CN104412109A - Cell culture and gradient migration assay methods and devices - Google Patents
Cell culture and gradient migration assay methods and devices Download PDFInfo
- Publication number
- CN104412109A CN104412109A CN201380018324.1A CN201380018324A CN104412109A CN 104412109 A CN104412109 A CN 104412109A CN 201380018324 A CN201380018324 A CN 201380018324A CN 104412109 A CN104412109 A CN 104412109A
- Authority
- CN
- China
- Prior art keywords
- cell
- combination
- aforementioned
- gradient
- micro
- 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.)
- Pending
Links
- 238000000034 method Methods 0.000 title claims abstract description 66
- 238000004113 cell culture Methods 0.000 title claims description 38
- 238000010232 migration assay Methods 0.000 title 1
- 210000004027 cell Anatomy 0.000 claims description 315
- 235000015097 nutrients Nutrition 0.000 claims description 41
- 239000000126 substance Substances 0.000 claims description 41
- 239000001963 growth medium Substances 0.000 claims description 39
- 239000000463 material Substances 0.000 claims description 37
- 230000010412 perfusion Effects 0.000 claims description 36
- 238000010586 diagram Methods 0.000 claims description 35
- 238000012258 culturing Methods 0.000 claims description 34
- 230000012292 cell migration Effects 0.000 claims description 30
- 238000003384 imaging method Methods 0.000 claims description 30
- 230000004087 circulation Effects 0.000 claims description 27
- 239000012530 fluid Substances 0.000 claims description 26
- 230000005012 migration Effects 0.000 claims description 25
- 238000013508 migration Methods 0.000 claims description 25
- 238000002474 experimental method Methods 0.000 claims description 21
- 239000007788 liquid Substances 0.000 claims description 21
- 238000009792 diffusion process Methods 0.000 claims description 18
- 238000004458 analytical method Methods 0.000 claims description 15
- 230000033001 locomotion Effects 0.000 claims description 15
- 239000003153 chemical reaction reagent Substances 0.000 claims description 14
- 230000008569 process Effects 0.000 claims description 14
- 239000013000 chemical inhibitor Substances 0.000 claims description 13
- 230000004044 response Effects 0.000 claims description 13
- 206010028980 Neoplasm Diseases 0.000 claims description 12
- 230000005484 gravity Effects 0.000 claims description 11
- 239000003814 drug Substances 0.000 claims description 10
- 210000002966 serum Anatomy 0.000 claims description 10
- 201000011510 cancer Diseases 0.000 claims description 9
- 230000032258 transport Effects 0.000 claims description 9
- 230000035605 chemotaxis Effects 0.000 claims description 8
- 230000007246 mechanism Effects 0.000 claims description 8
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 7
- 239000001301 oxygen Substances 0.000 claims description 7
- 229910052760 oxygen Inorganic materials 0.000 claims description 7
- 238000005204 segregation Methods 0.000 claims description 7
- 230000011664 signaling Effects 0.000 claims description 7
- 230000000694 effects Effects 0.000 claims description 6
- 230000010261 cell growth Effects 0.000 claims description 5
- 230000037361 pathway Effects 0.000 claims description 5
- 235000003642 hunger Nutrition 0.000 claims description 4
- 230000001394 metastastic effect Effects 0.000 claims description 4
- 206010061289 metastatic neoplasm Diseases 0.000 claims description 4
- 230000037351 starvation Effects 0.000 claims description 4
- 238000012360 testing method Methods 0.000 claims description 4
- 206010027476 Metastases Diseases 0.000 claims description 3
- 230000009087 cell motility Effects 0.000 claims description 3
- 230000008859 change Effects 0.000 claims description 3
- 210000004748 cultured cell Anatomy 0.000 claims description 3
- 229940079593 drug Drugs 0.000 claims description 3
- 230000001575 pathological effect Effects 0.000 claims description 3
- 230000029663 wound healing Effects 0.000 claims description 3
- 230000003466 anti-cipated effect Effects 0.000 claims description 2
- 239000005667 attractant Substances 0.000 claims description 2
- 230000031902 chemoattractant activity Effects 0.000 claims description 2
- 238000011161 development Methods 0.000 claims description 2
- 239000000975 dye Substances 0.000 claims description 2
- 239000005556 hormone Substances 0.000 claims description 2
- 229940088597 hormone Drugs 0.000 claims description 2
- 238000011534 incubation Methods 0.000 claims description 2
- 238000003973 irrigation Methods 0.000 claims description 2
- 230000002262 irrigation Effects 0.000 claims description 2
- 238000012544 monitoring process Methods 0.000 claims description 2
- 239000011148 porous material Substances 0.000 claims description 2
- 238000013139 quantization Methods 0.000 claims description 2
- 238000003860 storage Methods 0.000 claims description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims 3
- 239000012091 fetal bovine serum Substances 0.000 claims 3
- 206010052428 Wound Diseases 0.000 claims 1
- 208000027418 Wounds and injury Diseases 0.000 claims 1
- 238000001514 detection method Methods 0.000 claims 1
- 239000003102 growth factor Substances 0.000 claims 1
- 230000028993 immune response Effects 0.000 claims 1
- 230000001788 irregular Effects 0.000 claims 1
- 230000013011 mating Effects 0.000 claims 1
- 230000001737 promoting effect Effects 0.000 claims 1
- 230000004936 stimulating effect Effects 0.000 claims 1
- 230000007704 transition Effects 0.000 claims 1
- 238000004519 manufacturing process Methods 0.000 abstract description 6
- 238000009650 gentamicin protection assay Methods 0.000 abstract 1
- 230000009545 invasion Effects 0.000 description 74
- 238000013461 design Methods 0.000 description 34
- 238000011068 loading method Methods 0.000 description 30
- 239000002609 medium Substances 0.000 description 15
- 239000007789 gas Substances 0.000 description 14
- 230000006870 function Effects 0.000 description 13
- 239000000243 solution Substances 0.000 description 13
- 230000007774 longterm Effects 0.000 description 8
- 238000003556 assay Methods 0.000 description 7
- 230000006399 behavior Effects 0.000 description 6
- 230000008901 benefit Effects 0.000 description 6
- 230000004709 cell invasion Effects 0.000 description 6
- 230000001413 cellular effect Effects 0.000 description 5
- 238000005516 engineering process Methods 0.000 description 5
- 108010082117 matrigel Proteins 0.000 description 5
- 239000002245 particle Substances 0.000 description 5
- 238000011160 research Methods 0.000 description 5
- 239000000758 substrate Substances 0.000 description 5
- 230000003321 amplification Effects 0.000 description 4
- 230000036755 cellular response Effects 0.000 description 4
- 230000006872 improvement Effects 0.000 description 4
- 239000012120 mounting media Substances 0.000 description 4
- 238000003199 nucleic acid amplification method Methods 0.000 description 4
- 238000012546 transfer Methods 0.000 description 4
- 239000003981 vehicle Substances 0.000 description 4
- 238000007808 Cell invasion assay Methods 0.000 description 3
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 description 3
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 description 3
- 241000508269 Psidium Species 0.000 description 3
- 230000033228 biological regulation Effects 0.000 description 3
- 238000012512 characterization method Methods 0.000 description 3
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- 210000002744 extracellular matrix Anatomy 0.000 description 3
- 230000003287 optical effect Effects 0.000 description 3
- 239000002953 phosphate buffered saline Substances 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- 210000001519 tissue Anatomy 0.000 description 3
- 230000000007 visual effect Effects 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 230000004888 barrier function Effects 0.000 description 2
- 230000000903 blocking effect Effects 0.000 description 2
- 239000001045 blue dye Substances 0.000 description 2
- 230000032823 cell division Effects 0.000 description 2
- 230000009194 climbing Effects 0.000 description 2
- 238000004891 communication Methods 0.000 description 2
- 230000004069 differentiation Effects 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 230000008694 endothelial dysfunction Effects 0.000 description 2
- 230000014509 gene expression Effects 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- 238000007689 inspection Methods 0.000 description 2
- 230000002452 interceptive effect Effects 0.000 description 2
- 230000007794 irritation Effects 0.000 description 2
- 230000004807 localization Effects 0.000 description 2
- 230000003211 malignant effect Effects 0.000 description 2
- 238000005497 microtitration Methods 0.000 description 2
- 239000002417 nutraceutical Substances 0.000 description 2
- 235000021436 nutraceutical agent Nutrition 0.000 description 2
- CXQXSVUQTKDNFP-UHFFFAOYSA-N octamethyltrisiloxane Chemical group C[Si](C)(C)O[Si](C)(C)O[Si](C)(C)C CXQXSVUQTKDNFP-UHFFFAOYSA-N 0.000 description 2
- 238000006116 polymerization reaction Methods 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 238000010561 standard procedure Methods 0.000 description 2
- 230000003068 static effect Effects 0.000 description 2
- 108010035532 Collagen Proteins 0.000 description 1
- 102000008186 Collagen Human genes 0.000 description 1
- 208000005189 Embolism Diseases 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 102000009123 Fibrin Human genes 0.000 description 1
- 108010073385 Fibrin Proteins 0.000 description 1
- BWGVNKXGVNDBDI-UHFFFAOYSA-N Fibrin monomer Chemical compound CNC(=O)CNC(=O)CN BWGVNKXGVNDBDI-UHFFFAOYSA-N 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- 206010064390 Tumour invasion Diseases 0.000 description 1
- 239000012190 activator Substances 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 238000005452 bending Methods 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 230000031018 biological processes and functions Effects 0.000 description 1
- 230000017531 blood circulation Effects 0.000 description 1
- 230000009400 cancer invasion Effects 0.000 description 1
- 230000008568 cell cell communication Effects 0.000 description 1
- 210000003850 cellular structure Anatomy 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 239000002975 chemoattractant Substances 0.000 description 1
- 229920001436 collagen Polymers 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000012937 correction Methods 0.000 description 1
- 239000006059 cover glass Substances 0.000 description 1
- 238000013480 data collection Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 125000000118 dimethyl group Chemical group [H]C([H])([H])* 0.000 description 1
- 239000004205 dimethyl polysiloxane Substances 0.000 description 1
- 235000013870 dimethyl polysiloxane Nutrition 0.000 description 1
- 230000007646 directional migration Effects 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 238000007876 drug discovery Methods 0.000 description 1
- 238000007877 drug screening Methods 0.000 description 1
- 229920001971 elastomer Polymers 0.000 description 1
- 230000008030 elimination Effects 0.000 description 1
- 238000003379 elimination reaction Methods 0.000 description 1
- 230000013020 embryo development Effects 0.000 description 1
- 230000007717 exclusion Effects 0.000 description 1
- 229950003499 fibrin Drugs 0.000 description 1
- 238000000799 fluorescence microscopy Methods 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 239000008246 gaseous mixture Substances 0.000 description 1
- 230000007045 gastrulation Effects 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 238000009434 installation Methods 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- 208000024312 invasive carcinoma Diseases 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 238000009533 lab test Methods 0.000 description 1
- 238000003475 lamination Methods 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 210000004379 membrane Anatomy 0.000 description 1
- 230000006386 memory function Effects 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 230000001537 neural effect Effects 0.000 description 1
- 230000001123 neurodevelopmental effect Effects 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 210000000496 pancreas Anatomy 0.000 description 1
- 230000035699 permeability Effects 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 238000004987 plasma desorption mass spectroscopy Methods 0.000 description 1
- 210000004224 pleura Anatomy 0.000 description 1
- 229920000435 poly(dimethylsiloxane) Polymers 0.000 description 1
- 229920001296 polysiloxane Polymers 0.000 description 1
- 239000004810 polytetrafluoroethylene Substances 0.000 description 1
- 229920001343 polytetrafluoroethylene Polymers 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 239000012266 salt solution Substances 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 229920005573 silicon-containing polymer Polymers 0.000 description 1
- 229920002379 silicone rubber Polymers 0.000 description 1
- 238000004088 simulation Methods 0.000 description 1
- 230000007480 spreading Effects 0.000 description 1
- 238000003892 spreading Methods 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 238000004448 titration Methods 0.000 description 1
- 231100000041 toxicology testing Toxicity 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 230000002792 vascular Effects 0.000 description 1
- 210000002845 virion Anatomy 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
- C12M41/46—Means for regulation, monitoring, measurement or control, e.g. flow regulation of cellular or enzymatic activity or functionality, e.g. cell viability
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/02—Form or structure of the vessel
- C12M23/16—Microfluidic devices; Capillary tubes
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Electro-optical investigation, e.g. flow cytometers
- G01N15/1484—Electro-optical investigation, e.g. flow cytometers microstructural devices
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
- G01N33/5029—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects on cell motility
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/08—Geometry, shape and general structure
- B01L2300/0809—Geometry, shape and general structure rectangular shaped
- B01L2300/0829—Multi-well plates; Microtitration plates
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/08—Geometry, shape and general structure
- B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
- B01L2300/0867—Multiple inlets and one sample wells, e.g. mixing, dilution
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/08—Geometry, shape and general structure
- B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
- B01L2300/0877—Flow chambers
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2400/00—Moving or stopping fluids
- B01L2400/04—Moving fluids with specific forces or mechanical means
- B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces
- B01L2400/0406—Moving fluids with specific forces or mechanical means specific forces capillary forces
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2400/00—Moving or stopping fluids
- B01L2400/04—Moving fluids with specific forces or mechanical means
- B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces
- B01L2400/0457—Moving fluids with specific forces or mechanical means specific forces passive flow or gravitation
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2400/00—Moving or stopping fluids
- B01L2400/04—Moving fluids with specific forces or mechanical means
- B01L2400/0475—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure
- B01L2400/0487—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure fluid pressure, pneumatics
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N2015/1006—Investigating individual particles for cytology
-
- G01N2015/1027—
Abstract
A number of novel improved microfluidic configurations and systems and methods of manufacture and operation for a microfluidic invasion assay system.
