CN103396946A - Biological reaction apparatus, preparation method and applications thereof - Google Patents
Biological reaction apparatus, preparation method and applications thereof Download PDFInfo
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Abstract
The present invention provides a biological reaction apparatus, a preparation method and applications thereof, wherein the biological reaction apparatus comprises: a first plate provided with first through holes and a second plate provided with second through holes, the first plate and the second plate are integrally connected, and the first through holes and the second through holes are corresponding one by one. The biological reaction apparatus can be used for cell culture and drug screening, particularly for achieving high throughout and automatic cell and drug loading, and can provide characteristics of time saving, labor saving, simpleness, convenience, and rapid drug screen compared with the traditional single-sample complex operation.
Description
Technical field
The present invention relates to biological field, particularly, the present invention relates to biological reaction apparatus and its preparation method and application.
Background technology
In vitro study cell and small molecules chemicals, extracellular matrix (ECM) protein molecular and with the interaction of other cells, embody further important meaning in fields such as molecular cytobiology, clinical diagnosis and medicament research and development gradually.The three-dimensional environment of cells in vivo growth can't well be simulated and reappear to the two-dimentional culture environment of traditional culture dish or porous plate.Therefore realize that in the three-dimensional microenvironment of external structure cell and other Some Circulating Factors or the repercussion study of cell on three-dimensional level are significant for accelerating further developing of biomedical and medicament research and development.And, along with proposition and the development of Personalized medicine concept, it has been recognized that the significance of individuation difference in medicament research and development.Single medicine kind and dosage thereof differ greatly for the result for the treatment of of Different Individual.Three-dimensional cell is cultivated to combine with Personalized medicine certainly will develop a kind of brand-new biology and medicament research and development pattern, promote greatly developing of biomedicine field.
Bulk information need to be obtained and analyze to modern biological medicine research, high-throughput (high-throughput) technology is arisen at the historic moment, and has greatly improved the efficiency of obtaining bioinformation and has reduced the cost of required reagent (as antibody, medicine), material (as extracellular matrix protein) and cell (as a limited number of primary cells) etc.
High-throughput platform technology commonly used is based on microwell plate (as 96,384 orifice plates) or chip (as gene, albumen, material and cell chip) form at present, carry out process of the test with automation operating system (mechanical manipulator, the volley of rifle fire, chip point sample system, individual point sample instrument etc.), realize trace sample research.But expensive automation operating system does not effectively reduce the total cost of research, need exploitation more efficient, load (loading) more easily
Method realizes the three-dimensional microscale arrangement of the high-throughput of medicine, material and cell chip.
Micro-meter scale processing (little processing) technology of being born in semi-conductor industry more and more is widely used in biomedical research to realize that it has power in the construction high-throughout three-dimensional microenvironment of patterning field for accurate control and the high-throughput arrangement spatially of molecule, material and cell.For example: the three-dimensional microenvironment of accurately controlling on space can be used to the outer bionic model of rebuilding body (as the multilayer physiological structure of simulated blood vessel, and the meticulous physiological structure of liver lobule); The three-dimensional microenvironment that high-throughput is arranged can be used to build three-dimensional medicine, material and cellular array chip.The micro-processing method of realizing the three-D pattern arrangement commonly used comprises: photolithography (photolithography), micro shaping technology (micro-molding), reticle pattern technology (stencil patterning), imprint lithography techniques (Imprint lithography), fluid photoetching technique (flow lithography) etc.The design of three-dimensional microenvironment need to relate to many factors, the mode (encapsulate, be encapsulated in type support in making support process) of the physico-chemical property (chemical property, hardness, degradation property, structural) of the source of biological example material (natural or synthetic), material, the biological activity of material (whether adhesion sites exists, inducing molecule whether exists) and medicine, material and cell encapsulation.
Nowadays, micro-processing technology mostly is confined to have engineering in the application of building three-dimensional microenvironment and realizes in making the laboratory of background, its widespread use in traditional biological, pharmacy and medical laboratory remains bottleneck technically, especially at the structure that relates to viable cell and have the three-dimensional microenvironment of bioactive material and medicine.For example, laboratory cell epimatrix material matrigel (matrigel), gelatin (collagen) commonly used is commercial gel protein, have good biological activity, biocompatibility, degradability, be the excellent material of the culturing cell of generally acknowledging, especially matrigel is the standard base material of cultivating embryonic stem cell (embryonic stem cell).Its plastic (gelation) mode is generally temperature transition (4 ℃～37 ℃), with conventional method, is difficult to realize three-dimensional patterned arrangement.Prepare mould by micro-processing technology, the microscale space constraint of dependence mould can realize the three-dimensional microscale arrangement of gel protein and cell.But research cost or operation skill difficulty have been increased again to a certain extent.
