CN103396946B - Biological reaction apparatus and its preparation method and application - Google Patents
Biological reaction apparatus and its preparation method and application Download PDFInfo
- Publication number
- CN103396946B CN103396946B CN201310359407.9A CN201310359407A CN103396946B CN 103396946 B CN103396946 B CN 103396946B CN 201310359407 A CN201310359407 A CN 201310359407A CN 103396946 B CN103396946 B CN 103396946B
- Authority
- CN
- China
- Prior art keywords
- hole
- plate
- cell
- reaction apparatus
- biological reaction
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 238000006243 chemical reaction Methods 0.000 title claims abstract description 51
- 238000002360 preparation method Methods 0.000 title abstract description 8
- 239000003814 drug Substances 0.000 claims abstract description 56
- 238000012216 screening Methods 0.000 claims abstract description 10
- 238000000034 method Methods 0.000 claims description 30
- 239000003292 glue Substances 0.000 claims description 20
- 235000015097 nutrients Nutrition 0.000 claims description 16
- 229920003229 poly(methyl methacrylate) Polymers 0.000 claims description 8
- 239000004926 polymethyl methacrylate Substances 0.000 claims description 8
- 238000004113 cell culture Methods 0.000 claims description 6
- 230000002629 repopulating effect Effects 0.000 claims description 5
- 239000007787 solid Substances 0.000 claims description 3
- 239000002253 acid Substances 0.000 claims 1
- 150000004702 methyl esters Chemical class 0.000 claims 1
- 229940079593 drug Drugs 0.000 abstract description 14
- 210000004027 cell Anatomy 0.000 description 79
- 239000000463 material Substances 0.000 description 23
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 15
- 108010010803 Gelatin Proteins 0.000 description 11
- 238000005516 engineering process Methods 0.000 description 11
- 239000008273 gelatin Substances 0.000 description 11
- 229920000159 gelatin Polymers 0.000 description 11
- 235000019322 gelatine Nutrition 0.000 description 11
- 235000011852 gelatine desserts Nutrition 0.000 description 11
- 238000011068 loading method Methods 0.000 description 9
- 230000004907 flux Effects 0.000 description 8
- 238000009792 diffusion process Methods 0.000 description 7
- 238000013461 design Methods 0.000 description 6
- 230000008569 process Effects 0.000 description 6
- 238000012545 processing Methods 0.000 description 6
- 238000011160 research Methods 0.000 description 6
- 238000012360 testing method Methods 0.000 description 6
- 238000002474 experimental method Methods 0.000 description 5
- 108090000623 proteins and genes Proteins 0.000 description 5
- 239000002202 Polyethylene glycol Substances 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 229920001223 polyethylene glycol Polymers 0.000 description 4
- 238000012827 research and development Methods 0.000 description 4
- 210000001519 tissue Anatomy 0.000 description 4
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 description 3
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 description 3
- 206010028980 Neoplasm Diseases 0.000 description 3
- 239000012620 biological material Substances 0.000 description 3
- 201000011510 cancer Diseases 0.000 description 3
- 238000007877 drug screening Methods 0.000 description 3
- 239000000499 gel Substances 0.000 description 3
- 238000001459 lithography Methods 0.000 description 3
- 108010082117 matrigel Proteins 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 238000012549 training Methods 0.000 description 3
- 102000000905 Cadherin Human genes 0.000 description 2
- 108050007957 Cadherin Proteins 0.000 description 2
- AOJJSUZBOXZQNB-TZSSRYMLSA-N Doxorubicin Chemical compound O([C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(=O)CO)[C@H]1C[C@H](N)[C@H](O)[C@H](C)O1 AOJJSUZBOXZQNB-TZSSRYMLSA-N 0.000 description 2
- 201000008808 Fibrosarcoma Diseases 0.000 description 2
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 239000012930 cell culture fluid Substances 0.000 description 2
- 230000010261 cell growth Effects 0.000 description 2
- 230000001413 cellular effect Effects 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 239000008367 deionised water Substances 0.000 description 2
- 229910021641 deionized water Inorganic materials 0.000 description 2
- 230000004069 differentiation Effects 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 210000001671 embryonic stem cell Anatomy 0.000 description 2
- 210000002744 extracellular matrix Anatomy 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 238000011081 inoculation Methods 0.000 description 2
- 238000010147 laser engraving Methods 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 238000001053 micromoulding Methods 0.000 description 2
- 210000000056 organ Anatomy 0.000 description 2
- 238000000206 photolithography Methods 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
- 230000019491 signal transduction Effects 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 238000003860 storage Methods 0.000 description 2
- 108010035532 Collagen Proteins 0.000 description 1
- 102000008186 Collagen Human genes 0.000 description 1
- 208000030453 Drug-Related Side Effects and Adverse reaction Diseases 0.000 description 1
- 241000403354 Microplus Species 0.000 description 1
- 206010070863 Toxicity to various agents Diseases 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 239000002390 adhesive tape Substances 0.000 description 1
- 229940009456 adriamycin Drugs 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- -1 antibody Substances 0.000 description 1
- 239000002246 antineoplastic agent Substances 0.000 description 1
- 229940041181 antineoplastic drug Drugs 0.000 description 1
- 238000003491 array Methods 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 229940044683 chemotherapy drug Drugs 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 238000003759 clinical diagnosis Methods 0.000 description 1
- 229920001436 collagen Polymers 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 239000012531 culture fluid Substances 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 239000006185 dispersion Substances 0.000 description 1
- 238000005538 encapsulation Methods 0.000 description 1
- 230000007705 epithelial mesenchymal transition Effects 0.000 description 1
- 238000001704 evaporation Methods 0.000 description 1