Description
The cross reference of related application
To be title be " MICROFLUIDIC CELL CULTURE SYSTEMS " to the application and the U.S. Patent application 13/011 submitted on January 21st, 2011, the continuation part application of 857, and its require title to be " MICROFLUIDIC CELL CULTURE ARRAYS " and on January 21st, 2010 submit to 61/297,278 right of priority.
To be title be " CELL CULTURE AND INVASION ASSAY METHOD AND SYSTEM " to the application and the U.S. Patent application 13/436 submitted on April 1st, 2012, the continuation part application of 992, and it requires that the title submitted on April 1st, 2011 is the right of priority of 61/471,103 of " CELL CULTURE AND INVASION ASSAY METHOD AND SYSTEMS ".The title that the application also requires on February 5th, 2013 to submit to is the right of priority of 61/761,227 of " CELL CULTURE AND INVASION ASSAY METHOD AND SYSTEM ".
Copyright notice
According to 37 C.F.R. 1.71 (e); applicant notices, a part of this disclosure comprises material protected by copyright (such as, but not limited to any other side of figure, device photo or this motion (its copyright protection is may be maybe available in any administrative area).Copyright owner does not oppose anyone facsimile copy by patent document or patent disclosure, because it occurs, in any case but retain all copyrights in other side in patent and trademark office's patent document or record.
Technical field
The present invention relates in various embodiments for using microfluidic system to the chemical examination of the relevant behavior of the invasion and attack behavior or other small items that detect cell, system and device.Special embodiment relate to can with various standard automated processing system, to use together with pouring into the active of nutrient culture media or passive loading and be provided for that analysis of cells is attacked, moved, the high-throughput polyvoltine of chemotaxis or other character tests the configuration of automatic system.
Technical background
In this motion (comprising any document submitted to together with the application) Anywhere to any work, announce thing, the discussion of sale or activity should not be understood to admit that any work like this forms prior art.Any activity in this article, work or announce the discussion of thing and do not admit such activity, work or announce thing to exist in any special administrative area or known.
It is important technology for the application in drug screening, tissue cultures, toxicity screening and biological study that micro-current controlled cell is cultivated, and the biological function of improvement, better quality can be provided based on the data of cell, the reagent consumption of minimizing and lower cost.High-quality molecule and cell sample preparation to various clinical, research and other application be important.Vitro samples (it closely represents its body internal characteristic) can be of value to molecule and the cell application of wide region potentially.Cell or other biological process that is upper or the chemically material (being such as coated with the pearl of various biomolecule) of activity, characterization, cultivation and visual becoming in drug discovery, medical diagnosis on disease and analysis and other treatment multiple and experimental work are paid attention to day by day.
Discuss in above-cited and relevant patented claim relate to microfluidic system, device, method and manufacture a lot of in.Although should not read special restriction from those applications herein to any claim presented, these documents merged provide the useful background material about specific embodiment.
A field interested in cell assays system is the chemical examination of the characteristic can determining cell migration.Important in the characterization of other cell of such chemical examination in the characterization of various types of malignant cell and also under various stimulation.
Some chemical examinations using microchamber or micro-fluidic are proposed.Other system uses the standard culture plate with various obstacle embolus to attempt to detect cell invasion.But current available system about to easy to use, high-throughput or automatically application necessary a lot of in be failed.
Cultivate relevant various configurations, method and system as above-cited work in early time and patented claim discuss with micro-current controlled cell, and this work and those announce things and be merged in by reference herein.
Summary of the invention
The present invention relates to and the microfluidic cell culture improved and system---especially for aggressive or otherwise metabolism or can the cultivation of kinetocyte and the system of analysis---relevant various parts, system and method.In an aspect, the present invention relates to microfluidic cell culture, the system and method for the novelty of the advantage of the above-mentioned invasion and attack having and be better than using many culturing room plate or micro-fluidic structure or migration or mobile chemical examination.In another aspect, the present invention relates to and cultivate in cellular system for multiple micro-current controlled cell cultivation and/or cellular invasiveness laboratory test report unit are integrated into various many cells, such as to comprising various standard orifice plate form (such as 96 hole SBS culture plates or other panel formula, it comprises and has 6,12,24,96,384 or 1536 sample wells and open bottom standard orifice plate, thus allows the micro-fluidic structure that is attached to as described herein) microtitration AND DEWATERING FOR ORIFICE STRUCTURE in novel structure and method.
In special embodiment and example, design feature comprises provides invasion and attack assay device with general format, its allow pipeline and to the connector of plate itself elimination, use passive segregation drive stream to maintain the ability of long-term continous pouring cell chulture, the ability to ability, effectively the treatment gel culture medium of the outlet opening of micro-fluidic plate and/or cell invasion viewport or culture hole execution Direct Analysis.
Although a lot of examples discussed in detail are in this article designed to combined standard or customization orifice plate uses, but the micro-fluidic structure of various configuration as described in this article and cultivate unit and system and method and also can be used independent of any orifice plate (such as in various integrated chip laboratory system), implements integrated chip laboratory system and is not configured to use in conjunction with orifice plate or other micro fluidic device various or system.
For purposes of clarity, device, method and concept are mentioned in this discussion in particular example.But the present invention and aspect thereof can be applicable to various types of device and system.Therefore be intended that the present invention not to be limited, except as provided in claims and equivalents.
In addition, be known in the art, all systems as described in this article and embodiment can comprise multiple different parts and different functions with modular fashion.Different embodiments of the invention can comprise the difference mixing of element and function, and various function can be grouped into the part of various element.For purposes of clarity, the present invention is described at the system aspects of the innovative combination comprising much different innovative components and innovative components and known elements.Should not infer, limit the invention to the combination of all innovative components listed in any exemplary embodiment comprised in this manual.Unless separately had concrete regulation in this article, any combination of element described herein should be understood to include each sub-portfolio of any subset of those elements and also comprise any sub-portfolio with any subset of those elements of other elements combination any described herein, as the technology practitioner in this area by understand.
In accompanying drawing below and some in describing in detail, in the important individual embodiment of multi-part device or system, describe the present invention.This should not be understood to limit various novel aspect of the present invention, and it uses the instruction provided to can be applicable to other situation a lot of herein.In accompanying drawing below and some in describing, describe the present invention comprising in a lot of specific example embodiment of the special parameter relevant with the size of structure, the pressure of liquid or volume, temperature, electrical value, duration etc.Except the occasion provided so in the following claims, these parameters are exemplarily provided and do not limit the present invention, and it comprises other device or system with different size.In order to provide the object of more suggestive description, specific known making step, cell treatment step, reagent, chemistry or mechanical technology and other known elements making system or manufacturing installation according to a particular embodiment of the invention can be included exemplarily be presented.Those of skill in the art will understand, and except separately having in this article and specifically mentioning, make various known substituting in the process that can describe in this article.
All references of quoting in this is submitted to, announcement thing, patent and patented claim are thus in order to all objects are all incorporated into by reference.
Accompanying drawing explanation
The file of this patent comprises at least one accompanying drawing performed with colour.The copy with this patent of color drawings is provided when asking and pay necessary expense by United States Patent and Trademark Office.
Fig. 1 (A) is the schematic diagram according to the micro-fluidic plate design of the example of specific embodiment, have 24 invasion and attack chemical examination unit on 96 orifice plates in this example, each unit comprises 4 holes in this example: inflow entrance, cell/gel entrance, invasion and attack room and flow export.(B) be illustrate that the schematic diagram of the details of unit is cultivated according to a particular embodiment of the invention one invasion and attack.
Fig. 2 is the photo being filled with the single stream unit of example of blue dyes by the image diagram obtained from top (A) and bottom (B), and wherein bottom image by climbing over plate and being acquired on top-down direction, makes the left side of ingate in two pictures.
Fig. 3 A-C be according to a particular embodiment of the invention being equipped with gel with a series of microphotos in region of the invasion and attack room after illustrating by the invasion and attack chemical examination operation of gradient and cell migration.
Fig. 4 A-B be illustrate according to a particular embodiment of the invention in assay system and device by the cancer cell invasion of gradient and the microphoto of cell migration.
The configuration that Fig. 5 A-B diagram example cell culturing room with multiple entrance according to a particular embodiment of the invention designs and operation.
The configuration of the cell culture chamber design that Fig. 6 A-C diagram example is according to a particular embodiment of the invention substantially rectangular and the design of gradient room and operation.
Fig. 7 diagram is used for the mechanical accompanying drawing of exemplary customized plate framework of micro-fluidic living cells imaging, and which illustrates 4 independently unit (such as going), for improvement of the large imaging window of optical device, air enter the/out extending space that improves between the vacuum-packed hole of manifold of mouth (such as adjacent to imaging window) and being used in this example.In this example, there are every unit 6 ingates when having 2 outlet openings (in this example, an outlet opening amplifies to hold more volume).
Fig. 8 diagram is according to 2 the cultivation cell boards having three inflow entrances, imaging window, cell entry and flow exports for each unit of specific embodiment.
Fig. 9 diagram is according to the 16 unit versions having the cultivation cell board of three inflow entrances, imaging window, cell entry and flow exports for each unit of specific embodiment.
Figure 10 diagram accompanying drawing with the example board manifold of gas loading line according to a particular embodiment of the invention.
Figure 11 A-B is that diagram has four independent schematic diagram and photos cultivating the example of the active control panel of unit according to specific embodiment, and each is cultivated unit and has 6 inflow entrances, culturing room and two flow exports.
Figure 12 A-C is that diagram has four independent diagrams of cultivating the further example culture plate of unit according to specific embodiment, each cultivate unit have can be used for putting into practice the one or more methods described in this article 6 inflow entrances, culturing room and two flow exports.
Figure 13 diagram according to specific embodiment being exposed to an example of the cell migration after stable gradient.
Figure 14 exemplarily illustrate according to specific embodiment be used for illustrate that gradient is drawn on the example X/Y cell migration of the impact of the signaling in microfluidic cell culture better.
Figure 15 exemplarily illustrates and moves according to the example cell of the function as the distance of advancing in microfluidic cell culture of specific embodiment.
Figure 16 exemplarily illustrates and exemplifies according to particular implementation the cell being exposed to stable gradient in microfluidic cell culture and to obtain than the signaling in stable media environment and draw faster.
Figure 17 illustrate according to specific embodiment to the exposure irritation cell of a gradient example towards the active migration of high concentration meeting point.
Figure 18 A-C illustrates the top view of the schematic diagram of example manifold according to a particular embodiment of the invention, side view and plan view.In this example, the air of eight pipe lines for compressing on the right, and each pipe line is configured to provide pressure to the row cell entry hole in micro-fluidic array.Leftmost in the drawings line is used for vacuum and the external vacuum ring be connected at manifold ambient.Each row in hole are generally connected to single pressure line, and the hole wherein on imaging area is skipped.
Figure 19 diagram example system and manifold for operating micro-fluidic plate according to a particular embodiment of the invention.
Figure 20 diagram there are additional gas line and object lens and the manifold in five active holes in the micro-fluidic plate being connected to culture apparatus be shown according to a particular embodiment of the invention.
Figure 21 is the block scheme that representative illustration logical unit is shown, wherein various aspect of the present invention can be embodied.