As mentioned above, for meeting the different research fields such as biology, pharmacy, medical science for accurate controlled and demand that high-throughout three-dimensional microenvironment strengthens day by day, but in the urgent need to a kind of platform technology of simple and easy widespread use with fast, harmless and high-throughput ground realize medicine, material and cell with and composition thereof the arrangement of three-D pattern microscale.Desirable little machining 3 D microenvironment high flux screening platform is tackled in personnel's easy handling of being familiar with conventional two-dimensional medicine, cell and investigation of materials, need not special professional technique and means (as micro-processing technology and special synthetic materials) and expensive equipment (as automatization, little processing units), to realize the accessible widespread use at traditional biological, pharmacy and medical field.
Summary of the invention
The present invention one of is intended to solve the problems of the technologies described above at least to a certain extent or provides at least a kind of useful business to select.For this reason, one object of the present invention is to propose a kind of biological reaction apparatus and its preparation method and application.
In one aspect of the invention, the present invention proposes a kind of biological reaction apparatus, according to embodiments of the invention, this biological reaction apparatus comprises: the first plate, and described the first plate has the first through hole; The second plate, described the second plate has the second through hole; The first plate be connected plate and connect by double faced adhesive tape, described the first through hole is corresponding one by one with described the second through hole.This organoid incubator is simple in structure, and complete function can high-flux parallel automatic loading cell, nutrient solution and medicine etc.
In addition, biological reaction apparatus according to the above embodiment of the present invention can also have following additional technical characterictic:
According to embodiments of the invention, described the first through hole comprises a plurality of through holes, and described the second through hole comprises a plurality of through holes.
According to embodiments of the invention, the diameter of described the first through hole is less than the diameter of the second through hole.
According to embodiments of the invention, described the first plate and the second plate are formed by polymethylmethacrylate, and described the first plate and the second plate connect as one by biocompatible glue.
In a second aspect of the present invention, the present invention proposes a kind of method for preparing above-mentioned biological reaction apparatus, the method comprises: provide the first plate, preparation the first through hole on described the first plate; Provide the second plate, preparation the second through hole on described the second plate; Described the first plate and described the second plate are connected as one, and wherein, described the first through hole is corresponding one by one with described the second through hole.
Aspect the 3rd of the present invention, the present invention proposes a kind of method of utilizing above-mentioned biological reaction apparatus culturing cell, the method comprises: described the first through hole and the second through hole are carried out hydrophilic treatment; The parallel gelatin that loads in described the first through hole; Continue parallel repopulating cell in described described the first through hole that is mounted with gelatin; The parallel nutrient solution that loads in described the second through hole.
Aspect the 4th of the present invention, the present invention proposes the purposes of above-mentioned biological reaction apparatus in culturing cell.
Aspect the 5th of the present invention, the present invention proposes the purposes of above-mentioned biological reaction apparatus in screening of medicaments.
Additional aspect of the present invention and advantage part in the following description provide, and part will become obviously from the following description, or by practice of the present invention, recognize.
Description of drawings
Above-mentioned and/or additional aspect of the present invention and advantage are from obviously and easily understanding becoming the description of embodiment in conjunction with following accompanying drawing, wherein:
Fig. 1 is the longitdinal cross-section diagram of biological reaction apparatus according to an embodiment of the invention.
Fig. 2 is the vertical view of biological reaction apparatus according to an embodiment of the invention.
Fig. 3 is the structural representation of medicine carrying chip according to an embodiment of the invention.
Fig. 4 utilizes biological reaction apparatus screening of medicaments Chinese traditional medicine diffusion front and back drug level distributed image.
Fig. 5 is the detected result that adopts biological reaction apparatus of the present invention and conventional two-dimentional porous plate to be at the cultivator fibrosarcoma cell.
