CN104774903A - Application of three-dimensional culture cell in screening of orthopaedic drugs - Google Patents
Application of three-dimensional culture cell in screening of orthopaedic drugs Download PDFInfo
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Abstract
To rapid apply a cell culture matrix simulating the internal environment of a human body to screening of orthopaedic drugs, the invention provides a three-dimensional culture medium and realizes the purpose of rapid drug screening through spinner cultivation to obtain considerable human mesenchymal stem cells, preparation of a three-dimensional culture matrix, crosslinking of the three-dimensional culture matrix, addition of drugs for three-dimensional culture and detection and screening of a variety of drugs. Through three-dimensional cell culture, the growth states of cells under different drug concentrations can be acquired, so a great amount of experimental data is provided for later clinical practice, and validity and toxicity of drugs can be more rapidly determined. Control of pathogen pollution in the process of drug screening is more easily realized; cell growth states and cell reaction can be observed for a long time; and data support is provided for analysis of side-effects in later drug therapy.
Description
Technical field
The present invention relates to a kind of method and screening matrix thereof of three-dimensional culturing cell, be specifically related to the application of the method in orthopaedics drug screening, belong to technical field of pharmaceutical biotechnology.
Background technology
Mass cell culture technique refers under artificial condition (setting ph, temperature, dissolved oxygen etc.), in the technology of bioreactor middle-high density mass propgation cell for the production of biological products.Current large scale culturing cell mainly adopts spinner culture system and stainless cylinder of steel culture systems.There is following problem in spinner culture: its finite surface area, and cell density is low; During cultivation, monitor and forecast envrionment conditions (pH, dissolved oxygen etc.) is unstable, affects quality product; Labour intensity is large, and take up room large, pollution rate is high.Stainless cylinder of steel is cultivated exists following problem: need complicated incumbent firms (CIP) or sterilizing in place (SIP) pipeline, the possibility of crossed contamination between technical process runs; Every production batch setup time is long, and producers' labour intensity is large; Stirring system shearing force is large, and cell injury is serious.These cells carry out drug screening, and direct and co-culture of cells, causes pericellular drug level completely the same.And in drug screening process, be all that cell and the medicine Dual culture adopting these cultural methods to obtain screens at first, can practical situation are medicines being by penetrating into human body, making different pericellular drug level difference.
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.
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 described in the utility model, 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 utility model 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.
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 the small shreds that diameter is about 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 utility model 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 probability, decreases 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 utility model 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.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 three-dimensional culturing cell method and the application in drug screening process.
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 medicine of screening.
Therefore, the invention provides the application of three-dimensional culturing cell in orthopaedics drug screening, 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, obtain human marrow mesenchymal stem cell through 800 ~ 1000rpm low-speed centrifugal 5-10min;
(2) three-dimensional matrix preparation and crosslinked: get the human marrow mesenchymal stem cell 10mL that step (1) obtains and put into 300mL culturing bottle, add the 1.2% thiolated hyaluronic acid aqueous solution of 100mL again, the 15% high-molecular weight polyethylene glycol diacrylate aqueous solution of 50mL is added again after stirring 5-10min, pour into immediately after carrying out Homogeneous phase mixing in 6 orifice plates, 28-32 DEG C of standing 12-20min, must contain the three-dimensional developing medium of human marrow mesenchymal stem cell;
(3) with medicine three-dimensional Dual culture: add the cell culture fluid containing different concns medicine in 6 orifice plates, at the CO of 37 DEG C
2cultivate in incubator;
(4) 6 orifice plates cultivated directly are put into inverted microscope and are observed, determine various medicine cell growth state under different concns by microscopy observation of cell state: when cultivating with the medicine of different concns after 2h;
(5) screening of medicaments Establishing: the concentration determining various medicine according to fractographic cell growth state, the 1.2% thiolated hyaluronic acid aqueous solution getting 200mL puts into 500mL culturing bottle, add the stem cell 10mL that step (1) obtains, be uniformly mixed 5-10min, the 15% high-molecular weight polyethylene glycol diacrylate aqueous solution of 100mL, pour into immediately after carrying out Homogeneous phase mixing in 96 orifice plates, 30 DEG C of standing 15min, add various medicine at the CO of 37 DEG C in 96 orifice plates
2cultivate in incubator, after 2h, directly 96 orifice plates cultivated are put into inverted microscope observe, 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.High-molecular weight polyethylene glycol diacrylate and thiolated hyaluronic acid can be bought from Guangzhou grace Ke peptide Pharmaceutical Technology Co., Ltd.