Figure 22 (table 1) diagram according to a particular embodiment of the invention can evaluated or medicine or other treatment can for the example of its tested disease, situation or state.
Embodiment
1. summarize
Definition
" particle " refers to biological cell, and shape is as mammal or bacterial cell, virion or liposome or other particle that can be chemically examined according to the present invention.Such particle has the minimum dimension between about 50-100 nm, and can be as big as 20 microns or larger.When for describing according to cell assays of the present invention, term " particle " and " cell " use interchangeably.
" microchannel " or " passage " or " circulation road " are commonly referred to as the micro-meter scale passage of the various parts for fluidly connecting system according to a particular embodiment of the invention and device.Microchannel has rectangle (such as square) or circular cross section usually, wherein side and depth dimensions respectively between 10 and 500 microns and between 10 and 500 microns in a preferred embodiment.The fluid flowed in microchannel can show micro-fluidic behavior.When microchannel for mentioning in microwell array device of the present invention, term " microchannel " and " passage " use interchangeably." circulation road " generally represents and is designed so that nutrient culture media, reagent or other fluid or gel and the passage that passes through of cell in certain embodiments." culture channel " or " cell chulture passage " generally represents that cell is designed to flow through and a part for the cell chulture structure also retained during cell chulture (although cell can be arranged in the special cultivation region of culture channel in certain embodiments)." air duct " generally represents the passage for the roughly micro-meter scale allowing gas (such as air, rich oxygen containing potpourri etc.) to pass through near circulation road or cultivation region." perfusion channel " is sometimes used to indicate and allows nutrient culture media to be filled into the circulation road of cultivation region and any perfusion path or structure.
" obstacle " or " Diffusion Barrier " or " perfusion obstacle " or " mass transfer obstacle " refer to the combination of solid construction and the path less than circulation road, and it generally makes circulation road be separated with cell chulture district or room.Path is generally than microchannel height and/or width little (such as about 5-50% or about 10%), and be designed in certain embodiments prevent cell, other cultivation article and gel shift in certain embodiments in circulation road, allow some fluids flowing of any combination by diffusion, perfusion or mass transfer mechanism, it generally has and to flow much higher fluid resistance than the fluid in circulation road simultaneously.In an example embodiment, obstacle has 4 microns high and otherwise along the path that the most of length of microchannel extends.In other embodiments, obstacle has approximately equally high with microfluidic channel but a lot of paths of about 4 microns wide.Obstacle also can allow cell or cell component through obstacle or other material or some migrations of being small enough to through the particle of path.
" micro fluidic device " refers to the device with various or the hole connected by micro-meter scale microchannel, and wherein fluid shows micro-fluidic behavior by its flowing through passage.
" microwell array " refers to the array of the two or more micropores formed on substrate.
" device " is widely used in this area and comprises the term of the meaning of wide region.Such as at its most the most uncomplicated level place of fundamental sum, " device " can represent the substrate of feature with such as passage, room and mouth simply.The level of complexity place increased, " device " also can comprise around described feature substrate or there is other layer of the micro-fluidic feature as one man or independently operated.At the level place that it is the most complicated, " device " can comprise and promote the global function substrate that the interactive object between the external world and the micro-fluidic feature of substrate coordinates.Such object can be variously referred to as holder, shell, housing or similar terms as discussed below.As used in this article, term " device " refers to any one in context these embodiments denotable or level of complexity.
" any combination " of element as used in this article or claim refers to the combination that element mentions thing or mentioned any Individual elements.
Microfluidic system provides powerful instrument to guide Bioexperiment.Recently, especially obtain universal based on elastomeric micro-fluidic due to its optical clarity, gas permeability and simple method for making.But, the additional step that the hole of the labour intensive passed through elastic body with the interface requirement of final user and pipeline and syringe pump are connected.
The present invention relates to the integrated micro-fluidic for various cultivation and chemical examination application.The invention still further relates to the manufacture method for using such plate to make the micro-fluidic of cell culture automation and parts and system.The advantage of specific embodiment comprises standard microtiter plate form, without pipeline cell chulture and the use of bionical microenvironment for chemically examining invasion and attack, migration or chemotaxis cell behavior.
The system (such as using 96 hole on-gauge plates) that standard technique and equipment for the treatment of standard microtiter plate operate according to a particular embodiment of the invention can be used, as in known in the art.Such as, realize liquid and/or gel with standard volumetric pipette mechanism or cell distributes, and can carry out and the cell chulture of existing incubator and plate reader compatibility and analysis.
According to another embodiment of the invention, novel loading cells system uses pneumatic manifolds and Pneumatic pressure to be placed in micro-cultivation region by cell.When adding this loading cells system, other automatic equipment existed for the treatment of standardized titration plate can be used to carry out the cultivation of full automation micro-current controlled cell and analyze.
In a further embodiment, segregation drive stream cultivates the nutrient culture media level error of configuration using between entrance and exit hole and design fluid resistance realizes the expectation flow rate in nL/min situation.This provides and nutrient culture media " passively " can be made to flow the remarkable advantage of long-time section (such as nearly 4 days), and not using huge external pump or pipe, it allows the easy foundation of the analysis of the one or more time periods after cultivation starts and the easy reading of invasion and attack chemical examination structure when attacking chemical examination.
In a further embodiment, the present invention relates to for allowing to adhere to and/or the long term time of invasion and attack or migrating cell passes the microfluidic system of control of the cell culture environment of microexamination.According to a particular embodiment of the invention, the invention provides the multiple micro-fluidic fluid chamber of the inspection allowing passage of time microexamination experiment and the cell invasion in the middle of other chemical examination.Micro-fluidic room uses perfusion obstacle to come isolated cell and circulation road and attack obstacle to study in culturing room and the invasion and attack character of attacking the cell between room.Example embodiment is formatted to standard orifice plate, and it allows liquid and cell/gel sample to use standard facility to be directly moved in suitable inlet reservoirs.
In certain embodiments, customize aerodynamic flow controller to can be used for switching in loading cells to cultivation region and between different exposure solution.Numerical software interface can be used for allowing user to specific input (pulse, inclined-plane etc.) along with the programming of time is exposed to sophisticated functions to make cell during passage of time imaging.
Dynamic response in living cells is the basis for phenomenon (such as bio signal process, Gene expression and regulation, differentiation and cell division).In certain embodiments, the present invention relates to control the system of cell micro-environment with the multiplexed format of current cell culture processes compatibility.High power fluorescent microscope can be used to carry out quantization cell response with the dynamic information obtaining having daughter cell resolution.This ability has a wide range of applications in cellular systems biology, and wherein dynamic unicellular response experiment is current is unpractiaca.Although major part when being exposed to an only nutrient culture media/reagent mixture or passive system completely can be used according to some invasion and attack chemical examination embodiments of specific embodiment, can use complicated reagent scheduling to utilize manifold as described herein to perform other invasion and attack chemical examination according to specific embodiment.
2. micro-fluidic culture systems and array
Application above-mentioned discusses the configuration of multiple different cell chulture and manufacturing technology.The part of the operation in cell chulture district and material are useful as the background of this discussion.In some examples wherein, one or more micro-cultivation region is connected to nutrient culture media or reagent passage via the grid (or spreading entrance or conduit) of fluid passage, and wherein grid comprises multiple intersection high fluid resistance perfusion path.In an example discussed, path in grid is in height about 1 to 4 μm, be about 25 to 50 μm and be about 5 to 10 μm on width in length, grid allow nutrient culture media or between reagent passage and cultivation region evenly diffusion, and allow easier manufacture and evenly diffusion.High fluid resistance ratio (ratio such as in the scope of about 10:1,20:1 to 30:1) the comparatively Zao application also discussed between microchamber and perfusion/diffusion paths or grid provides the lot of advantages of cell chulture, such as: the size exclusion of (1) cell; (2) location of the cell in microchamber inside; (3) the unified fluid environment being used for Growth of Cells is promoted; (4) ability of the array of microchamber or cultivation region is configured; (4) what make is easy, and (5) do not have the manipulation of the reagent of extensive valve network.Illustrate example, wherein according to a particular embodiment of the invention, lattice-shaped perfusion obstacle than cultivation region much shorter or close to identical height or at identical At The Height, and can which illustrates the various configurations of culture apparatus in addition.
3. invasion and attack chemical examination unit
In a particular embodiment, the present invention also comprises the micro-fluidic plate for the chemical examination of 3D cancer cell invasion.In specific example implementation, plate uses the standard 96 orifice plate form with 4 holes connected by microfluidic channel to create each other flowing and invasion and attack chemical examination unit (every plate has such as 24 unit in a particular embodiment).In certain embodiments, flow by such as other local capillary force of discussing and segregation drive in this article, it allows plate operate in standard culture case and does not have outside to be connected after the initial introducing of cell and nutrient culture media.In certain embodiments, the cell in 3D gel receives in culturing room by device of the present invention.Culturing room is separated with invasion and attack room by invasion and attack obstacle, and both is all separated with circulation road by one group of such as micro-fluidic hole of 8x8 micron xsect or path (in this article sometimes referred to as invasion and attack obstacle), thus imitates the internal milieu of tumor invasion.
Fig. 1 (A) is the schematic diagram according to the micro-fluidic plate design of the example of specific embodiment, unit are chemically examined in its 24 invasion and attack had in this example on 96 orifice plates, and each unit comprises 4 holes in this example: inflow entrance, cell/gel entrance, invasion and attack room and flow export.In this embodiment, contact with microchannel with the liquid in flow export at inflow entrance, cell/gel entrance.Hole on invasion and attack room keeps empty in order to better image quality.The basal surface of plate is glass slide.Every plate has 24 to flow unit (each unit is that 4 holes are taken advantage of in 1 hole, and 8x12 orifice plate is formed 8x3 array).
Turn back to the schematic diagram shown in Figure 1A-B, this figure provides three amplification levels.F7 illustrates an invasion and attack room according to specific embodiment details with the district of amplifying most of the special hole site of instruction in example 96 orifice plate is marked as in Figure 1A-B.This invasion and attack chemical examination/cultivation region can be understood to include 5 main region.
Cell/gel loads the bottom place that passage is illustrated in figure.According to specific embodiment, be blended in cell in gel (such as matrigel, collagen, fibrin etc.) by capillary flow or use as described herein other initiatively or passive loading attachment be loaded onto in foot passage.In operation, passage is designed such that gel-filled loading passage and also fills invasion and attack obstacle and attack the part or all of of room, but by perfusion obstacle.In an example embodiment, loading passage is 550 μm and is in height 50 μm on width.
According to specific embodiment, load passage and be separated with invasion and attack room by invasion and attack obstacle.In specific example, invasion and attack obstacle is made up of the network of the passage being similar to 50x8x8 μm of (LxWxH) size.These are in certain embodiments or become and be filled with gel or liquid, and imitate endothelium dysfunction in the tissue.It is indoor that invasive carcinoma cell can move to invasion and attack through the narrow passage of invasion and attack obstacle.Invasion and attack room is in this example approximately 4.8 x 0.5 x .05 mm in size (LxWxH), and for invasion and attack or from loading the number count of cell of channel migration by invasion and attack obstacle.During chemical examination operation, the cell in this room is by microscope or other device are counted and are quantized the invasion and attack index with determining hole manually or automatically.
Perfusion obstacle is the network of the passage of the size of 100x4x2 μm (LxWxH) in certain embodiments, and it is separated invasion and attack room and circulation road.Narrow xsect prevents cell and gel through inculcating obstacle.Nutrient culture media is (with the medicine carried in the medium, comprise chemical inhibitor, dyestuff or attacking in chemical examination or other material used in cell chulture) cross over the diffusion of perfusion obstacle and form gradient to attacking cell, imitate the tumor environment in vascular system.
100x50 μm of (WxH) circulation road carries from inlet bore by attacking the fluid of room, and is emptied to outlet bore.From the nutraceutical diffusion feeder cell of the stream by perfusion obstacle.This passage stimulates the blood flow in health.In specific example embodiment, segregation drive flow rate is set to ~ 20 μ l/ days, and it allows be greater than the continuous stream experiment of 3 days and do not refill hole.
As above state, the size provided herein cultivates unit for example.According to various specific embodiment, any size being suitable for special nutrient culture media or cultivation article can use according to other instruction provided herein.
4. invasion and attack chemical examination plate
According to specific embodiment, invasion and attack chemical examination unit as above is configured in standard culture orifice plate and runs while allowing multiple invasion and attack to chemically examine experiment.Multiple chemical examinations that these experiments can comprise single object---identical or different tissue sample---, the multiple chemical examinations from different object, and chemical examination cell being exposed to different nutrient culture media, hormone or other stimulus, medicine, chemical inhibitor etc. can be comprised.