- 230000008020 evaporation Effects 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 238000001879 gelation Methods 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 238000009434 installation Methods 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 230000003211 malignant effect Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000011664 nicotinic acid Substances 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 238000010899 nucleation Methods 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 238000011017 operating method Methods 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 210000003463 organelle Anatomy 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 238000010422 painting Methods 0.000 description 1
- 238000000059 patterning Methods 0.000 description 1
- 238000001259 photo etching Methods 0.000 description 1
- 229920003023 plastic Polymers 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 239000011120 plywood Substances 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 238000003672 processing method Methods 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 238000012797 qualification Methods 0.000 description 1
- 239000000718 radiation-protective agent Substances 0.000 description 1
- 238000007790 scraping Methods 0.000 description 1
- 239000004065 semiconductor Substances 0.000 description 1
- 238000007493 shaping process Methods 0.000 description 1
- 238000004088 simulation Methods 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 229910000033 sodium borohydride Inorganic materials 0.000 description 1
- 239000012279 sodium borohydride Substances 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 229920002994 synthetic fiber Polymers 0.000 description 1
- 230000009967 tasteless effect Effects 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 230000007704 transition Effects 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 238000009777 vacuum freeze-drying Methods 0.000 description 1
Landscapes
- Apparatus Associated With Microorganisms And Enzymes (AREA)
Abstract
The present invention proposes biological reaction apparatus and its preparation method and application, wherein, biological reaction apparatus includes：First plate, the first plate has first through hole；Second plate, the second plate has the second through hole；First plate and the second plate are connected as one, and first through hole is corresponded with the second through hole.The biological reaction apparatus can be used for culture cell and screening medicine, can realize that high-flux parallel loads cell and medicine automatically especially with the biological reaction apparatus, more traditional single sample complex operations are more time saving and energy saving, succinct convenient, being capable of quick screening drugs.
Description
Technical field
The present invention relates to biological field, specifically, the present invention relates to biological reaction apparatus and its preparation method and application.
Background technology
In vitro study cell and small-molecule chemical medicine, extracellular matrix（ECM）Protein molecular and with other cells
Interaction gradually embodies further important meaning in fields such as molecular cytobiology, clinical diagnosis and medicament research and developments.Pass
The culture dish of system or the two-dimentional culture environment of porous plate can not simulate and reappear the three-dimensional environment of internal cell growth well.Cause
This builds three-dimensional microenvironment and realizes cell and the repercussion study of other Some Circulating Factors or cell in three-dimensional level in vitro
For accelerating the further development of biomedical and medicament research and development significant.And with carrying for Personalized medicine concept
Go out and develop, it has been recognized that individuation difference is in the significance of medicament research and development.Single medicine species and its dosage pair
Differed greatly in the therapeutic effect of Different Individual.Three-dimensional cell culture is combined with Personalized medicine certainly will develop one kind entirely
New biology and medicament research and development pattern, promote greatly developing for biomedicine field.
Modern biotechnology medicine research needs to obtain and analyze bulk information, high flux（high-throughput）Technology should
Transport and give birth to, substantially increase the efficiency for obtaining biological information and reduce required reagent（Such as antibody, medicine）, material（As carefully
Extracellular matrix protein）And cell（Such as a limited number of primary cells）Deng cost.
Conventional high flux platform technology is based on microwell plate at present（Such as 96,384 orifice plates）Or chip（Such as gene, albumen, material
Material and cell chip）Form, uses automation operating system（Manipulator, the volley of rifle fire, chip spotting system, personal point sample instrument etc.）Perform
Process of the test, realizes that trace sample is studied.But, expensive automation operating system does not have the assembly of effectively reduction research
This is, it is necessary to develop more efficient, more easy load（loading）
Method arranges chip come the three-dimensional minute yardstick of high flux for realizing medicine, material and cell.
It is born in the micro-meter scale processing of semi-conductor industry（Micro Process）Technology is increasingly widely used in biological doctor
Learn to realize accurate control and high flux arrangement for molecule, material and cell spatially in research, it is building pattern
Changing high-throughout three-dimensional microenvironment field has power.For example：The three-dimensional microenvironment spatially accurately controlled can by with
To rebuild external bionic model（Such as the multilayer physiological structure of simulated blood vessel, and the fine physiological structure of lobuli hepatis）；High pass
The three-dimensional microenvironment of amount arrangement can be used to build three-dimensional medicine, material and cellular array chip.Conventional realization is three-dimensional
The micro-processing method of patterned arrangement includes：Photolithography（photolithography）, micro-molding 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 needs to be related to many factors, for example, give birth to
The source of thing material（It is natural or synthesis）, material physico-chemical property（It is chemical property, hardness, degradability, structural）, material
Bioactivity（Attachment sites presence or absence, inducing molecule presence or absence）And the mode of medicine, material and cell encapsulation（
Make in stent procedures and encapsulate, be encapsulated on molded support）.