Embodiment
Below describe embodiments of the invention in detail, the example of described embodiment is shown in the drawings, and wherein same or similar label represents same or similar element or the element with identical or similar functions from start to finish.Be exemplary below by the embodiment that is described with reference to the drawings, be intended to for explaining the present invention, and can not be interpreted as limitation of the present invention.
in description of the invention, it will be appreciated that, term " " center ", " vertically ", " laterally ", " length ", " width ", " thickness ", " on ", D score, " front ", " afterwards ", " left side ", " right side ", " vertically ", " level ", " top ", " end " " interior ", " outward ", " clockwise ", orientation or the position relationship of indications such as " counterclockwise " are based on orientation shown in the drawings or position relationship, only the present invention for convenience of description and simplified characterization, rather than device or the element of indication or hint indication must have specific orientation, with specific orientation structure and operation, therefore can not be interpreted as limitation of the present invention.
In addition, term " first ", " second " only are used for describing purpose, and can not be interpreted as indication or hint relative importance or the implicit quantity that indicates indicated technical characterictic.Thus, one or more these features can be expressed or impliedly be comprised to the feature that is limited with " first ", " second ".In description of the invention, the implication of " a plurality of " is two or more, unless otherwise expressly limited specifically.
In the present invention, unless otherwise clearly defined and limited, broad understanding should be done in the terms such as term " installation ", " being connected ", " connection ", " fixing ", for example, can be to be fixedly connected with, and can be also to removably connect, or connect integratedly; Can be mechanical connection, can be also to be electrically connected to; Can be directly to be connected, also can indirectly be connected by intermediary, can be the connection of two element internals.For the ordinary skill in the art, can understand as the case may be above-mentioned term concrete meaning in the present invention.
Below with reference to Fig. 1, the biological reaction apparatus of the embodiment of the present invention is described in detail, particularly, this biological reaction apparatus comprises: the first plate 10, the first plates 10 have the first through hole 11; The second plate 20, the second plates 20 have the second through hole 21; The first plate 10 and the second plate 20 connect as one, and the first through hole 11 is corresponding one by one with the second through hole 21.According to a particular embodiment of the invention, the corresponding center of circle line that refers to the first through hole overlaps with the center of circle line of the second through hole one by one.According to a particular embodiment of the invention, the first through hole is suitable for the loading solid cell culturing bracket, such as being gelatin etc., and repopulating cell in the through hole of gelatin will be housed further.The second through hole 21 is suitable for loading nutritive medium, and the first through hole is corresponding one by one with the second through hole, so that the nutritive medium in each second through hole 21 can be supplied with the cell of the first through hole 11 interior plantations corresponding with it.
According to a particular embodiment of the invention, the first through hole 11 on above-mentioned the first plate 10 comprises a plurality of through holes, and the second through hole 21 on the second plate 20 equally also comprises a plurality of through holes.According to a particular embodiment of the invention, the diameter of the first through hole 11 is less than the diameter of the second through hole 21.When loading nutritive medium in repopulating cell, the second through hole in the first through hole, can guarantee enough nutritive medium supports thus.In the system of micro-meter scale, easily cause the rapid evaporation of liquid due to the amount of liquid rareness, make the nutrition that cell obtains run off very soon, therefore the diameter of the second through hole 11 is designed to greater than the first through hole 21, can be so that nutrient solution be enough supported the growth of cell in the first through hole at short notice.
According to a particular embodiment of the invention, the first plate and the second plate are formed by polymethylmethacrylate, and the first plate 10 and the second plate 20 connect as one by biocompatible glue, and wherein, the first plate 10 is ice glue support plate.Can improve thus the cell survival rate while utilizing this biological reaction apparatus culturing cell.The biocompatible glue that relates in the present invention, for without base material transparent double face glue, is usually used in optical research, and is in Biochemical Research, tasteless nontoxic.By biocompatible glue, the first plate and the second plate adhering junction are integrated, make the through hole of every a pair of correspondence all can be considered independent reflection unit, be independent of each other, can realize thus arranging simultaneously different experiment conditions in same device.
In another aspect of this invention, the present invention proposes a kind of method for preparing above-mentioned biological reaction apparatus, the method comprises: provide the first plate, preparation the first through hole on the first plate; Provide the second plate, preparation the second through hole on the second plate; The first plate and the second plate are connected as one, and wherein, the first through hole is corresponding one by one with the second through hole.According to a particular embodiment of the invention, the method that adopts the present invention to prepare biological reaction apparatus can prepare as biological reaction apparatus as described in Fig. 1,2.