Although illustrate 4 holes chemical examination unit on 96 orifice plates, different cell size and different culture plate sizes also can embody the present invention, as from the discussion provided herein with will be clearly in the relevant application be incorporated to.
Fig. 2 is the photo being filled with the example single current unit of blue dyes by the image diagram obtained from top (A) and bottom (B), and wherein bottom image by climbing over plate and being acquired on top-down direction, makes the left side of ingate all in two pictures.
5. exemplary operations
Fig. 3 A-C be according to a particular embodiment of the invention being equipped with gel with a series of microphotos in region of the invasion and attack room after illustrating by the invasion and attack chemical examination operation of gradient and cell migration.The matrigel mixed with fluorescent dye (redness) is loaded into by capillary flow and loads in passage and be polymerized 15 minutes under 37C.Fig. 3 A illustrates 40 times of amplifications of invasion and attack room, its part gel-filled loading passage, invasion and attack obstacle being shown and attacking room.Fig. 3 B illustrates 200 times of amplifications of invasion and attack obstacle.Polymeric gel can be inner and in sight in invasion and attack room at invasion and attack obstacle.Fig. 3 C illustrates 200 times of amplifications of perfusion obstacle, and it illustrates that gel can not cross narrow passage network.As will be understood further from instruction herein, " gel " can have the various viscosity of being down to fluid viscosity in specific embodiment and specific test.In certain embodiments, the gel viscosity that obstacle allows relative broad range used according to the invention is poured into.
Fig. 4 A-B be illustrate according to a particular embodiment of the invention in assay system and device by the cancer cell invasion of gradient and the microphoto of cell migration.In this example, HT-1080 aggressive mankind chest cancer cell to be loaded in 3D matrigel and to be perfused with the nutrient culture media comprising 10% serum.Fig. 4 A illustrates that the cell and then after the loading and polymerization of gel is positioned on the bottom side of invasion and attack obstacle.Fig. 4 B illustrates the cell after with 24 hours of perfusion cultures of serum (HT-1080 invasion and attack known signals) that comprise nutrient culture media, some cells in cell through matrigel and the migration of invasion and attack obstacle to occupy invasion and attack room.Image is obtained with phase contrast under amplifying at 40 times.
In a further embodiment, various strategy can be used for removing some cells in all cells in invasion and attack room, to carry out other analysis.According to specific embodiment, the present invention also by providing culture environment to promote this, until they are removed in the invasion and attack room maintaining cell.
In a further embodiment, by such as other local air flue of describing and pore promote the oxygen diffusion that enters through the material limiting microfluidic channel (such as silicone elastomer dimethyl silicone polymer (PDMS)) structure in cultivation region in this article.
As in other local discussion, various amendment can be carried out to cell chulture district as above.Various configuration is possible to perfusion obstacle (such as lattice-shaped access structure).To imply that other changes herein for there are the those of skill in the art of the instruction provided.
Disclosed structure also can be suitable for being used in the system in the more or less hole on the plate of standard microtitration orifice plate or Complete customization or part customization above, the system described in such as other example in quoted document and in this article.
Plate as described herein and system can with the cell chulture district such as to describe in above-cited patented claim with attack room and micro-fluidic flow structure other configure together with use.In the design of an amendment, the cell chulture district provided is the cell culture chamber of rectangle in essence.Cell culture chamber has cell entry on the right and exit passageway, and flow export on the right.In this example, cell pathway is paired, and wherein center is loaded for stream of cells, and exporting being used as stream of cells on either side.
Once cell is loaded, after any aggressive cell has sufficient time to move through invasion and attack obstacle, invasion and attack chemical examination is as proceeding of summarizing above.
The configuration that Fig. 5 A-B diagram example cell culturing room with multiple entrance according to a particular embodiment of the invention designs and operation.This example comprises the cell/gel with intersection hatch perfusion via design and pours into obstacle and attack obstacle as discussed above.The hatch design that intersects allows cell in gel-type vehicle to flow in room and allows the perfusion of nutrient culture media.Although the hatch perfusion obstacle that intersects is preferred at present in some designs, also realize the culturing room that there is different perfusion obstacles or do not pour into obstacle according to specific embodiment.Outlet on the same side of obstacle and entrance is included at the parameatal stream of nutrient culture media.Fig. 5 A illustrates general embodiment, and its middle outlet and inlet opens are illustrated in the right.Fig. 5 B illustrates the access road on the left side and the exit passageway on the right, and this is configured in use in some example system of orifice plate as described herein and is more suitable for.This figure also provides the detailed example size of sample design according to a particular embodiment of the invention.Therefore in a further embodiment, cell culture chamber is modified to allow more easily cultivating of the cell in 3D gel-type vehicle.In this design, perfusion obstacle is separated as directed cell chulture district and circulation road.Obstacle is designed to 3D gel to remain in culturing room.Obstacle is coupled with above-described 3 passage cells/gel entry design the key character of the performance being to provide improvement.By having inflow entrance/outlet separately on every side of obstacle, fluid gel may be positioned in culturing room, and not make its choked flow passage.
Invasion and attack obstacle as above be placed on by figure dotted line indicate region in, and for make cell enter with culturing room with invasion and attack room be separated, as from instruction herein by understand.In the alternative embodiment, can perfusion channel be provided, make them only adjacent to invasion and attack room.
As in other local discussion, in certain embodiments, the invention provides the biological cell cultivation of responsive gel entrapment culture matrix (such as Matrigel, Geltrex, ossein etc.) of such as serviceability temperature and the 3D gel environment of invasion and attack chemical examination.Example gel is liquid under 4C, and it is such as polymerized under room temperature or 37C.In an exemplary method, cell mixes with the cell suspending liquid on ice at first.Then solution be moved in cell entry hole, and be transported in micro-fluidic room and cultivation and invasion and attack room via capillary flow.In specific example, plate is kept at room temperature.Flow rate allows enough cell/gel solutions to fill culturing room completely before the polymerization, and cell does not enter in invasion and attack room owing to attacking the size of path between fluid flow periods simultaneously.Perfusion obstacle prevents any gel solution from draining in circulation road.When gel warms, it is aggregated in semi-solid quality, and wherein cell is embedded in cultivation region.Flowing through of nutrient culture media in circulation road is attacked room and is diffused in cell culture chamber by gel, and trophocyte is to cultivate, simultaneously for aggressive cell provides attractant to move to invasion and attack room through invasion and attack obstacle.This novel designs allows the 3D alphael culture system that the invention provides in micro fluidic device, avoids the problem making gel blocking circulation road simultaneously.
In the example shown in Fig. 5 B, blue region instruction air stream, and be optional and be not present in all embodiments.Grey area instruction has the circulation road of the example height of about 40 μm, and red color area instruction has cell chulture and the invasion and attack district of the example height of about 200 μm, and the instruction of green district has the perfusion obstacle of the example height of about 2 μm.Yellow invasion and attack obstacle generally has the height identical with cultivation region (such as 200 μm) or similar height, but will have and attack blocking structure as above.
Once cell is loaded, have time enough to pass invasion and attack after obstacle moves at any aggressive cell, what invasion and attack chemical examination was just as above summarized proceeds.
3D gel systems
In the example system being sometimes called as 3D:M in this article, the multiple Perfusion Imaging of cell can be performed in 3D gel-type vehicle.Example board comprises and 24 of cell/gel selected as user can be housed independently cultivate unit.In example system, every a line (A-H) of plate comprises 3 and completely independently flows unit (each stream unit has 4 holes), and it is made up of nutrient culture media entrance (such as row 1,5,9), cell chulture/invasion and attack/imaging hole (such as row 2,6,10), cell/gel entrance (row 3,7,10) and outlet (row 4,8,12).Oxygen diffusion passage (blueness) provides gas to shift to cell.Entrance is designed to allow nutrient culture media to flow to cell via segregation drive process continuously with 40 μ l/ days.In this example, each room is 1.5 x 0.5 mm in size, has the height of 200 μm.Perfusion obstacle guarantees the even nutrients transfer by gel-type vehicle, and bottom thin cover glass, (170 μm) allow optimal imaging quality.Invasion and attack obstacle is provided in the separation of cultivating between region and invasion and attack region.Can load as the 3D gel in such a system of the execution above and described in the reference be incorporated to.
Other place as is in this article discussed, and various novel micro-current controlled cell culturing room can be integrated with hole titer plate device according to a particular embodiment of the invention with any one in the micro-fluidic structure be associated, and uses in grand cell culture assay as usual.Provided below is a lot of specific example, although the present invention includes for other system integrated with micro fluidic device.
In this design, each is cultivated unit and is made up of 4 hole sites.First hole is used for perfusion cultures base, and the second hole is used for cell entry, and the 3rd hole is used for making the imaging of micro-fluidic room, and the 4th hole is outlet.Cell disorders/perfusion channel by celluar localization to cellular regions, and improve during continuous perfusion culture nutrients transport.Cell entry enables cell via gravity or Surface Tension Method by quick load to the low fluid resistance of outlet pathway, and does not have external cellular load mechanism.The high fluid resistance of perfusion Inlet flow passages allows the long-term continous pouring via the nutrient culture media of run by gravity, and without any external pump mechanism.Invasion and attack barrier operations is to make, through cultured cells and invasion and attack region disconnecting, to chemically examine to carry out invasion and attack.
In example particular system, Cytology Lab is designed to imitate space organizational environment, and wherein cell is embedded into or covers in physiological cells epimatrix (ECM), and is fed from the capillary channel of continous pouring via diffusion.Cell micro-environment makes it possible to carry out the long term growth in such as 200 micron thickness gel layers.Oxygenate passage maintains enough gas transport, and allows high-quality cell imaging bottom glass cover-slip.Standard layout allows senior microfluidic cell just to operate as typical 96 orifice plates.Gravity-driven perfusion design eliminates the needs connected pump or pipeline, as mentioned above.
In example system, the anticipated number of every unit cell is about 500 cells.Example irrigation rate is 40 ul/ days for individual unit.Cytology Lab volume is 150 nL, and chamber size is 1.5x0.5x0.2 mm.Gas diffusion membrane is the 50 um silicone with basal surface #1.5 thick lid slide.
Second obstacle that also invasion and attack district can be used to be separated with cultivation region for the open top micro-current controlled cell culturing room of continous pouring is modified.
6. example gradient culturing room
The configuration of the cell culture chamber design that Fig. 6 A-C diagram example is according to a particular embodiment of the invention substantially rectangular and the design of gradient room and operation.In an exemplary design, the cell chulture district provided is the cell culture chamber of rectangle in essence.As in Fig. 6 A and Fig. 6 B, illustrated cell culture chamber has the cell entry and exit passageway E2 that illustrate on the right and the flow export E1 also illustrated on the right.In this example, cell pathway is paired, and wherein center is loaded for stream of cells, and exporting being used as stream of cells on either side.The inflow entrance of multiple separation is illustrated in the left side, is marked as A1, A2, B1, B2, C1, C2, and inflow entrance has comb mesh pattern to prevent from being blocked by cell in this example design.Oxygen diffusion passage is illustrated as around room.Outlet E1 provides the outlet of the fluid stream of partly isolating with culturing room.
Fig. 6 B illustrates the loading cells of cultivation unit as shown in Figure 6A.Cell is loaded via lower resistance fluid path (having the higher drag in flow path).Cell is prevented from choked flow path by resistance ratios (cell preferentially flows to cell outlet instead of circulation road).Passage in this special embodiment be arranged such that cell enter with cell out passage on the right side of room.This causes unique feature, wherein cell flow to into indoor, turn through 180 degree, and flow out, as shown in by Fig. 6 B to enter into the sharply bending streamline of cell out path from cell illustrated.Therefore, according to a particular embodiment of the invention, cell is loaded (via capillary force) and from the right passage in top and bottom out from the right passage in one or more center.Very small amount of stream is towards side outlet passage directed (the longer more unbending streamline shown in Fig. 6 B exits at the left hand edge of room).Effluent is not important for loading cells, but with helping cell to be more uniformly distributed in room.Due to the low viscosity of stream, cell is parked on room floor naturally, and without any need for physical obstacle.Cell outlet path helps to make loading become symmetrical, and increases the quantity being loaded into indoor cell.This load mechanism can be used for loading cell, particulate, pearl, gel, having the gel etc. of cell.