Nowadays, micro-processing technology is mostly confined to manufacture the reality of background with engineering in the application for building three-dimensional microenvironment
Test in room and realize, it remains bottleneck technically in traditional biological, pharmacy and the extensive use of medical laboratory, especially existed
It is related to living cells and material and the structure of the three-dimensional microenvironment of medicine with bioactivity.For example, the conventional cell in laboratory
Extracellular matrix materials matrigel（matrigel）, gelatin（collagen）It is the gel protein of commercialization, is lived with good biology
Property, biocompatibility, degradability, be the excellent material of generally acknowledged culture cell, especially matrigel is culture embryonic stem cell
（embryonic stem cell）Standard substrate material.Its plastic（gelation）Mode is usually temperature transition（4 DEG C~
37℃）, three-dimensional patterned arrangement is difficult to realize with conventional method.Mould is prepared by micro-processing technology, by mould
Microscale spatial limitation can realize the three-dimensional minute yardstick arrangement of gel protein and cell.But add grind again to a certain extent
Study carefully cost or operation skill difficulty.
As described above, to meet the different research fields such as biology, pharmacy, medical science for controllable precise and high-throughout three
Tie up the increasingly enhanced demand of microenvironment, in the urgent need to it is a kind of it is simple, can wide variety of platform technology with quick, lossless and height
Realize medicine, material and cell and the three dimensional patterned minute yardstick arrangement of its mixture flux.Preferable micro Process is three-dimensional
Microenvironment high flux screening platform tackle it is easily operated in being familiar with conventional two-dimensional medicine, cell and the personnel of investigation of materials, without
Special professional technique and means（Such as micro-processing technology and special synthetic material）And expensive equipment（As automation, it is micro- plus
Construction equipment）, to realize the accessible extensive use in traditional biological, pharmacy and medical domain.
The content of the invention
It is contemplated that at least solving one of above-mentioned technical problem to a certain extent or providing at a kind of useful business
Industry is selected.Therefore, it is an object of the present invention 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, and embodiments in accordance with the present invention should
Biological reaction apparatus includes：First plate, first plate has first through hole；Second plate, second plate has the second through hole；
First plate and the second plate are by two-sided gemel connection, and the first through hole is corresponded with second through hole.The organelle culture
Device is simple in construction, full-featured, can high-flux parallel load cell, nutrient solution and medicine etc. automatically.
In addition, biological reaction apparatus according to the above embodiment of the present invention can also have technical characteristic additional as follows：
Embodiments in accordance with the present invention, the first through hole includes multiple through holes, and second through hole includes multiple through holes.
Embodiments in accordance with the present invention, the diameter of the first through hole is less than the diameter of the second through hole.
Embodiments in accordance with the present invention, first plate and the second plate are formed by polymethyl methacrylate, and described first
Plate and the second plate are connected as one by biocompatible glue.
In the second aspect of the present invention, the present invention proposes a kind of method for preparing above-mentioned biological reaction apparatus, this method
Including：First plate is provided, first through hole is prepared on first plate；Second plate is provided, second is prepared on second plate
Through hole；First plate and second plate are connected as one, wherein, a pair of the first through hole and second through hole 1
Should.
In the third aspect of the invention, the present invention proposes a kind of above-mentioned biological reaction apparatus culture cell of utilization
Method, this method includes：The first through hole and the second through hole are subjected to hydrophilic treated；The loaded in parallel in the first through hole
Gelatin；Continue parallel repopulating cell in the first through hole for being mounted with gelatin；In second through hole and luggage
Culture nutrient solution.
In the fourth aspect of the invention, the present invention proposes use of the above-mentioned biological reaction apparatus in culture cell
On the way.
In the fifth aspect of the invention, the present invention proposes use of the above-mentioned biological reaction apparatus in screening medicine
On the way.
The additional aspect and advantage of the present invention will be set forth in part in the description, and will partly become from the following description
Obtain substantially, or recognized by the practice of the present invention.
Brief description of the drawings
The above-mentioned and/or additional aspect and advantage of the present invention will become from description of the accompanying drawings below to embodiment is combined
Substantially and be readily appreciated that, wherein：
Fig. 1 is the longitdinal cross-section diagram of biological reaction apparatus according to an embodiment of the invention.
Fig. 2 is the top view of biological reaction apparatus according to an embodiment of the invention.
Fig. 3 is the structural representation of load medicine core piece according to an embodiment of the invention.
Fig. 4 is to utilize drug concentration profile picture before and after drug diffusion in biological reaction apparatus screening medicine.
Fig. 5 is
Testing result.
Embodiment
Embodiments of the invention are described below in detail, the example of the embodiment is shown in the drawings, wherein from beginning to end
Same or similar label represents same or similar element or the element with same or like function.Below with reference to attached
The embodiment of figure description is exemplary, it is intended to for explaining the present invention, and be not considered as limiting the invention.