According to one embodiment of present invention, the first through hole comprises a plurality of through holes, and the second through hole comprises a plurality of through holes.According to a particular embodiment of the invention, each through hole wherein all can be used as an independent biological reaction apparatus, thus can this biological reaction apparatus a plurality of biological respinses space simultaneously, utilize this biological reaction apparatus walk abreast automatic loading cell and medicine with can realizing high-throughput, the cultivation of experiment three-dimensional cell, and drug screening.
According to a particular embodiment of the invention, the first through hole of preparation and the method for the second through hole are not subjected to special standby restriction, according to a particular embodiment of the invention, can adopt the method for the technology such as laser-engraving technique, the processing of rapid shaping machinery to prepare through hole, only need to design pattern with computer design software, removed complicated Mold Making flow process from, the rapid processing of implement device, and further improve the qualification rate of through hole.
According to one embodiment of present invention, the diameter of the first through hole is less than the diameter of the second through hole.According to a particular embodiment of the invention, the first concrete through hole and the radius of the second through hole can be respectively 0.5mm and 0.8mm, pass through size design, distance of center circle on device between Kong Yukong and the size of single hole can reach with the size of 384 porous plates of traditional commerce agrees with mutually, greatly increased thus the broad applicability of this device, can realize the slitless connection with existing porous plate test set, the use in convenient vast laboratory and detection.
According to one embodiment of present invention, the first plate and the second plate are formed by polymethylmethacrylate, and described the first plate and the second plate connect as one by biocompatible glue, and wherein, the first plate is ice glue support plate.According to a particular embodiment of the invention, the dimensions of the first plate and the second plate can design according to specific needs, for example can be 76 * 26cm
2(with commercially available slide specification, being consistent), thus can be so that follow-uply detect it.According to a particular embodiment of the invention, the thickness of the first plate and the second plate also is not particularly limited, according to a particular embodiment of the invention, the thickness of the first plate can be identical with the thickness of the second plate, concrete thickness can be 0.5～1mm, can conveniently to the cell sample in installing, carry out microscopic examination thus.
According to a particular embodiment of the invention, the quantity of the through hole on each plate also and be not particularly limited, according to concrete example of the present invention, can be 3 * 8 or 6 * 16, can improve thus its efficiency or raising that is used for culturing cell and utilize its screening of medicaments efficiency.
The present invention proposes a kind of method of utilizing above-mentioned biological reaction apparatus culturing cell more on the one hand of the present invention, according to a particular embodiment of the invention, the method can comprise: the first through hole and the second through hole are carried out hydrophilic treatment; The parallel gelatin that loads in the first through hole; Continue parallel repopulating cell in being mounted with the first through hole of gelatin; The parallel nutrient solution that loads in the second through hole.Utilize thus the method can effectively realize the blocking dimensional culture of cell, and by the bonding independent processing that realizes each unit of two plates.
In still another aspect of the invention, the present invention proposes the purposes of above-mentioned biological reaction apparatus in culturing cell.Utilize thus this biological reaction apparatus can significantly improve the efficiency of culturing cell, realize high-flux parallel automatic loading cell and to its effective cultivation.Utilize the relatively traditional single sample complex operations of this biological reaction apparatus culturing cell can be more time saving and energy saving, succinctly easily and fast.Utilize simultaneously this biological reaction apparatus culturing cell can also realize fast, efficiently, application of sample accurately, realize the cultivation of three-dimensional cell.Can reduce simultaneously the use to cell, cell culture fluid, cultivation consumptive material, cost is significant for saving.For example use this device to carry out drug screening when operation of 96 flux, because the materials such as cell all load by the loaded in parallel mode, consuming time only have half a minute, and while using 96 orifice plates to carry out manual operation, will spend 5～10 minutes at least.