Fig. 6 C illustrates that the use of the design such as in Fig. 6 A-B to set up stable gradient in cell culture chamber design according to a particular embodiment of the invention.Example design in Fig. 6 C only somewhat different than the design of Fig. 6 A-B, and is applied to other design to the operator scheme that one of these two kinds designs herein describe.In the example of Fig. 6 C, cell pathway is azygous.Three azygous inflow entrances are illustrated in the left side, and these also have comb mesh pattern to prevent and are blocked by cell.Oxygen diffusion passage is generally placed near room, although do not illustrate in this drawing.Fig. 6 C also illustrate according to a particular embodiment of the invention in micro fluidic device as above by making 2(or more) plant solution flowing in culturing room, create gradient.
7. movable control pours into plate
Fig. 7 diagram is used for the mechanical accompanying drawing of exemplary customized plate framework of micro-fluidic living cells imaging, and which illustrates 4 independently unit (such as going), for improvement of the large imaging window of optical device, air enter the/out extending space that improves between the vacuum-packed hole of manifold of mouth (such as adjacent to imaging window) and being used in this example.In this example, there are every unit 6 ingates when having 2 outlet openings (in this example, an outlet opening amplifies to hold more volume).
Fig. 8 diagram has 2 of three inflow entrances, imaging window, cell entry and flow exports to cultivate cell board according to every unit of specific embodiment.
Fig. 9 diagram has 16 unit versions of the cultivation cell board of three inflow entrances, imaging window, cell entry and flow exports according to every unit of specific embodiment.
Figure 10 diagram accompanying drawing with the example board manifold of gas loading line according to a particular embodiment of the invention.
Figure 11 A-B is that diagram has four independent schematic diagram and photos cultivating the example of the active control panel of unit according to specific embodiment, and each is cultivated unit and has 6 inflow entrances, culturing room and two flow exports.Figure 11 A illustrates to have the plate design that 4 are independently flowed unit (row of plate), and it has 6 entrance solution, open chamber or other culturing room, outlet and run by gravity passage.Figure 11 B illustrates four open chamber (green circle), its have entrance stream on open chamber and under outlet flow.This design allows stream to be delivered to exit passageway from entrance is logical, and does not overflow open chamber.The availability of multiple liquid or reagent inlet provides living cells imaging and other experiment in cell biology and chemically examines particularly preferred system.In such a system, research can study the cultivation of pancreas or other organ cell or cancer cell, to determine how they make response to different medicines or via other stimulus that entrance is introduced.In the specific embodiment of this design, gravity hole also provides and promoted to maintain (such as feeding) cell before experiment is performed.Plate can be sealed to pneumatic manifolds, thus allows to control the pressure-driven of 6 entrance solution.This allows experiment, and wherein solution changes fast on cell.Due to the large resistance area between entrance and culturing room, the pressure driven flow up to 10 PSI is possible, thus causes the pressure being less than 1/1000 of input pressure near room.
Figure illustrate there are 4 separate units (OK), 6 upstream entrance (A1-A6, B1-B6, C1-C6, D1-D6), the central imaging window with four culturing room, large outlet hole are (oval, A7, B7, C7, D7) and the layout of active control panel of gravity perfusion hole (last row, A8, B8, C8, D8).
Figure 12 A-C is that diagram has four independent figure cultivating another example culture plate of unit according to specific embodiment, each cultivate unit have can be used for putting into practice the one or more methods described in this article 6 inflow entrances, culturing room and two flow exports.Have 4 independently culturing room (such as A-D) at this plate sold first in June, 2010, each culturing room has run by gravity entrance (1), four solution inlet (2-5), cell entry (6) and two shared outlet openings (7 and 8).Culturing room as corresponding in process in the every a line (A-D) in hole.B () all four culturing room are positioned under single imaging window to minimize the high travel distance amplifying phase objective.(c) room by the perfusion Obstacles Constraints on top and bottom margin with separation chamber and circulation road.Ingate 2 and 3 makes medium flow in upper channel, and 4 and 5 make medium flow through lower channel.Flow through upper and lower passage by making the nutrient culture media of heterogeneity simultaneously and set up gradient.Due to continous pouring, stable gradient can be maintained the time period (> 2 days) of prolongation.
8. application is explained, the cell migration in the stable gradient in micro-fluidic culture apparatus
Generally, cell migration is directed and stimulate by the reciprocation of cell and extracellular matrix (ECM), flanking cell or chemical inhibitor.During embry ogenesis, cell migration participates in scope from Gastrulation to neurodevelopmental nearly all Form genesis process.In adult's organism, cell migration contributes to physiology and pathologic conditions, and is important on the therapeutic development affecting wound healing and metastases.Finally, by analyzing the mechanism cellular response of migration correctives (inhibitor or activator) being understood to migration.Therefore, for the quantification of migrating cell and visual technology, life science has been become important.
Most widely accepted cell migration chemical examination is Bo Yideng (Boyden) the room chemical examination of use two Room porous plate, and the film wherein in each hole provides porous interfacial layer between the two chambers.Chemical inhibitor is placed in lower room, and system is allowed to balance, wherein it is desirable that, gradient will be formed between upper hole and lower opening.But in fact, very steep gradient can be formed along the single shaft on the surface perpendicular to film, cause the difference lower than expection in the chemical attractants agent concentration between upper hole and lower opening.As a result, the method is not suitable for making specific cellular response relevant with special gradient characteristics (that is, slope, concentration, Time evolution etc.) and being not suitable for studying many gradient signals integration.In addition, gradient is not highly stable under " static " cell chulture situation, thus stops living cells imaging.
According to specific embodiment, to the sufficiently stable diffusion gradient quantitatively limited of long-term living cells imaging during the such as cell culture chamber shown in superincumbent figure or system also can be used for being created in the process of a couple of days.According to so micro-fluidic gradient culturing room of the plate of specific embodiment described herein and method, the chemical inhibitor of the precision controlling on whole perfusion obstacle is spread and can create spatial gradient in cultivation region.
According to specific embodiment, the inflow entrance of culturing room and go out interruption-forming and maintain the continuous stream " infinite source/meeting point " that stable concentration gradient distributes several days.In some example system in superincumbent example system, the change in gradient direction opens and closes gradient and switches between gradient and single solution expose is possible.
According to other embodiment, the long-term living cells imaging in stable chemical inhibitor gradient can be used for other cell that research malignant cell maybe can move or move.In a lot of examples be discussed below, the impact of serum gradient on metastatic chest cancer cell migration distance, speed and chemotaxis degree according to specific embodiment is characterized in detail.
Serum gradient is on the impact of metastatic chest cancer cell migration distance, speed and chemotaxis degree
In an example experiment, MDA-MB-231 (HTB-26, ATCC) mankind's chest cancer system, obtained from metastatic pleura spilling position is maintained at complete medium (by the Du Shi improved culture medium (DMEM) that 1O °/ov/v hyclone (FBS), 1% nonessential amino acid, 2 mM Glus and microbiotic (full EMD micropore) are supplementary), and go down to posterity routinely to guarantee that logarithmic phase grows by Trypsin Induced (TrypLE Select, GIBCO).
For experiment, cell by trypsinized, to be gathered in the crops by centrifuge method and in complete medium Eddy diffusion.Each cell sample of 10 uL mixes with the guava ViaCount reagent (EMD micropore) of 190 uL and cultivates 5 minutes in room temperature (rt).Sample data is acquired and uses guava ViaCount software (EMD micropore) analyzed on guava easyCyte HT instrument.
Figure 13 diagram according to specific embodiment being exposed to an example of the cell migration after stable gradient.In this specific example, illustrate using one of above-described multiple gradient culturing room to be exposed to MDA-MB-231 cell migration after stable FBS gradient.This example illustrates the presentation graphics (10 x magnification) from Cytology Lab, and wherein cell is cultivated and is exposed to the 0-10% FBS gradient set up along Y-axis (vertical plane) in complete medium.The image that top panel is caught after being included in loading cells for 24 hours.Centre panel illustrates the cell loaded latter 96 hours; Incubation time is divided into three days in complete medium, after be the serum starvation (without FBS) of 24 hours.Be apparent that from image, cell expansion all occurred three day interim with mobile.Image in bottom panel is acquired after gradient is introduced for 12 hours.
Figure 13 illustrates the migration in response to single MDA-MB-231 cell of FBS gradient in the process of three days.Cell expansion and movement all occurred three day interim between top row and centre row.Image in bottom panel is introduced in gradient and is acquired for latter 12 hours.Generally, for those cultures being exposed to FBS gradient, there is the obvious movement of the top obstacle of cell along Y-axis towards Cytology Lab.On the contrary, the control cell cultivated when not having space serum gradient shows less movement with much more random direction character.Frame (being numbered 1-8) in middle and bottom panel is for identifying specific cell and showing their total movements in 12 hours frames.Generally speaking, the cell being exposed to FBS gradient tends to preferentially move towards the source of higher FBS concentration.
Figure 14 exemplarily illustrate according to specific embodiment be used for illustrate that gradient is drawn on the example X/Y cell migration of the impact of the signaling in microfluidic cell culture better.In this example, this drawing is used to be shown in the X/Y migration drawing of the stable gradient (the FBS gradient such as on MDA-MB-231) in micro-fluidic cultivation unit on the impact of signaling.Is four drawing shown in this particular example: (a) 0/0 FBS(without gradient), (b) 10/10 FBS(without gradient), (c) 10/0 bottom FBS(to top gradient), and (d) 0/10 FBS(top-to-bottom gradient).For this special example, each data acquisition obtains from 50 representative cells from Single cell culture room, and for initial 36 picture frames of passage of time video (12 hours), these 50 representative cells are tracked.In each is drawn, black line and red line specify the cell having " upwards " or " downwards " movement only relative to Y-axis respectively.
Image J software is for following the tracks of the transport property of respective cells under various condition of culture.In some examples in these examples, monitored 12 hours altogether of 50 cells; Result is presented in the X/Y drawing of Figure 14.Analysis shows: (1) cell tends to than the degree moving much less when being exposed to nutrients gradient in stable nutrient culture media, and (2) are when gradient is established in culturing room, and cell migration is obviously more guided.
In order to distinguish chemotaxis towards FBS and non-directional migration, we and y-axis (being parallel to gradient) analyze the movement (Fig. 6) in x direction (perpendicular to gradient) with being moved apart.Significant chemotaxis is only by the cell display being exposed to FBS gradient (10/0 FBS A-C and 0/10 FBS).Total movement that cell display in the FBS that space is constant is very little.
Figure 15 exemplarily illustrates and moves according to the example cell of the function as the distance of advancing in microfluidic cell culture of specific embodiment.In this illustration, the MDA-B-231 cell migration of the function as advanced distance is shown.For each condition in six conditions (for each condition, a n=50 representative cell), the mean distance that moved (by μm in units of) be measured as the function of total distance (a) and Euclidean distance (b), wherein the latter represents the straight line of position from start to end.
Use the migration distance calculated from migration drawing, relative to the gross migration Distance geometry Euclidean distance of gradient condition shown in Figure 15 A.As shown, compare with the cell in stable environment, cultivate the little increase that there is total distance of advancing for gradient.Enjoyably, at 0/0(serum starvation) and 10/10(complete medium) there are differences hardly between condition.On the contrary, when clean (Euclid) distance is evaluated in Figure 15 B, big-difference is found between these two conditions (stable relative to gradient).In this case, the cell being exposed to FBS gradient must originate from from it than those cell migrations in constant nutrient culture media state away from 3 times, position.Such discovery hint cell is initiatively sought to be rich in nutraceutical nutrient culture media at the high FBS concentration end place of gradient, and therefore shows directional mobility greatly.
Figure 16 exemplarily illustrates and exemplifies according to particular implementation the cell being exposed to stable gradient in microfluidic cell culture and to obtain than the signaling in stable media environment and draw faster.This example illustrates in some cases, and the cell being exposed to gradient (such as FBS) obtains faster than those signalings in stable media environment.Difference in the migration velocity of this diagrammatic representation in this example in the middle of four condition of culture (μm/min).Computing velocity is carried out based on moved average total distance.
Usually, cell moves in the room establishing FBS gradient with speed higher a little.Illustriously, at one of four gradient cultures (10/0 FBS(Q)) in migrating cell show much bigger migration velocity than other example.From the observation carried out at this experimental session, there are two factors, it can contribute to the cell migration accelerated potentially: (1) cell tends to move farther in the culture of lower cell density, and the member of (2) cell mass tends to move faster than the counter pair of isolation.This rear response is caused by the localization cell-cell communication in microenvironment potentially, reports in the research as the relation above between analysis of cells between reciprocation and migration rate.