In the description of the invention, it is to be understood that term " " center ", " longitudinal direction ", " transverse direction ", " length ", " width ",
" thickness ", " on ", " under ", "front", "rear", "left", "right", " vertical ", " level ", " top ", " bottom " " interior ", " outer ", " up time
The orientation or position relationship of the instruction such as pin ", " counterclockwise " are, based on orientation shown in the drawings or position relationship, to be for only for ease of
The description present invention and simplified description, rather than indicate or imply that the device or element of meaning must have specific orientation, Yi Te
Fixed azimuth configuration and operation, therefore be not considered as limiting the invention.
In addition, term " first ", " second " are only used for describing purpose, and it is not intended that indicating or implying relative importance
Or the implicit quantity for indicating indicated technical characteristic.Thus, define " first ", the feature of " second " can express or
Implicitly include one or more this feature.In the description of the invention, " multiple " are meant that two or more,
Unless otherwise specifically defined.
In the present invention, unless otherwise clearly defined and limited, term " installation ", " connected ", " connection ", " fixation " etc.
Term should be interpreted broadly, for example, it may be being fixedly connected or being detachably connected, or be integrally connected；It can be machine
Tool is connected or electrically connected；Can be joined directly together, can also be indirectly connected to by intermediary, can be two members
Connection inside part.For the ordinary skill in the art, above-mentioned term can be understood in this hair as the case may be
Concrete meaning in bright.
The biological reaction apparatus of the embodiment of the present invention is described in detail below with reference to Fig. 1, specifically, the biological respinse
Device includes：First plate 10, the first plate 10 has first through hole 11；Second plate 20, the second plate 20 has the second through hole 21；First
The plate 20 of plate 10 and second is connected as one, and first through hole 11 is corresponded with the second through hole 21.According to the specific implementation of the present invention
Example, one-to-one corresponding refers to that the center of circle line of first through hole is overlapped with the center of circle line of the second through hole.According to the specific implementation of the present invention
Example, first through hole is suitable to loading solid cell culturing bracket, such as can be gelatin, and further will be equipped with the through hole of gelatin
Interior repopulating cell.Second through hole 21 is suitable to load nutrient solution, and first through hole and the second through hole are corresponded, so as to each second logical
Nutrient solution in hole 21 can supply the cell of plantation in corresponding first through hole 11.
According to a particular embodiment of the invention, the first through hole 11 on above-mentioned first plate 10 includes multiple through holes, the second plate
The second through hole 21 on 20 equally also includes multiple through holes.According to a particular embodiment of the invention, the diameter of first through hole 11 is small
In the diameter of the second through hole 21.Thus when loading nutrient solution in repopulating cell in first through hole, the second through hole, it ensure that foot
Enough nutrient solutions are supported.In the system of micro-meter scale, because amount of liquid rareness easily causes the rapid evaporation of liquid so that cell
Obtained nutrition is lost in quickly, therefore is that, more than first through hole 21, can cause nutrient solution by the diameter design of the second through hole 11
Support the growth of cell in first through hole enough in a short time.
According to a particular embodiment of the invention, the first plate and the second plate are formed by polymethyl methacrylate, the first plate 10
Connected as one with the second plate 20 by biocompatible glue, wherein, the first plate 10 is ice glue support plate.It is possible thereby to improve utilization
Cell survival rate during the biological reaction apparatus culture cell.The biocompatible glue being related in the present invention is transparent double without base material
Face glue, is usually used in optical research, Biochemical Research, tasteless nontoxic.The first plate and the second plate are bonded by biocompatible glue
Connect as one so that every a pair of corresponding through holes can be considered individually reflection unit, be independent of each other, thus may be implemented in same
Different experiment conditions are set simultaneously in one device.
In another aspect of this invention, the present invention proposes a kind of method for preparing above-mentioned biological reaction apparatus, the party
Method includes：First plate is provided, first through hole is prepared on the first panel；Second plate is provided, the second through hole is prepared on the second plate；Will
First plate and the second plate are connected as one, wherein, first through hole is corresponded with the second through hole.According to the specific implementation of the present invention
Example, the method for preparing biological reaction apparatus using the present invention can prepare the biological reaction apparatus as described in Fig. 1,2.
According to one embodiment of present invention, first through hole includes multiple through holes, and the second through hole includes multiple through holes.According to
The specific embodiment of the present invention, each through hole therein can be as an independent biological reaction apparatus, it is possible thereby to the life
Thing reaction unit can be while multiple biological respinse spaces, can be realized parallel automatic with high throughput using the biological reaction apparatus
Cell and medicine are loaded, the culture of three-dimensional cell, and drug screening is tested.
According to a particular embodiment of the invention, the first through hole of preparation and the method for the second through hole be not by special standby limit
System, according to a particular embodiment of the invention, the method system that the technologies such as laser-engraving technique, rapid shaping machinery processing can be used
Standby through hole, only need to design good pattern with computer design software, eliminate the Mold Making flow of complexity, realize that the quick of device adds
Work, and further improve the qualification rate of through hole.