The three-dimensional cell of the little ice glue of gelatin that forms based on bio-reactor according to a particular embodiment of the invention, is cultivated array chip and can be promoted cell aggregation to form the three-dimensional microscale tissue (being called for short little tissue) in an analogue body.This little the organizing of three-dimensional cell not only can avoid the common oxygen of three-dimensional tissue and nutritive substance to transmit problem of non-uniform, and can better the residing three-dimensional environment of analogue body inner cell, guarantee that cultured cells has between the matrix morphology that is similar to cell in the in-vivo tissue organ, cell mutual function and signal transduction mechanism etc.Due to the microscale characteristic of three-dimensional rack, this device can be saved a large amount of cells, medicine and cell culture fluid.For example with 5 * 10
6The cell seeding density inoculating cell of/ml is in device, only needs 100 μ l, and namely 5 * 10
5Individual cell, can realize the cell inoculation of 96 supports, and compared to the inoculation of conventional three-dimensional cell, this consumption is only the inoculum size of a support., for meeting the cell cultures in 96 holes, only need 200 μ l nutrient solutions in addition,, compared to the minimum dosage of the 96 every hole 100 μ l of orifice plate, saved 48 times.And medicine is loaded into each microscale is cultivated only needs at most 1 μ l in unit amount of liquid, can significantly save thus precious medical compounds.
, in a fifth aspect of the present invention, the present invention proposes the purposes of above-mentioned biological reaction apparatus in screening of medicaments.Utilize this biological reaction apparatus can be effective to screening of medicaments, owing to utilizing above-mentioned bio-reactor to can be good at the residing three-dimensional environment of analogue body inner cell, therefore guaranteed that cultured cells has mutual function and signal transduction mechanism etc. between the matrix morphology that is similar to cell in the in-vivo tissue organ, cell, make and utilize this cell, for drug screening, reaction is more accurately arranged, make and utilize this bioreactor culture cell to have better predictability for screening of medicaments.When for example cancer cell is grown in three-dimensional porous rack, part differentiation (partial differentiation) can occur, be that an epithelium-matter transforms (epithelial-mesenchymal transition), cancer cells can progressively be divided into malignant state, change as cellular form, surface of cell membrane E-cadherin is expressed and is reduced, the N-cadherin is expressed to be increased, and the activation of other genetic expressions in nucleus, thereby cause cancer cells to reduce chemotherapy drug susceptibility, resistance increases.
Below with reference to specific embodiment, present invention is described, need to prove, these embodiment are only descriptive, and do not limit the present invention in any way.
The preparation method of embodiment 1 biological reaction apparatus:
Select the thick PMMA plate of 0.5mm (two sides is all with protective membrane) as ice glue support plate; according to the size of mentioning in embodiment in mapping software the design apparatus sketch (the device size is 76 * 26mm; on ice glue support plate, total hole is 96, and array arrangement is 6 * 16, and bore dia is designed to 1mm).Then graphic file is transferred in the laser engraving machine sequence of control, carries out die-cut, the PMMA ice glue support plate that cuts out is rinsed well with deionized water, and dries in 60 ℃ of baking ovens.Pollute hydrophobic PMMA surface when in this process, PMMA two surface protective films do not take to prevent the pre-gathering solutions of follow-up loading biological material support off.It is standby that the mould for preparing can be put into clean sealed bag room temperature preservation.
When the preparation dimensional culture is used biomaterial scaffolds, in advance above-mentioned mould is carried out hydrophilic treatment with plasma cleaner, the time, 1min got final product.The selection of biomaterial is very extensive, and polyoxyethylene glycol commonly used and gelatin etc. all can be used to prepare the support in this device.Take the gelatin support as example, the aqueous gelatin solution of preparation 4%, be placed in precooling on ice, then add glutaraldehyde solution, make the final concentration of glutaraldehyde reach 0.1%, then get 200 μ l and drip to mould one end, with thin plate (as thin slide), drop evenly is coated with and scrapes in die surface, solution can be filled in array hole on mould naturally.After the mould of desired number all is mounted with material solution, it is placed in cryogenic refrigerator (12～-20 ℃) as early as possible, place 12h so that react completely.Take out afterwards the mould be loaded with the premolding support, be submerged in the sodium borohydride solution of 0.1M and soaked 10 minutes, with in and unreacted aldehyde radical completely, reduce the autofluorescence of support, then use the deionized water rinsing mould two to three times.After cleaning process finishes, mould is immersed in the water and is frozen into ice, be placed in Freeze Drying Equipment and process 12h, ice crystal in support distils, produce the duct that is interconnected of tens to 100 microns in support, can take out mould this moment, is placed in dry environment and stores, so far, the biological reaction apparatus preparation is completed.