Figure 17 illustrate according to specific embodiment to the exposure irritation cell of a gradient example towards the active migration of high concentration meeting point.In this example, stimulate MDAMB-231 cell towards the active migration of high concentration meeting point to the exposure of FBS gradient.Migration index is provided in X(perpendicular to gradient) and Y(be parallel to gradient) measurement of the movement (origin positions relative to them) of cell on direction.For each chemical examination in six chemical examinations, the value of two planar movements is all shown (each mean value representing 50 cells followed the tracks of individually in individual cells indoor).
Example is set up
Before above-described particular experiment, the environment temperature in cell chulture indoor uses the micro-incubator controller of CellASIC ONIX, temperature correction plate and DirecTemp monitoring temperature software (EMD micropore) to be calibrated to 37 DEG C.Cytology Lab uses phosphate buffered saline (PBS) (PBS) solution to snug, and phosphate buffered salt solution is sucked out from the hole 1,6,7 and 8 of CellASIC plate, and 10 uL nutrient culture media are moved in hole 6.This process repeats the full chamber unit on microplate.Plate is vacuum sealed to F84 manifold.By using CellASIC ONIX FG software, nutrient culture media is configured to pour into 2 minutes with 0.25 psi from hole 6.
For loading cells, the MDA-MB-231 cell of trypsinized is with 2 x 10
6the speed Eddy diffusion of/mL is in complete medium.Nutrient culture media is from the PTFE ring sucking-off hole 1 and 6.Hole 1 10 uL nutrient culture media replace, and hole 6 cell suspending liquid of 10 uL replaces.Plate is vacuum sealed to F84 manifold and is placed on the platform of EVOS fl inverted microscope (advanced microscope inspection group) to monitor loading process.CellASIC ONIX FG software is used to load cell by flow from hole 1 and 6 with 0.4 psi 0.3 minute (18 seconds) simultaneously.Plate is placed on standard 37
#c/5% C0
2within in incubator one hour, before pouring at automatic nutrient culture media, cell is allowed to adhere to.
For these example experiment, each experiment relates to three different stages---and complete medium feeding is (from ingate 2 and 4, flow 48 hours with 1 psi), serum starvation (from ingate 3 and 5, flowing 24 hours with 1 psi) and be exposed to 0-10% FBS gradient.In order to be based upon the gradient in culturing room, 300 uL complete mediums and complete medium subtract FBS and are loaded onto respectively in ingate 2 and 4, and flow with 1 psi simultaneously.In particular instances, at 24 hours later, being directed through of gradient switches source aperture and reverses to pour into.In this case, complete medium subtracts FBS and complete medium is loaded onto in hole 3 and 5 respectively, and flows with 1 psi simultaneously.The nutrient culture media using CellASIC ONIX microfluidic system to come in this system pours into.
Under 10 x magnifications, use EVOS fl inverted microscope to catch image.During the FBS gradient part of each experiment, the execution time passes imaging; Image is caught with 20 minutes intervals in the length of gradient open-assembly time.In conjunction with manually tracking (NIH) and chemotaxis instrument (ibid.) plug-in unit use image J software (NIH) to analyze the image chemically examined from MDA-MV-231 cell migration.At each occurrence, 50 representative cells are followed the tracks of for the transport property on a series of 36 images (in cultivation 12 hours) of whole continuous print.
The real-time living cells imaging of experiment shows during the cell migration research of Altitude control can be used for observing and studies in the migration and invasion process becoming the cell related in the mechanism on the basis of pathological phenomenon (such as wound healing and metastases).
9. pneumatic manifolds
Although gravity or passive loading are effective to some microfluidic cell culture and be expect in certain embodiments, as the special pneumatic manifold in this article and described in above-cited application can mate with plate, and Pneumatic pressure can be applied to cell entry district, to carry out loading cells and to carry out the cultivation during invasion and attack chemical examination.
According to another embodiment of the invention, novel loading cells system uses pneumatic manifolds and Pneumatic pressure to be placed in micro-cultivation region by cell.When adding this loading cells system, other automation equipment that there is cleanup standard titer plate can be used to carry out full automation micro-current controlled cell and to cultivate and analyze.
In a further embodiment, the present invention relates to the microfluidic system allowing to control to pass the cell culture environment of microexamination for the long term time of attached cell.Because continue towards the trend of " systems biology ", study dynamic behaviour in other living cells individual and improve the function of high-throughput living cells screening and economy and will become and become more and more important.According to a particular embodiment of the invention, the invention provides the multiple micro-fluidic flow chamber of the passage of time microexamination experiment allowed in the middle of other chemical examination.Micro-fluidic room uses artificial endothelium dysfunction to be separated to make cell with circulation road.Device is formatted to standard orifice plate, thus allows liquid and cell sample to use standard set-up to be directly moved in suitable inlet reservoirs.Customization aerodynamic flow controller then for by loading cells to cultivating in region and switching between different exposure solution.Numerical software interface can be used for allowing user to specific input (pulse, inclined-plane etc.) along with the programming of time to be exposed to sophisticated functions by cell during passage of time imaging.
Dynamic response in living cells is the basis for phenomenon (such as bio signal process, Gene expression and regulation, differentiation and cell division).In certain embodiments, the present invention relates to control the system of cell micro-environment with the multiplexed format of current cell culture processes compatibility.Cellular response can use high power fluorescence microscopy to quantize the dynamic information obtaining having subcellular definition.This ability is widely used in cell system.
Figure 18 A-C illustrates the top view of the schematic diagram of example manifold according to a particular embodiment of the invention, side view and plan view.In this example, the air of eight pipe lines for compressing on the right, and each pipe line is configured to provide pressure to the row in the cell entry hole in micro-fluidic array.Leftmost in the drawings line is used for vacuum and the external vacuum ring be connected at manifold ambient.Each row in hole are generally connected to single pressure line, and the hole wherein on imaging region is skipped.Manifold is placed on the top of other configuration of standard orifice plate or plate.Rubber washer is between plate and manifold, and its mesopore mates with manifold (not shown).Vacuum line creates vacuum in chamber between the holes, thus plate and manifold is kept together.Pressure be applied to hole with by liquid driven in microfluidic channel (not shown).Use the typical pressure of 1 psi, therefore vacuum strength is enough to maintain gas-tight seal.There are 9 pipe lines to pressure controller in one example: 8 lines are used for pressurized air, and 1 line is used for vacuum (Far Left).In specific example embodiment, each row is connected to single pressure line.Row on cell imaging district are skipped.
Supercharging loading cells in system is according to a particular embodiment of the invention found in the culture of the cell (such as solid tumor, liver, muscle etc.) that preparation is assembled effective especially.Supercharging loading cells also allows the structure with elongated cultivation region to be effectively loaded.Allow the present invention to utilize quite simple two entry design for the plenum manifold of loading cells and the use for pouring into the passive stream operating and attack chemical examination, and do not need the ingate added as used in other design and/or valve.
The manifold of amendment
In a further embodiment, plate manifold comprises and has specific gaseous environment (such as 5% CO for being immersed in by cell
2) micro fluidic device in additional " gas line ".Other example comprises oxygen and nitrogen controls, but any gaseous mixture can be sent to cell.Gas is flow in the seal bore on cell chulture district by manifold, and in the micro-fluidic air duct that the hole in micro fluidic device enables gas flow to specifies, as mentioned above.Gas transmissive device layers (PDMS) allowed gas to be diffused in nutrient culture media before exposed cell.By making gas continuous flow through micro-fluidic plate, stable gaseous environment is maintained.
This is provided for controlling gaseous environment micro-fluidic plate to be placed on the option means in incubator.In the manifold of this amendment, manifold can be used for creating independent of surrounding air " micro-incubator ".
Figure 19 diagram example system and manifold for operating micro-fluidic plate according to a particular embodiment of the invention.
10. automated system
Because plate is designed to be used in the instrument meeting SBS and processes, various " ready-made " machine can be used for creating automated system.This schematic diagram illustrates this example how to be done.Micro-fluidic plate is moved to another from a platform by robots arm's (sheet processor).Robotization incubator is memory board at suitable temperature and gaseous environment, to carry out the long-term perfusion via run by gravity.Liquid (nutrient culture media, medicine, laboratory reagent etc.) is assigned to ingate by pipettor, and removes liquid from outlet opening.Plate reader is used for chemical examination.Loading cells device is alternatively for introducing cell in micro-fluidic chemical examination when testing and starting.Particularly, loading cells device is not generally " ready-made ", and by the designation hole that Pneumatic pressure is applied to array board to cause stream to operate.Standard or customized computer software can be used to make operation complete.
Basic process comprises: 1) removed from incubator by plate, 2) via pipettor, liquid is removed from outlet opening, 3) from " plate lamination " mobile nutrient culture media/medicament storage plate, 4) via pipettor, liquid is transferred to micro-fluidic plate from nutrient culture media/medicine plate, 5) micro-fluidic plate is placed in incubator, 6) repetition is carried out to each plate, 7) repeat after the time interval of specifying (such as 24 hours).
96 orifice plate standards allow microfluidic system to use standard technique and device to operate.Such as, Liquid distribution standard volumetric pipette machinery realizes, and cell chulture and analysis and existing incubator and plate reader compatible.The loading cells system that customization is set up can be used for using air pressure as above to load cell.Segregation drive stream cultivates the expectation flow rate that the nutrient culture media level error of configuration using between entrance and exit hole and design fluid resistance realize in nL/min situation.This provides the remarkable advantage that can make " passively " the long-time section of media flow (such as reaching 4 days), and does not use huge external pump.
Integrated system
For cell and the Collection and analysis of other data and the compiling for database of the present invention, the integrated system stored and access generally comprises digital machine, it has the software of the instruction set comprised for sequence search and/or analysis, high-throughput sample control software design alternatively, image analysis software, collect data interpretation software, for solution to be transferred to the robot controlling armature being operationally linked to digital machine of destination (such as pick-up unit) from source, for subject data is input to digital machine or be used for control analysis operation or undertaken by robot controlling armature high-throughput sample transfer input media (such as computer keyboard).Alternatively, integrated system also comprise valve, concentration gradient, fluid multiple passage and/or for as described in microchamber other micro-fluidic structure interactive.
Use easy the to be available computing hardware resource of standard operation system may be utilized and be modified according to the instruction provided herein, the PC(Intel x86 such as used in integrated system of the present invention or DOS, OS2, WINDOWS, WINDOWS NT, WINDOWS95, WINDOWS98, LINUX of compatible Pentium chip or even Macintosh, Sun or PCs are by enough).Current techniques in software engineering is enough to the method allowing to realize on the computer systems instructing herein.Therefore, in certain embodiments, one group of logical order (instruction of software or hardware encoding) of one or more methods during the present invention can comprise for performing as instructed in this article method.Such as, for providing the software of data and/or statistical study standard programming language (such as Visual Basic, Fortran, Basic, Java etc.) can be used to construct by technician.Such software also can utilize multiple statistics programming language, tool box or storehouse to construct.
Figure 21 illustrates that can be understood to can from the information instrument (or digital device) 700 of the logical device of medium 717 and/or the network port 719 reading command, and the network port 719 can be connected to the server 720 with mounting medium 722 alternatively.Equipment 700 can use those instructions to come direct server or client logic hereinafter, as understood in the art, to embody aspect of the present invention.The logical device that can embody a type of the present invention is as the computer system as shown in 700, and it comprises CPU 707, optional input media 709 and 711, disc driver 715 and optional monitor 705.Mounting medium 717 or the mounting medium on port 719 722 can be used for such System Programming and can represent magnetic disc type light or magnetic medium, tape, solid-state dynamically or static memory etc.In certain embodiments, the present invention can be presented as the software be recorded on this mounting medium whole or in part.Communication port 719 also can be used for initial reception and is used for such System Programming and the instruction that can represent the communication connection of any type.
Various programmed method and algorithm---comprise genetic algorithm and neural network---and can be used for and perform Data Collection, associate and the aspect of memory function and other desired function, as described herein.In addition, numeral or simulation system (such as numeral or analog computer system) can control other function multiple, the display of such as input and output file and/or control.Software for performing electrical analysis method of the present invention is also included within computer system of the present invention.
Other embodiment
Although describe the present invention in various specific embodiment, intention is not that the present invention is limited to these embodiments.Amendment in spirit of the present invention will be obvious for those of skill in the art.
What understand is, example described herein and embodiment are in order to illustrative object, and imply by instruction herein those of skill in the art according to its various amendment or change, and by the scope of the spirit and scope and claim that are included in the application.