According to one embodiment of present invention, the diameter of first through hole is less than the diameter of the second through hole.According to the present invention's
The radius of specific embodiment, specific first through hole and the second through hole can be respectively 0.5mm and 0.8mm, by size design,
The size of distance of center circle and single hole on device between Kong Yukong can reach the size phase contract with 384 porous plates of traditional commerce
Close, thus considerably increase the broad applicability of the device, the slitless connection with existing porous plate detection device can be achieved, more
Facilitate the use and detection in vast laboratory.
According to one embodiment of present invention, the first plate and the second plate are formed by polymethyl methacrylate, and described first
Plate and the second plate are connected as one by biocompatible glue, wherein, the first plate is ice glue support plate.According to the specific reality of the present invention
Apply example, the dimensions of the first plate and the second plate can be designed according to specific needs, for example, can be 76 × 26cm2（With it is commercially available
Slide specification is consistent）, it is possible thereby to be easy to subsequently detect it.According to a particular embodiment of the invention, the first plate
It is not particularly restricted with the thickness of the second plate, according to a particular embodiment of the invention, the thickness of the first plate can be with the second plate
Thickness it is identical, specific thickness can be 0.5~1mm, it is possible thereby to more facilitate in device cell sample carry out it is micro-
Observation.
According to a particular embodiment of the invention, the quantity of the through hole on each plate is also not particularly restricted, according to this hair
Bright specific example, can be 3 × 8 or 6 × 16, it is possible thereby to improve its efficiency or raising profit that are used to cultivate cell
Drug efficiency is screened with it.
In another aspect of the invention, the present invention proposes a kind of side using above-mentioned biological reaction apparatus culture cell
Method, according to a particular embodiment of the invention, this method can include：First through hole and the second through hole are subjected to hydrophilic treated；
Loaded in parallel gelatin in first through hole；Continue parallel repopulating cell in the first through hole for be mounted with gelatin；In the second through hole
Loaded in parallel nutrient solution.Thus the blocking dimensional culture of cell can be effectively realized using this method, and by the viscous of two plates
Close the independent process for realizing each unit.
In still another aspect of the invention, the present invention proposes purposes of the above-mentioned biological reaction apparatus in culture cell.
Thus the efficiency of culture cell can be significantly improved using the biological reaction apparatus, realizes that high-flux parallel loads cell simultaneously automatically
To its effective culture.Using the relatively conventional single sample complex operations of the biological reaction apparatus culture cell can it is more time saving and energy saving,
Succinctly easily and fast.Simultaneously using the biological reaction apparatus culture cell can also realize it is quick, efficient, be accurately loaded, it is real
The culture of existing three-dimensional cell.It can reduce simultaneously to cell, cell culture fluid, the use for cultivating consumptive material, for saving cost tool
It is significant.For example when the drug screening operation of 96 flux is carried out with the device, because the materials such as cell are all by parallel
Load mode is loaded, and takes only half a minute, and when being operated manually with 96 orifice plates, at least to spend 5~10 points
Clock.
According to a particular embodiment of the invention, the Three-dimensional cell culture battle array of the micro- ice glue of gelatin based on bioreactor formation
Row chip can promote the three-dimensional microscale tissue that cell aggregation is formed in an analogue body（Abbreviation micro-assembly robot）.Three-dimensional cell
This micro-assembly robot can not only avoid the common oxygen of three-dimensional tissue and nutriment transmits problem of non-uniform, and can be preferably
Three-dimensional environment residing for analogue body inner cell, it is ensured that the cell of culture has the skeleton shape similar to cell in in-vivo tissue organ
Mutual function and signal transduction mechanism etc. between state, cell.Due to the microscale nature of three-dimensional rack, the device can be saved
Substantial amounts of cell, medicine and cell culture fluid.For example with 5 × 106/ ml cell seeding density inoculating cell is in device, only
Need 100 μ l, i.e., 5 × 105Individual cell, you can the cell inoculation of 96 supports is realized, compared to the inoculation of conventional three-dimensional cell, the use
Amount is only the inoculum concentration of a support.It is in addition the cell culture in 96 holes of satisfaction, it is only necessary to 200 μ l nutrient solutions, compared to 96 holes
Minimum dosage of the plate per the μ l of hole 100, saves 48 times.And medicine is loaded into each minute yardstick culture unit at most only needs 1 μ l
Amount of liquid, thus can save significantly on preciousness medical compounds.
In the fifth aspect of the present invention, the present invention proposes purposes of the above-mentioned biological reaction apparatus in screening medicine.
Can be effective for screening medicine, due to can be good at analogue body using above-mentioned bioreactor using the biological reaction apparatus
Three-dimensional environment residing for inner cell, therefore ensure that the cell of culture has the skeleton shape similar to cell in in-vivo tissue organ
Mutual function and signal transduction mechanism etc. between state, cell so that being used for drug screening using the cell has more accurately anti-
Should so that be used to screen medicine with more preferable predictive using the bioreactor culture cell.For example cancer cell is three
When being grown in dimension porous support, it may occur that partial differentiation（partial differentiation）, i.e. epithelial-mesenchymal conversion
（epithelial-mesenchymal transition）, cancer cell can progressively be divided into malignant state, and such as cellular morphology changes
Become, the expression of cell membrane surface E- cadherins is reduced, the expression increase of N- cadherins, and other gene tables in nucleus
The activation reached, so as to cause cancer cell to chemotherapy drug susceptibility reduction, resistance to the action of a drug increase.