The concrete structure of the biological reaction apparatus for preparing by aforesaid method is as follows:
This device minute up and down is two-layer, all adopts the modular size design, and outward appearance is consistent with the standard microscope slide, long 76mm, wide 26mm.The array hole design is consistent with commercially available 384 orifice plates, is 6 * 16 arrays.Upper strata is that nutrient solution stores plate, and bore dia is 1.6mm, and lower plywood is ice glue support plate, and bore dia is 1mm, and on two plates, the distance of center circle in hole is 4.5mm, by biocompatibility double faced adhesive tape good bond.
Embodiment 2 utilizes the working method of biological reaction apparatus culturing cell
At first device is carried out ultraviolet-ray sterilizing and process, prevent that in experiment, cell sample is contaminated.Be prepared into the cell suspension of desired concn, every milliliter 1～5 * 10 with being used in advance the cell of testing
6Individual cell.The cell suspension of drawing 100～120 μ l is added drop-wise to an end of ice glue support plate, with clean aseptic thin slide, evenly is coated with the surface of cell scraping suspension in plate, and the ice glue on plate can be with the cell automatic absorbing wherein, and so far, cell loads and completes.
Then get 200 μ l cell culture fluids, an end that is added drop-wise to the device another side is the end that nutrient solution stores plate, with clean aseptic thin slide, all scrapes in plate surperficially with being coated with equally, completes the loading of nutrient solution.Finally the reaction unit that has loaded is placed in the abundant container of humidity, 37 ℃ of cellar cultures get final product.
The purposes of embodiment 3 biological reaction apparatus in screening of medicaments
, if carry out the screening experiment of cell drug toxicity,, because each culture system in device is separate, can conveniently carry out the interpolation of different concns, different sorts drug molecule.
If experiment is equipped with the high-throughput service platform, can be directly to each reaction member corresponding kind of interpolation in device and the medicine of respective concentration.
If common laboratory detects use, the Simple medicament that drug solution first can be added drop-wise to the present invention's preparation loads on chip, and the chip (Fig. 3) that then will be mounted with medicine is buckled in the nutrient solution storage plate one side of device, and medicine namely can slowly diffuse into cell culture system.
Medicine loads the preparation method of chip: the medicine carrying chip in this embodiment adopts the conventional photolithographic method to make, material adopts the polyoxyethylene glycol material of Photocrosslinkable, molecular weight 4000, concentration 10%w/t, be equipped with simultaneously the light trigger I2959 of 0.5%w/t, UV-irradiation is 35 seconds under the covering of photomask.Clean postradiation polyethylene glycol gel chip with clear water afterwards ,-20 ℃ freezing 6～7 hours, vacuum lyophilization got final product about 12 hours.
The summary of medicine carrying chip structure: the polyoxyethylene glycol support on this chip adopts the cylindrical array pattern equally, and diameter is designed to 1mm, and spacing 4.5mm between cylinder is consistent with the cell loading chip size in this invention, convenient experiment.
Detected result:
Be respectively drug diffusion process front and back drug level distributed image shown in two figure of Fig. 4 left and right.The simulation results medicine can reach balance about 4.8 hours, and can find out also that from the right figure of Fig. 4 medicine still keeps concentration gradient after the experience diffusion.Table 1 has been enumerated the loading concentration before drug diffusion, Theoretical Calculation concentration and the actual measurement concentration after diffusion.Can find out obviously that through contrast the actual dispersion process of medicine and theoretical prediction have very good corresponding relation.
Table 1
| Medicine loads concentration (μ g/ml) | 97.66 | 195.3 | 390.6 | 781.3 | 1562.5 | 3125 | 6250 |
| Concentration (μ g/ml) after the theoretical prediction diffusion | 16.76 | 33.52 | 67.03 | 134.1 | 268.1 | 534.6 | 1069 |
| The actual rear concentration (μ g/ml) of diffusion that records | 16.80 | 33.48 | 66.90 | 133.83 | 267.6 | 535.8 | 1071 |
After drug effect for some time (be generally 24h or more than), to install and directly be put in the 50ml centrifuge tube (if use medicine to load chip, remove and get final product), centrifugal 2 minutes of 1000rpm, liquid in removal device, then evenly be coated with and scrape on installing according to the reagent (solution as blue in Armagh) that adds the same method of cell culture fluid and will detect cytoactive, 37 ℃ were reacted 1～2 hour, to install taking-up, be placed on testing stand, the detected value of each unit in the microplate reader proofing unit, the relative reactivity of cell under calculating different pharmaceutical condition.