All announcement things that are that quote in this article or that submit to together with this motion, patent and patented claim---comprise as the submitted any reference of the part of information disclosure statement---and being all incorporated into by reference.
Claims (63)
1., for using micro-current controlled cell culture systems to cultivate and monitor a method for cell, comprising:
One or more groups system is incorporated in the Cytology Lab of described micro-current controlled cell culture systems;
One or more groups cell described is cultivated in described Cytology Lab;
One or more microfluidic flow passages is used to create in described Cytology Lab and control gradient;
Wherein said Cytology Lab described gradient cultivation and to create and control period is provided to described cell does not interrupt light path;
In described micro-fluidic culture systems, catch multiple images of described cell with control period between the culture period of described gradient;
Infosystem is used to analyze the one or more cells in the sequence of multiple image, to determine the transport property of described one or more cell.
2. the method for claim 1, also comprises:
One or more microfluidic flow passages in wherein said microfluidic flow passages is associated with one or more obstacles of restrictive cell movement while being introduced by material in described Cytology Lab.
3. as claim 1 to aforementioned claim any combination as described in method, also comprise:
Control the gradient parameter of one or more material.
4. as claim 1 to aforementioned claim any combination as described in method, also comprise:
The different time periods control gradient parameter with provide the gradient of steady state (SS) or change or both.
5. as claim 1 to aforementioned claim any combination as described in method, also comprise:
During image capture, use the micro-fluidic control formed cell culture condition and chemical inhibitor gradient to determine the accurate dynamic quantization of cell migration in response to stimulating.
6. as claim 1 to aforementioned claim any combination as described in method, also comprise:
Move towards the chemotaxis of material and non-directional by distinguishing with the movement analyzed with being moved apart on x direction (such as perpendicular to gradient) on y direction (being such as parallel to gradient).
7. as claim 1 to aforementioned claim any combination as described in method, also comprise:
Determine the gross migration Distance geometry Euclidean distance relative to gradient condition.
8. as claim 1 to aforementioned claim any combination as described in method, also comprise:
Determine the impact on the migration trend of the cell in one or more in the tissue of tumour, wound, development, immune response and other biosystem of signaling molecule, growth factor or other material, other biosystem described can be characterized relative to competent cell migration.
9. as claim 1 to aforementioned claim any combination as described in method, also comprise:
Cultured cell in three different stages:
Complete material feeding;
Serum starvation; And
Be exposed to chemical inhibitor (such as fetal bovine serum (FBS)) gradient.
10. as claim 1 to aforementioned claim any combination as described in method, also comprise:
To reverse after the selected time period gradient orientation.
11. methods as claimed in claim 10, the wherein said selected time period is the time being enough to determine cell migration or other character on a direction of gradient, such as 3,6,12,24 hours or any reasonable time section.
12. as claim 1 to aforementioned claim any combination as described in method, also comprise:
Capture time transition diagram picture during gradient part or cultivation part the numeral of storage figure picture are to analyze;
Wherein image during gradient open-assembly time or capture time or both with expect interval be captured;
Infosystem is used to analyze described image;
Wherein follow the tracks of one or more other cells for the transport property in whole continuous print series or other image series.
13. methods as claimed in claim 12, also comprise:
Wherein expect that interval is such as about 20 minutes intervals or any numeral between 1 minute and 48 h apart;
Wherein other cell individual comprises about 10, about 20, about 30, about 40, about 50, about 60-100 or about 100-5, the one or more cells in 000 representative cell;
Wherein said image series is the rule of 36-300 image during 10-36 the image of 8-16 such as in cultivation hour period, in cultivation 24 hours or any expectation during any expectation incubation time section or irregular image capture.
14. as claim 1 to aforementioned claim any combination as described in method, wherein one or more other unicellular (such as MDA-MB-231) are monitored in response to the gradient in the process of long-time section (time period of such as about 6 hours to about 6 days or the time period of about 3 days) in addition.
15. as claim 1 to aforementioned claim any combination as described in method, be also determined to become the mechanism on the pathological phenomenon basis of such as wound healing and metastases from migration.
16. as claim 1 to aforementioned claim any combination as described in method, also comprise:
Analysis of cells transport property is carried out with the equivalent cell being exposed to nutrients or other gradient by the cell compared in stable nutrient culture media.
17. as claim 1 to aforementioned claim any combination as described in method, also comprise:
Analysis of cells transport property is carried out by comparing cell migration, to determine that when more obviously directed cell migration is, thus the response of instruction to the gradient in culturing room.
18. as claim 1 to aforementioned claim any combination as described in method, also comprise:
Come in response to gradient determination cell migration by the cell compared in constant material, and determine that whether the cell in described gradient is substantially more mobile further from (about 3 times or about 1.5 times to 10 times) its position of originating from than the cell in constant material state.
19. as claim 1 to aforementioned claim any combination as described in method, also comprise:
Come in response to described gradient determination cell migration with the speed of the cell in described gradient by comparing the speed of the cell in constant material.
20. as claim 1 to aforementioned claim any combination as described in method, also comprise:
The image being used in certain time period acquisition after gradient causes carrys out the considerable time section (such as 4 hours to 6 day time period) that cultured cell one section allows cell expansion and movement.
21. as claim 1 to aforementioned claim any combination as described in method, also comprise:
Configuration micro fluidic device, it comprises: first passage; Be separated the first obstacle of described first passage and Cytology Lab; Be separated the second obstacle of described Cytology Lab and second channel; Described first passage is configured to comprise the first material; Described second channel is configured to comprise the second material;
Cell in described Cytology Lab is exposed to the gradient of described cytotaxis to described first material in described Cytology Lab of described first obstacle.
22. as claim 1 to aforementioned claim any combination as described in method, also comprise:
Configuration micro fluidic device, it comprises:
A. Cytology Lab;
B. for making the first material flow to first-class entrance in described Cytology Lab;
C. for making the second material flow to second entrance in described Cytology Lab;
D. at least one cell entry;
The cell in described Cytology Lab is made to be exposed to the continous-stable gradient of described first material and described second material.
23. as claim 1 to aforementioned claim any combination as described in method, also comprise:
Any combination according to claim 26 to 46 configures micro fluidic device, makes the cell in described Cytology Lab be exposed to controlled gradient.
24. as claim 1 to aforementioned claim any combination as described in method, also comprise:
According to any combination configuration microfluidic system of claim 47 to 55, the cell in the multiple Cytology Labs in multiple micro fluidic device is made to be exposed to one or more controlled gradient.
25. 1 kinds, for using micro-current controlled cell culture systems to cultivate and monitor the method for cell, comprising:
Monitoring cell (such as metastatic mankind chest cancer cell line MDA-MB-231) is to determine their movement in response to the spatial variations in serum-concentration;
Detect in the positive dirction of described gradient along Y-axis towards the movement of the cell of the top obstacle of described Cytology Lab for the cell (such as FBS) being exposed to gradient;
Monitor the one or more control cells cultivated when not having space serum gradient have much more random direction character less movement with detection;
The cell being wherein exposed to gradient is confirmed as preferentially moving towards the source of higher gradient concentration; And wherein cell expansion both occurred 4 hours to 6 day interims with mobile, image certain time period after gradient is introduced is acquired.
26. 1 kinds of micro fluidic devices, comprising:
A. first passage:
B. the first obstacle of described first passage and Cytology Lab is separated;
C. the second obstacle of described Cytology Lab and second channel is separated;
D. described first passage is configured to comprise the first material;
D. described second channel is configured to comprise the second material;
E. the cell in cell retaining zone is made to be exposed to the gradient of described cytotaxis to described first material in described Cytology Lab of described first obstacle.
27. 1 kinds of micro fluidic devices, comprising:
A. Cytology Lab;
B. for making the first material flow to first-class entrance in described Cytology Lab;
C. for making the second material flow to second entrance in described Cytology Lab;
D. at least one cell entry;
E. the cell in described Cytology Lab is made to be exposed to the continous-stable gradient of described first material and described second material.
28. micro fluidic devices according to claim 27, in addition wherein:
Described first-class entrance or described second entrance or both comprise allow material to flow in described Cytology Lab and no thoroughfare described first-class entrance or described second entrance to multiple perfusion paths of the cell pathway of described outdoor.
29. micro fluidic devices according to claim 27, also comprise:
F. for making the 3rd material flow to the 3rd inflow entrance in described Cytology Lab.
30. arrive the micro fluidic device described in any combination of aforementioned claim according to claim 26, in addition wherein:
Described device is configured to make the stream in one or more circulation road to be driven by capillary force or gravity or active pneumatic manifolds or its any combination.
31. according to claim 26 to aforementioned claim any combination described in micro fluidic device, in addition described first-class passage or second passage or both be separated with described Cytology Lab by the one group of micro-fluidic hole of xsect or path being measured as about 8x8 micron.
32. arrive the micro fluidic device described in any combination of aforementioned claim according to claim 26, wherein contact with circulation road with the liquid in outlet opening in first passage hole with second channel hole in addition.
33. arrive the micro fluidic device described in any combination of aforementioned claim according to claim 26, and the hole in addition wherein on culturing room keeps empty in order to better image quality, and the basal surface of plate is microslide.
34. according to claim 26 to aforementioned claim any combination described in micro fluidic device, wherein one or more circulation roads are about 550 μm and be in height 50 μm on width.
35. arrive the micro fluidic device described in any combination of aforementioned claim according to claim 26, and wherein one or more obstacles are made up of the network of the path of about 50x8x8 μm (LxWxH).
36. arrive the micro fluidic device described in any combination of aforementioned claim according to claim 26, wherein one or more obstacles are made up of the network of the path of about 100x4x2 μm (LxWxH), and the narrow xsect of other wherein said path prevents cell from passing described obstacle, and any material of other wherein nutrient culture media and/or such as chemical inhibitor, dyestuff or other material is crossed over the diffusion of described obstacle and is formed gradient to described cell.
37. according to claim 26 to aforementioned claim any combination described in micro fluidic device, wherein said room is approximately 4.8x0.5x.05mm and for the number count of cell to migration in size (LxWxH).
38. according to claim 26 to aforementioned claim any combination described in micro fluidic device, wherein cell in the chamber manually or micrometron be counted and be quantized.
39. arrive the micro fluidic device described in any combination of aforementioned claim according to claim 26, wherein one or more circulation roads are about 100x50 μm (WxH), and at least one circulation road transports fluid along obstacle from inlet bore and described fluid is emptied to outlet bore, thus material is allowed to be spread from described circulation road by described obstacle.
40. arrive the micro fluidic device described in any combination of aforementioned claim according to claim 26, wherein said device is configured in orifice plate and runs while allowing one or more chemical examination to test, and described one or more chemical examination experiment comprises the chemical examination making cell be exposed to different material, hormone or other stimulus, medicine, chemical inhibitor etc.
41. arrive the micro fluidic device described in any combination of aforementioned claim according to claim 26, also comprise the structure of the culture environment be provided in the described room maintaining described cell and the oxygen diffusion passing through the material limiting the passage promoted by air flue and pore.
42. arrive the micro fluidic device described in any combination of aforementioned claim according to claim 26, material in addition wherein in one or more circulation road or the stream of liquid are diffused into described cell chulture indoor and nourish described cell to cultivate, and provide the attractant to cell simultaneously.
43. arrive the micro fluidic device described in any combination of aforementioned claim according to claim 26, and wherein each room is about 1.5x0.5mm in size in addition, has the height of about 200 μm.
44. arrive the micro fluidic device described in any combination of aforementioned claim according to claim 26, and wherein the anticipated number of every unit cell is about 500 cells.
45. arrive the micro fluidic device described in any combination of aforementioned claim according to claim 26, and wherein example irrigation rate is about 40 ul/ days.
46. arrive the micro fluidic device described in any combination of aforementioned claim according to claim 26, and wherein example cell room volume is about 150 nL, and chamber size is about 1.5x0.5x0.2mm.
47. 1 kinds of microfluidic system, comprising:
A. according to the one or more devices of claim 26 to any combination of aforementioned claim;
B. there is the micro-fluidic plate at least three holes be associated with described one or more device;
C. wherein at least one hole is arranged so that chemical inhibitor is incorporated into described first passage or described second channel by it.
48. 1 kinds of microfluidic system, comprising:
A. according to the one or more devices of claim 26 to any combination of claim 46;
The activity b. with at least three holes of each device be connected in described device controls micro-fluidic plate;
C. wherein at least one hole is configured to make it be incorporated into by chemical inhibitor in described first passage or described second channel; And
D. for controlling the manifold of pressure at least one hole being connected at least one device and/or content during cell chulture.