Below with reference to specific embodiment, present invention is described, it is necessary to which explanation, these embodiments are only description
Property, without limiting the present invention in any way.
The preparation method of the biological reaction apparatus of embodiment 1：
Select 0.5mm thickness PMMA plates（Two sides carries diaphragm）As ice glue support plate, according to what is mentioned in embodiment
Size designs device sketch in mapping software（Device size is that 96, hole, array arrangement are had on 76 × 26mm, ice glue support plate
For 6 × 16, bore dia is designed as 1mm）.Then graphic file is transferred in laser engraving machine control program, carries out mould and cut
Cut, the PMMA ice glue support plates cut out are rinsed well with deionized water, and dried in 60 DEG C of baking ovens.PMMA two during being somebody's turn to do
Surface protective film is not taken off to prevent from polluting hydrophobic PMMA surface during the pre-gathering solutions of follow-up loading biological material support.Prepare
Mould can be put into room temperature preservation in clean sealed bag standby.
When preparing dimensional culture biomaterial scaffolds, above-mentioned mould is subjected to hydrophilic place with plasma cleaner in advance
Reason, time 1min.The selection of biomaterial is very extensive, and conventional polyethylene glycol and gelatin etc. can be adopted to prepare the device
In support.By taking gelatin support as an example, 4% aqueous gelatin solution is prepared, precooling on ice is placed in, then adds glutaraldehyde solution, make
The final concentration of glutaraldehyde reaches 0.1%, then takes 200 μ l to drip to mould one end, uses thin plate（Such as thin slide）Drop is uniformly applied and scraped
In die surface, solution can be filled in the array hole on mould naturally.Treat that the mould of requirement is mounted with material solution
Afterwards, it is placed in low temperature refrigerator as early as possible（- 12~-20 DEG C）, 12h is placed so that reaction is complete.Take out afterwards and be loaded with preformation
The mould of type support, is submerged into 0.1M sodium borohydride solution and soaks 10 minutes, to neutralize the complete aldehyde radical of unreacted, reduces
The autofluorescence of support, then with deionized water rinsing mould two to three times.After cleaning process terminates, mould is immersed in the water and is frozen into
Ice, is placed into freeze dryer and handles 12h, and the ice crystal in support distils, and tens to 100 microns of generation is mutual in the bracket
The duct of connection, can now take out mould, be placed in dry environment storage, so far, and prepared by biological reaction apparatus completes.
The concrete structure of the biological reaction apparatus prepared by the above method is as follows：
Two layers above and below the device point, designed using modular size, outward appearance is consistent with standard microscope slide, it is long
76mm, wide 26mm.Array hole design is consistent with commercially available 384 orifice plate, is 6 × 16 arrays.Upper strata is that nutrient solution stores plate, hole
A diameter of 1.6mm, lower plywood is ice glue support plate, and bore dia is that the distance of center circle in hole on 1mm, two plates is 4.5mm, passes through biofacies
Capacitive double faced adhesive tape good bond.
Embodiment 2 utilizes the operating method of biological reaction apparatus culture cell
Device is subjected to ultraviolet-ray sterilizing processing first, prevents that cell sample is contaminated in experiment.It will be used in advance real
The cell suspension of concentration, every milliliter 1~5 × 10 needed for the cell tested is prepared into6Individual cell.The cell for drawing 100~120 μ l hangs
Drop is added to one end of ice glue support plate, and cell scraping suspension is uniformly applied in the ice on the surface of plate, plate with clean sterile thin slide
Glue can be by cell automatic absorbing wherein, so far, and loading cells are completed.
Then 200 μ l cell culture fluids are taken, one end i.e. nutrient solution for being added drop-wise to device another side stores one end of plate, equally
The loading that nutrient solution is completed in plate surface is scraped with painting with clean sterile thin slide.Finally the reaction unit loaded is put
In the abundant container of humidity, 37 DEG C of cellar cultures.
Purposes of the biological reaction apparatus of embodiment 3 in screening medicine
If carrying out the screening experiment of cell drug toxicity, because each cultivating system is separate in device, can conveniently it enter
The addition of row various concentrations, variety classes drug molecule.
If experiment is equipped with high flux operating platform, the corresponding species of each reaction member addition that can be directly into device with
And the medicine of respective concentration.
If common laboratory detection is used, drug solution can be first added drop-wise to Simple medicament loading chip prepared by the present invention
On, it then will be loaded with the chip of medicine（Fig. 3）The nutrient solution storage plate one side of device is buckled in, medicine is that can slowly diffuse into carefully
Born of the same parents' cultivating system.