Fig. 5 has shown the comparing result of apparatus of the present invention and conventional two-dimentional porous plate detected result in cultivator fibrosarcoma cell system.can obviously be found out by Fig. 5, in apparatus of the present invention, the resistance of cell obviously improves, under the dimensional culture state, the broad-spectrum anti-cancer drug Zorubicin is 130.02 μ M to human fibrosarcoma cell's half-inhibition concentration, and under the porous plate training mode of conventional two-dimensional, the half-inhibition concentration of this medicine only has 9.55 μ M, can not truly reflect the antiradiation drug reaction of cell in original three dimensional growth environment at the lower half-inhibition concentration that two-dimentional training mode records, this is with the drug-fast bibliographical information of dimensional culture pattern raising tumour cell is basically identical in recent years, therefore also proved the accurate reliability of this device.
In the description of this specification sheets, the description of reference term " embodiment ", " some embodiment ", " example ", " concrete example " or " some examples " etc. means to be contained at least one embodiment of the present invention or example in conjunction with specific features, structure, material or the characteristics of this embodiment or example description.In this manual, the schematic statement of above-mentioned term not necessarily referred to identical embodiment or example.And the specific features of description, structure, material or characteristics can be with suitable mode combinations in any one or more embodiment or example.
Although the above has illustrated and has described embodiments of the invention, be understandable that, above-described embodiment is exemplary, can not be interpreted as limitation of the present invention, those of ordinary skill in the art is not in the situation that break away from principle of the present invention and aim can change above-described embodiment within the scope of the invention, modification, replacement and modification.
Claims (11)
1. a biological reaction apparatus, is characterized in that, comprising:
The first plate, described the first plate has the first through hole;
The second plate, described the second plate has the second through hole;
The first plate and the second plate connect as one, and described the first through hole is corresponding one by one with described the second through hole.
2. biological reaction apparatus according to claim 1, is characterized in that, described the first through hole comprises a plurality of through holes, and described the second through hole comprises a plurality of through holes.
3. biological reaction apparatus according to claim 2, is characterized in that, the diameter of described the first through hole is less than the diameter of the second through hole.
4. biological reaction apparatus according to claim 1, is characterized in that, described the first plate and the second plate are formed by polymethylmethacrylate, and described the first plate and the second plate connect as one by biocompatible glue.
5. a method for preparing the described biological reaction apparatus of claim 1-4 any one, is characterized in that, comprising:
Provide the first plate, preparation the first through hole on described the first plate;
Provide the second plate, preparation the second through hole on described the second plate;
Described the first plate and described the second plate are connected as one,
Wherein, described the first through hole is corresponding one by one with described the second through hole.
6. method according to claim 5, is characterized in that, described the first through hole comprises a plurality of through holes, and described the second through hole comprises a plurality of through holes.
7. method according to claim 6, is characterized in that, the diameter of described the first through hole is less than the diameter of the second through hole.
8. method according to claim 5, is characterized in that, described the first plate and the second plate are formed by polymethylmethacrylate, and described the first plate and the second plate connect as one by biocompatible glue.
9. a method of utilizing the described biological reaction apparatus culturing cell of claim 1-4 any one, is characterized in that, comprising:
Described the first through hole and the second through hole are carried out hydrophilic treatment;
The parallel gelatin that loads in described the first through hole;
Continue parallel repopulating cell in described described the first through hole that is mounted with gelatin; And
The parallel nutrient solution that loads in described the second through hole.
10. the purposes of the described biological reaction apparatus of claim 1-4 any one in culturing cell.
11. the purposes of the described biological reaction apparatus of claim 1-4 any one in screening of medicaments.
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| CN104480010A (en) * | 2014-11-28 | 2015-04-01 | 清华大学 | Biological reaction device and application thereof |
| CN105108817A (en) * | 2015-09-09 | 2015-12-02 | 通用生物系统（安徽）有限公司 | Manufacturing method for 384 hole solid-phase synthesis reaction device used for nucleic acid synthesis |
| CN106153891A (en) * | 2015-04-09 | 2016-11-23 | 清华大学 | Three dimensional biological marker detection device, preparation method and the method for detection biomarker |
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| CN1678909A (en) * | 2002-08-28 | 2005-10-05 | 法国原子能委员会 | Device for measuring the electrical activity of biological elements and its applications |
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