Described PBS solution refers to sodium chloride nacl 0.4g, potassium primary phosphate KH
2pO
40.024g, Sodium phosphate dibasic Na
2hPO
40.164g, potassium chloride (KCl) 0.01g, sodium bicarbonate NaHCO
30.3g water is made into 100mL.
The nutrient solution of described human marrow mesenchymal stem cell adopts low sugar DMEM substratum.The cell culture fluid preparation of different concns medicine: low sugar DMEM substratum and different pharmaceutical proportioning combine and obtain.
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. outward appearance after extracellular matrix 3D forming materials;
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) is separated
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 25cm
2culturing bottle, adds containing 10% foetal calf serum low sugar DMEM nutrient solution to 6mL.
Embodiment 2: the cultivation of human marrow mesenchymal stem cell (hBMSCs) with go down to posterity
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, divide by 1:3 and be filled to new culturing bottle, supply nutrient solution and complete and go down to posterity.Get forth generation hBMSCs as experimental cell, passage cell nutrient solution obtains human marrow mesenchymal stem cell through 800 ~ 1000rpm low-speed centrifugal 5-10min.
Embodiment 3: 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 stem cell 10mL, be uniformly mixed 8min, 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 20min, is cross-linked to form three-dimensional cell culture substrate gradually, sees Fig. 1.
Embodiment 4: utilize stereoscopic culture cell screening medicine
By embodiment 3 obtain the three-dimensional cell culture substrate of 6 orifice plates, add the low sugar DMEM cell culture fluid containing different concns chlorogenic acid wherein, at the CO of 37 DEG C
2cultivate in incubator, after 2h, directly 6 orifice plates cultivated are put into inverted microscope observe, determine various medicine cell growth state under different concns, see Fig. 2, Fig. 3.The concentration of various medicine is determined according to fractographic cell growth state, the 1.2% thiolated hyaluronic acid aqueous solution getting 200mL puts into 500mL culturing bottle, add the stem cell 10mL that embodiment 2 obtains, be uniformly mixed 5min, the 15% high-molecular weight polyethylene glycol diacrylate aqueous solution of 100mL, pour into immediately after carrying out Homogeneous phase mixing in 96 orifice plates, 30 DEG C of standing 15min, in 96 orifice plates, add various medicine at the CO of 37 DEG C
2cultivate in incubator, after 2h, directly 96 orifice plates cultivated are put into inverted microscope observe, the validity of the various medicine of rapid screening and toxicity.
Claims (4)
1. the invention provides the application of three-dimensional culturing cell in orthopaedics drug screening, 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, obtain human marrow mesenchymal stem cell through 800 ~ 1000rpm low-speed centrifugal 5-10min;
(2) three-dimensional matrix preparation and crosslinked: get the human marrow mesenchymal stem cell 10mL that step (1) obtains and put into 300mL culturing bottle, add the 1.2% thiolated hyaluronic acid aqueous solution of 100mL again, the 15% high-molecular weight polyethylene glycol diacrylate aqueous solution of 50mL is added again after stirring 5-10min, pour into immediately after carrying out Homogeneous phase mixing in 6 orifice plates, 28-32 DEG C of standing 12-20min, must contain the three-dimensional developing medium of human marrow mesenchymal stem cell;
(3) with medicine three-dimensional Dual culture: add the cell culture fluid containing different concns medicine in 6 orifice plates, at the CO of 37 DEG C
2cultivate in incubator;
(4) 6 orifice plates cultivated directly are put into inverted microscope and are observed, determine various medicine cell growth state under different concns by microscopy observation of cell state: when cultivating with the medicine of different concns after 2h;
(5) screening of medicaments Establishing: the concentration determining various medicine according to fractographic cell growth state, the 1.2% thiolated hyaluronic acid aqueous solution getting 200mL puts into 500mL culturing bottle, add the stem cell 10mL that step (1) obtains, be uniformly mixed 5-10min, the 15% high-molecular weight polyethylene glycol diacrylate aqueous solution of 100mL, pour into immediately after carrying out Homogeneous phase mixing in 96 orifice plates, 30 DEG C of standing 15min, add various medicine at the CO of 37 DEG C in 96 orifice plates
2cultivate in incubator, after 2h, directly 96 orifice plates cultivated are put into inverted microscope observe, 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 0.4g, potassium primary phosphate KH
2pO
40.024g, Sodium phosphate dibasic Na
2hPO
40.164g, potassium chloride (KCl) 0.01g, 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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| WO2021098098A1 (en) * | 2019-11-18 | 2021-05-27 | 孛朗孚有限公司 | Sulfhydryl modified hyaluronic acid, preparation method therefor and use thereof |
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Application publication date: 20150715 |