Medicine loads the preparation method of chip：Load medicine core piece in this embodiment is made using conventional photolithographic method, material
Using the polyethylene glycol material of Photocrosslinkable, molecular weight 4000, concentration 10%w/t, while being equipped with 0.5%w/t light trigger
I2959, ultraviolet light 35 seconds under the covering of photomask.The polyethylene glycol gel chip cleaned afterwards with clear water after irradiation ,-
20 DEG C freeze 6~7 hours, vacuum freeze drying 12 hours or so.
Carry the summary of medicine core chip architecture：Polyethylene glycol support on the chip equally uses cylindrical array pattern, and diameter is set
The loading cells chip size being calculated as between 1mm, cylinder in spacing 4.5mm, with the invention is consistent, convenient experiment.
Testing result：
It is respectively drug concentration profile picture before and after drug diffusion process shown in the figures of Fig. 4 or so two.Simulation results medicine
Thing can reach balance at 4.8 hours or so, and it is also seen that medicine still keeps dense after experience diffusion from Fig. 4 right figures
Spend gradient.Table 1 lists the loading concentration before drug diffusion, the theoretical calculation concentration after diffusion and actual measurement concentration.Through pair
Than this it appears that the actual dispersion process of medicine and theoretical prediction have very good corresponding relation.
Table 1
| Medicine loads concentration（μg/ml） | 97.66 | 195.3 | 390.6 | 781.3 | 1562.5 | 3125 | 6250 |
| Concentration after theoretical prediction diffusion（μg/ml） | 16.76 | 33.52 | 67.03 | 134.1 | 268.1 | 534.6 | 1069 |
| Concentration after actually measured diffusion（μg/ml） | 16.80 | 33.48 | 66.90 | 133.83 | 267.6 | 535.8 | 1071 |
Treat medicine effect a period of time（Generally 24h or more）Afterwards, device is directly put in 50ml centrifuge tubes（If making
Chip, removal are loaded with medicine）, 1000rpm centrifugations 2 minutes, the liquid in removal device, then according to addition cell training
The same method of nutrient solution will detect the reagent of cytoactive（Such as the blue solution of Armagh）Uniform apply is scraped on device, 37 DEG C of reactions 1
~2 hours, device is taken out, be placed on testing stand, the detected value of each unit in ELIASA detection means calculates different pharmaceutical
Under the conditions of cell relative activity.
Fig. 5 shows that apparatus of the present invention are cultivating testing result during human fibrosarcoma cell is with conventional two-dimentional porous plate
Comparing result.By Fig. 5 it will be evident that in apparatus of the present invention, the resistance to the action of a drug of cell is significantly improved, in dimensional culture state
Under, broad-spectrum anti-cancer drug adriamycin is 130.02 μM to the half-inhibition concentration of human fibrosarcoma cell, and in conventional two-dimensional
Porous plate training mode under, the half-inhibition concentration of the medicine only has 9.55 μM, two-dimentional training mode measure it is relatively low
Half-inhibition concentration can not truly reflect antiradiation drug reaction of the cell in original three dimensional growth environment, and this is trained with three-dimensional in recent years
The document report that the pattern of supporting improves drug resistant tumor cells is basically identical, therefore also demonstrates the accurate reliability of the device.
In the description of this specification, reference term " one embodiment ", " some embodiments ", " example ", " specifically show
The description of example " or " some examples " etc. means to combine specific features, structure, material or the spy that the embodiment or example are described
Point is contained at least one embodiment of the present invention or example.In this manual, to the schematic representation of above-mentioned term not
Necessarily refer to identical embodiment or example.Moreover, specific features, structure, material or the feature of description can be any
One or more embodiments or example in combine in an appropriate manner.
Although embodiments of the invention have been shown and described above, it is to be understood that above-described embodiment is example
Property, it is impossible to limitation of the present invention is interpreted as, one of ordinary skill in the art is not departing from the principle and objective of the present invention
In the case of above-described embodiment can be changed within the scope of the invention, change, replace and modification.
Claims (7)
1. a kind of biological reaction apparatus, it is characterised in that be formed by following structures：
First plate, first plate has first through hole；
Second plate, second plate has the second through hole；
First plate and the second plate are connected as one, and the first through hole is corresponded with second through hole,
Wherein, the first through hole includes multiple through holes, and second through hole includes multiple through holes,
Solid cell culture support is mounted with each first through hole, the solid cell culture support is ice glue,
Nutrient solution is loaded in each second through hole,
The size of the biological reaction apparatus is 76 × 26cm2,
Multiple through holes that the first through hole and the second through hole include are in 3 × 8 or 6 × 16 branches simultaneously respectively,
The radius of the first through hole and the second through hole is respectively 0.5mm and 0.8mm.
2. biological reaction apparatus according to claim 1, it is characterised in that first plate and the second plate are by poly- methyl-prop
E pioic acid methyl ester is formed, and first plate and the second plate are connected as one by biocompatible glue.
3. a kind of method of the biological reaction apparatus prepared described in claim 1 or 2, it is characterised in that including：
First plate is provided, first through hole is prepared on first plate；
Second plate is provided, the second through hole is prepared on second plate；
First plate and second plate are connected as one,
Wherein, the first through hole is corresponded with second through hole,
The first through hole includes multiple through holes, and second through hole includes multiple through holes,
The diameter of the first through hole is less than the diameter of the second through hole.
