaping rear outward appearance of orthopaedics medicine rapid screening substrate material;
Fig. 2 .hBMSCs matrix material aspect graph under 100X times of opticmicroscope;
3 weeks 150X times of electron-microscope scanning figure cultivated by Fig. 3 .hBMSCs matrix material;
3 weeks 1500X times of electron-microscope scanning figure cultivated by Fig. 4 .hBMSCs matrix material.
Technique effect:
1, cell growth state under different pharmaceutical concentration can be obtained by three-dimensional cell cultures, for later phase clinical provides lot of experimental data, judge validity and the toxicity of medicine more fast.
2, breakneck acceleration is fast, with short production cycle, and equipment is simple, and floor space is few, can save human and material resources etc., be convenient to factorial praluction.
3, this cell culture processes more can simulate human internal environment, and rely on macromolecular being cross-linked to provide stereoscopic-state completely, cell can make full use of again the hyaluronic acid in being cross-linked simultaneously, and method is simple, and production cost is low, can be mass, and using value is high.
4, more easily prevent and treat pathogen contamination in drug screening process, the long-time observation of cell growth conditions of energy and cell response, for side effect during later stage pharmacological agent provides Data support.
Embodiment
Below, the present invention will be further detailed by embodiment, but it is not limited to any one or similar example of these embodiments.
Embodiment 1: human marrow mesenchymal stem cell (hBMSCs) separation and ientification
After collecting marrow blood with the centrifuge tube containing 0.25%EDTA anti-freezing liquid, density gradient centrifugation is adopted to be separated hBMSCs.Get 15mL centrifuge tube, lower floor adds Ficoll parting liquid, and upper strata slowly adds marrow blood gently, makes the two have obvious layering, Ficoll parting liquid: marrow blood=3: 4.Centrifugal: 400g, 40min, speed rises soon falls slowly.Divide 4 layers after centrifugal, middle cloud floating layer is stem cell, carefully draws to another 15mL centrifuge tube with 1mL trace rifle.Centrifugal: 1200 turns/min, 5min.Abandon supernatant, add appropriate DMEM, blow and beat into single cell suspension.Be dispensed into 25cm2 culturing bottle, add containing 10% foetal calf serum low sugar DMEM nutrient solution to 6mL.HBMSCs cultivates with containing 10% foetal calf serum low sugar DMEM nutrient solution, at the bottom of growing to bottle about 80% full time, abandon nutrient solution, wash twice with PBS, add appropriate 0.25% containing EDTA tryptic digestion, after 3 minutes, Microscopic observation cell feeler is retracted and dropping situations, add and stop digestion containing blood serum medium, be transferred to 15mL centrifuge tube, centrifugal 5 minutes, rotating speed 1200 revs/min.Abandon supernatant, add and be about 6mL containing serum DMEM, under blowing and beating about 50-100 gently, make it to become single cell suspension, be filled to new culturing bottle by 1: 3 point, supply nutrient solution and complete and go down to posterity.Get forth generation hBMSCs as experimental cell.
Embodiment 2: the preparation of stereoscopic culture matrix
15g high-molecular weight polyethylene glycol diacrylate (PEGDA) is used 100mLPBS solubilize, form 15% aqueous solution, by 2.4g thiolated hyaluronic acid 200mLPBS solubilize, get 100mL thiolated hyaluronic acid solution, put into 300mL culturing bottle, add hBMSCs forth generation cell culture fluid 50mL, 2-4min is left standstill after mixing, add the 15% high-molecular weight polyethylene glycol diacrylate aqueous solution of 50mL again, pour into immediately after carrying out Homogeneous phase mixing in 6 orifice plates, 28-32 DEG C of standing 12-20min, obtains orthopaedics drug screening matrix, sees Fig. 1.
Embodiment 3: utilize stereoscopic culture cell screening medicine
By embodiment 2 obtain orthopaedics drug screening matrix, add the chlorogenic acid of different concns wherein, at the CO of 37 DEG C
2cultivate in incubator, after 2h, directly the human marrow mesenchymal stem cell of cultivation is put into inverted microscope observe, determine various medicine cell growth state under different concns, the validity of the various medicine of rapid screening and toxicity, be shown in Fig. 2, Fig. 3.
Claims (4)
1. the invention provides the orthopaedics medicine rapid screening method based on bone chondrocyte, its concrete steps comprise:
(1) spinner culture obtains a large amount of cell: adopt stem cell conventional culture methods to obtain the nutrient solution containing a large amount of human marrow mesenchymal stem cell;
(2) three-dimensional matrix is prepared and is cross-linked: the 1.2% thiolated hyaluronic acid aqueous solution getting 100mL puts into 300mL culturing bottle, add the nutrient solution 50mL that step (1) obtains, 2-4min is left standstill after mixing, add the 15% high-molecular weight polyethylene glycol diacrylate aqueous solution of 50mL again, pour into immediately after carrying out Homogeneous phase mixing in 6 orifice plates, 28-32 DEG C of standing 12-20min, obtains orthopaedics drug screening matrix;
(3) orthopaedics drug screening Establishing: the various medicines adding different concns in orthopaedics drug screening matrix, at the CO of 37 DEG C
2cultivate in incubator;
(4) microscopy observation of cell state: when cultivating with the various medicines of different concns, after 2h, directly the human marrow mesenchymal stem cell of cultivation is put into inverted microscope observe, determine various medicine cell growth state under different concns, the validity of the various medicine of rapid screening and toxicity.
2. method according to claim 1, the described 15% high-molecular weight polyethylene glycol diacrylate aqueous solution and the 1.2% thiolated hyaluronic acid aqueous solution, refer to mass volume ratio, the 15% high-molecular weight polyethylene glycol diacrylate aqueous solution is obtained with 15g high-molecular weight polyethylene glycol diacrylate 100mLPBS solubilize, 2.4g thiolated hyaluronic acid 200mLPBS solubilize is obtained the 1.2% thiolated hyaluronic acid aqueous solution, and above-mentioned various solution is through the degerming acquisition sterile solution of membrane filtration.
3. method according to claim 1, described PBS solution refers to sodium chloride nacl 0.9g, potassium primary phosphate KH
2pO
40.024g, Sodium phosphate dibasic Na
2hPO
40.164g, sodium bicarbonate NaHCO
30.3g water is made into 100mL.
4. method according to claim 1, described medicine refers to the water-soluble or fat-soluble medicines such as small organic molecule, microbiotic, polypeptide protein class medicine.
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