4. method according to claim 3, it is characterised in that first plate and the second plate are by polymethyl methacrylate
Formed, first plate and the second plate are connected as one by biocompatible glue.
5. a kind of method of the biological reaction apparatus culture cell described in utilization claim 1 or 2, it is characterised in that including：
The first through hole and the second through hole are subjected to hydrophilic treated；
The loaded in parallel ice glue in the first through hole；
Continue parallel repopulating cell in the first through hole for being mounted with ice glue；And
The loaded in parallel nutrient solution in second through hole.
6. purposes of the biological reaction apparatus in culture cell described in claim 1 or 2.
7. purposes of the biological reaction apparatus in screening medicine described in claim 1 or 2.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310359407.9A CN103396946B (en) | 2013-08-16 | 2013-08-16 | Biological reaction apparatus and its preparation method and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310359407.9A CN103396946B (en) | 2013-08-16 | 2013-08-16 | Biological reaction apparatus and its preparation method and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103396946A CN103396946A (en) | 2013-11-20 |
| CN103396946B true CN103396946B (en) | 2017-07-28 |
Family
ID=49560666
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310359407.9A Active CN103396946B (en) | 2013-08-16 | 2013-08-16 | Biological reaction apparatus and its preparation method and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103396946B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104480010B (en) * | 2014-11-28 | 2017-06-06 | 清华大学 | Biological reaction apparatus and its application |
| CN106153891B (en) * | 2015-04-09 | 2018-08-28 | 清华大学 | Three dimensional biological marker detection device, preparation method and the method for detecting biomarker |
| CN105108817B (en) * | 2015-09-09 | 2016-09-14 | 通用生物系统（安徽）有限公司 | A kind of manufacture method of the 384 hole solid phase synthesis devices for nucleic acid synthesis |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1330154A (en) * | 2000-06-27 | 2002-01-09 | 南京益来基因医学有限公司 | Cell microarray chip and its preparing process |
| FR2844052B1 (en) * | 2002-08-28 | 2005-07-01 | Commissariat Energie Atomique | DEVICE FOR MEASURING THE ELECTRIC ACTIVITY OF BIOLOGICAL ELEMENTS AND ITS APPLICATIONS |
| CN203065491U (en) * | 2012-09-28 | 2013-07-17 | 江苏吉锐生物技术有限公司 | Cell culture device |
-
2013
- 2013-08-16 CN CN201310359407.9A patent/CN103396946B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN103396946A (en) | 2013-11-20 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Yu et al. | Microfluidic bioprinting for organ-on-a-chip models | |
| Zhang et al. | Advances in organ-on-a-chip engineering | |
| Mi et al. | The crossing and integration between microfluidic technology and 3D printing for organ-on-chips | |
| Li et al. | Improving bioprinted volumetric tumor microenvironments in vitro | |
| CN111218404A (en) | Bionic multi-organ chip and preparation method and application thereof | |
| Hu et al. | Microfluidic technologies for vasculature biomimicry | |
| WO2007124481A2 (en) | Bioprinting three-dimensional structures onto microscale tissue analog devices for pharmacokinetic study and other uses | |
| Ahmed | Organ-on-a-chip microengineering for bio-mimicking disease models and revolutionizing drug discovery | |
| Park et al. | Reconstruction of in vivo-like in vitro model: enabling technologies of microfluidic systems for dynamic biochemical/mechanical stimuli | |
| CN103396946B (en) | Biological reaction apparatus and its preparation method and application | |
| CN112143642B (en) | Vascularized tumor micro-fluidic organ chip for in vitro culture and preparation method thereof | |
| WO2019237061A1 (en) | A 3d bioprinted perfusable platform for high-throughput disease modeling and drug screening | |
| Cui et al. | Pulsed microfluid force-based on-chip modular fabrication for liver lobule-like 3D cellular models | |
| CN108641931A (en) | A kind of digitlization microarray organ chip and its application | |
| CN101451105A (en) | Construction method of blood capillary model and microsystem chip thereof | |
| TW202303146A (en) | Bio-chips | |
| CN208762505U (en) | A kind of digitlization microarray organ chip | |
| US20230203417A1 (en) | Microfluidic device | |
| CN116478819B (en) | Microfluidic system for constructing three-dimensional organ microenvironment model, and preparation method and application thereof | |
| Mai et al. | MatriGrid® Based Biological Morphologies: Tools for 3D Cell Culturing | |
| Yu et al. | Vascularized organoid-on-a-chip: design, imaging, and analysis | |
| CN220166205U (en) | Organ-like co-culture chip | |
| CN117721019A (en) | Vascularized organ chip and application thereof | |
| Luo et al. | Development of Organs-on-Chips and Their Impact on Precision Medicine and Advanced System Simulation | |
| Wang et al. | Developing Liver Microphysiological Systems for Biomedical Applications |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20180928 Address after: 100084 KJ202-119 two, 6 building, 1 Zhongguancun East Road, Haidian District, Beijing. Patentee after: Beijing Hua Ting Biotechnology Co., Ltd. Address before: 100084 Haidian District 100084-82 mailbox in Beijing Patentee before: Tsinghua University |
|
| TR01 | Transfer of patent right |