49. systems according to claim 48, also comprise:
Except at least one hole described, wherein pressure is controlled by described manifold, at least one hole be configured to receive fluid and via passive segregation drive stream by fluid perfusion in described device.
50. arrive the system described in any combination of aforementioned claim according to claim 48, also comprise:
At least three ingates be associated with each device in described one or more device and an outlet opening.
51. arrive the system described in any combination of aforementioned claim according to claim 48, also comprise:
At least three inlet bore be associated with each device in described one or more device, at least one cell entry hole, at least one cell outlet hole and at least one imaging/viewing window or hole.
52. 1 kinds of micro fluidic devices, comprising:
A. according to the one or more devices of claim 26 to any combination of claim 47;
The activity b. with at least three ingates of each device be connected in described device controls micro-fluidic plate;
C. wherein at least one hole is configured to make it chemical inhibitor is incorporated into described first passage or described second channel; And
D. for controlling pressure at least one hole being connected at least one device and or the manifold of content during cell chulture.
53. systems according to claim 52, also comprise:
At least three ingates be associated with each device in described one or more device and an outlet opening.
54. arrive the system described in any combination of aforementioned claim according to claim 52, also comprise:
At least four inlet bore be associated with each device in described one or more device and an outlet opening.
55. arrive the system described in any combination of aforementioned claim according to claim 52, also comprise:
At least four devices in described device, each device is associated with at least two inlet bore, at least one outlet opening and imaging hole or region.
56. arrive the system described in any combination of aforementioned claim according to claim 48, also comprise:
Comprise the orifice plate of one or more micro fluidic device, each device is associated with multiple ingate; And
For mating with described plate and allowing the pneumatic manifolds that the pressure-driven of the solution in described ingate controls.
57. systems according to claim 56, also comprise:
For controlling the automatic digital controller with logic processor of the pressure in described hole via described manifold.
58. arrive the system described in any combination of aforementioned claim according to claim 56, also comprise:
Wherein said multiple liquid or reagent inlet hole make it possible to the controlled experiment of any combination of the method for carrying out according to claim 1 to 25.
59. arrive the system described in any combination of aforementioned claim according to claim 56, also comprise:
Wherein multiple liquid or reagent inlet hole make it possible to how carry out about cell to different medicines or the controlled experiment making response via other stimulus that described entrance is introduced.
60. arrive the system described in any combination of aforementioned claim according to claim 56, also comprise:
The gravity hole be associated with one or more device, it is used for promoting to maintain cell before described plate is attached to described manifold and experiment is performed.
61. arrive the system described in any combination of aforementioned claim according to claim 56, wherein multiple liquid or reagent inlet hole make it possible to carry out about cell how to different medicines or the controlled experiment that responds via other stimulus that described entrance is introduced, and wherein solution is changed quickly on described cell.
62. arrive the system described in any combination of aforementioned claim according to claim 56, also comprise:
For being captured in the digital image capture device of the image of the cell in one or more Cytology Lab;
For reading caught image with to cell count or determine respective cells or indivedual cell of group or the information handling system of the migration of both.
63. systems according to claim 62, wherein said information handling system controls the pressure driven flow of described manifold.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610259619.3A CN106399094A (en) | 2012-04-01 | 2013-02-06 | Cell culture and gradient migration assay methods and devices |
Applications Claiming Priority (5)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US13/436,992 | 2012-04-01 | ||
| US13/436,992 US9637715B2 (en) | 2005-07-07 | 2012-04-01 | Cell culture and invasion assay method and system |
| US201361761227P | 2013-02-05 | 2013-02-05 | |
| US61/761,227 | 2013-02-05 | ||
| PCT/US2013/024999 WO2013151616A1 (en) | 2012-04-01 | 2013-02-06 | Cell culture and gradient migration assay methods and devices |
Related Child Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610259619.3A Division CN106399094A (en) | 2012-04-01 | 2013-02-06 | Cell culture and gradient migration assay methods and devices |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN104412109A true CN104412109A (en) | 2015-03-11 |
Family
ID=49300898
Family Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610259619.3A Pending CN106399094A (en) | 2012-04-01 | 2013-02-06 | Cell culture and gradient migration assay methods and devices |
| CN201380018324.1A Pending CN104412109A (en) | 2012-04-01 | 2013-02-06 | Cell culture and gradient migration assay methods and devices |
Family Applications Before (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610259619.3A Pending CN106399094A (en) | 2012-04-01 | 2013-02-06 | Cell culture and gradient migration assay methods and devices |
Country Status (5)
| Country | Link |
|---|---|
| EP (1) | EP2834649A4 (en) |
| JP (2) | JP2015517804A (en) |
| CN (2) | CN106399094A (en) |
| SG (3) | SG10201607963SA (en) |
| WO (1) | WO2013151616A1 (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105062890A (en) * | 2015-09-11 | 2015-11-18 | 温州梅塔光学科技有限公司 | Single cell experiment device |
| CN105062879A (en) * | 2015-07-26 | 2015-11-18 | 江苏大学附属医院 | Apparatus for cell multidirectional migration experiment |
| CN109682880A (en) * | 2017-10-18 | 2019-04-26 | 株式会社岛津制作所 | Mass spectrometer apparatus for management of information |
| WO2022134159A1 (en) * | 2020-12-25 | 2022-06-30 | 中国科学院广州生物医药与健康研究院 | Chip for integrated tumor cell behavior experiments |
Families Citing this family (18)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR2992206B1 (en) * | 2012-06-21 | 2014-07-18 | Oreal | COSMETIC COMPOSITION COMPRISING AN OIL, HYDROPHOBIC SILICA AEROGEL PARTICLES AND A HYDROCARBON RESIN |
| SG11201506339RA (en) | 2013-02-22 | 2015-09-29 | Genzyme Corp | Microcarrier perfusion culturing methods and uses thereof |
| EP2958988A2 (en) | 2013-02-22 | 2015-12-30 | Genzyme Corporation | Microcarrier perfusion culturing methods and uses thereof |
| EP3112450A4 (en) * | 2014-02-25 | 2017-11-08 | Kyoto University | Microfluid device and three-dimensional microculture method for cell |
| TWI743024B (en) * | 2014-06-06 | 2021-10-21 | 美商健臻公司 | Perfusion culturing methods and uses thereof |
| TWI707949B (en) | 2014-06-09 | 2020-10-21 | 美商健臻公司 | Seed train processes and uses thereof |
| CN107567495A (en) | 2014-12-22 | 2018-01-09 | 建新公司 | The method for cultivating mammalian cell |
| NL2016404B1 (en) | 2016-03-09 | 2017-09-26 | Mimetas B V | Double tubular structures. |
| AU2017286096B2 (en) | 2016-06-15 | 2022-01-20 | Mimetas B.V. | Cell culture device and methods |
| US11161111B2 (en) * | 2016-06-29 | 2021-11-02 | Miltenyi Biotec B. V. & Co. KG | Multilevel disposable cartridge for biological specimens |
| WO2018234163A1 (en) * | 2017-06-16 | 2018-12-27 | İni̇ti̇o Biyomedikal Mühendislik Danişmanlik Sanayi Ve Ticaret Limited Şirketi | Method for determining cell migration and invasion |
| NL2020518B1 (en) | 2018-03-02 | 2019-09-12 | Mimetas B V | Device and method for performing electrical measurements |
| CN108641931A (en) * | 2018-04-04 | 2018-10-12 | 浙江大学 | A kind of digitlization microarray organ chip and its application |
| CN111057639B (en) * | 2019-11-29 | 2022-06-17 | 中国医科大学 | Circulating tumor cell separation and enrichment system and method based on micro-fluidic chip |
| WO2022009663A1 (en) * | 2020-07-06 | 2022-01-13 | 株式会社ニコン | Migration ability calculation device, migration ability evaluation method, and computer program for causing computer to execute migration ability evaluation method |
| WO2022018743A1 (en) * | 2020-07-22 | 2022-01-27 | INDIAN INSTITUTE OF TECHNOLOGY MADRAS (IIT Madras) | A system and method for simultaneous live-cell imaging and growing |
| NL2028424B1 (en) | 2021-06-10 | 2022-12-20 | Mimetas B V | Method and apparatus for forming a microfluidic gel structure |
| CN113648287B (en) * | 2021-07-30 | 2023-05-05 | 深圳华源再生医学有限公司 | Cell loading device and preparation method thereof |
Family Cites Families (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8129179B2 (en) * | 2002-08-27 | 2012-03-06 | Vanderbilt University | Bioreactors with an array of chambers and a common feed line |
| WO2005059088A1 (en) * | 2003-12-19 | 2005-06-30 | University Of Waterloo | Cultured cell and method and apparatus for cell culture |
| CA2586400A1 (en) * | 2004-11-11 | 2006-05-18 | Agency For Science, Technology And Research | Cell culture device |
| US9388374B2 (en) * | 2005-07-07 | 2016-07-12 | Emd Millipore Corporation | Microfluidic cell culture systems |
| WO2009062095A1 (en) * | 2007-11-07 | 2009-05-14 | Bellbrook Labs, Llc | Microfluidic device having stable static gradient for analyzing chemotaxis |
| US20130189671A1 (en) * | 2010-08-19 | 2013-07-25 | Jan H. Hoh | Cell Culture System and Method of Use Thereof |
-
2013
- 2013-02-06 CN CN201610259619.3A patent/CN106399094A/en active Pending
- 2013-02-06 CN CN201380018324.1A patent/CN104412109A/en active Pending
- 2013-02-06 SG SG10201607963SA patent/SG10201607963SA/en unknown
- 2013-02-06 SG SG11201405210QA patent/SG11201405210QA/en unknown
- 2013-02-06 SG SG10201803961VA patent/SG10201803961VA/en unknown
- 2013-02-06 EP EP13772405.0A patent/EP2834649A4/en not_active Withdrawn
- 2013-02-06 JP JP2015503203A patent/JP2015517804A/en active Pending
- 2013-02-06 WO PCT/US2013/024999 patent/WO2013151616A1/en active Application Filing
-
2017
- 2017-02-06 JP JP2017019804A patent/JP2017093467A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105062879A (en) * | 2015-07-26 | 2015-11-18 | 江苏大学附属医院 | Apparatus for cell multidirectional migration experiment |
| CN105062890A (en) * | 2015-09-11 | 2015-11-18 | 温州梅塔光学科技有限公司 | Single cell experiment device |
| CN109682880A (en) * | 2017-10-18 | 2019-04-26 | 株式会社岛津制作所 | Mass spectrometer apparatus for management of information |
| CN109682880B (en) * | 2017-10-18 | 2022-01-07 | 株式会社岛津制作所 | Information management device for mass spectrometer |
| WO2022134159A1 (en) * | 2020-12-25 | 2022-06-30 | 中国科学院广州生物医药与健康研究院 | Chip for integrated tumor cell behavior experiments |
Also Published As
| Publication number | Publication date |
|---|---|
| SG11201405210QA (en) | 2014-09-26 |
| EP2834649A1 (en) | 2015-02-11 |
| WO2013151616A1 (en) | 2013-10-10 |
| SG10201607963SA (en) | 2016-11-29 |
| SG10201803961VA (en) | 2018-07-30 |
| CN106399094A (en) | 2017-02-15 |
| JP2015517804A (en) | 2015-06-25 |
| JP2017093467A (en) | 2017-06-01 |
| EP2834649A4 (en) | 2015-12-09 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104412109A (en) | Cell culture and gradient migration assay methods and devices | |
| US11034925B2 (en) | Cell culture and invasion assay method and system | |
| US10179897B2 (en) | Cell culture and gradient migration assay methods and devices | |
| US9637715B2 (en) | Cell culture and invasion assay method and system | |
| Tomasi et al. | Individual control and quantification of 3D spheroids in a high-density microfluidic droplet array | |
| US9388374B2 (en) | Microfluidic cell culture systems | |
| Sivagnanam et al. | Exploring living multicellular organisms, organs, and tissues using microfluidic systems | |
| Wlodkowic et al. | Wormometry‐on‐a‐chip: Innovative technologies for in situ analysis of small multicellular organisms | |
| CN104245917B (en) | Miniature culture system and method for Microfluidic cell culture | |
| US10202569B2 (en) | Radial microfluidic devices and methods of use | |
| US20160340631A1 (en) | Layered microfluidic array | |
| US20090215104A1 (en) | Bioreactor Device, and Method and System for Fabricating Tissues in the Bioreactor Device | |
| US10744505B2 (en) | Microfluidic device for in vitro 3D cell culture experimentation | |
| CA2852950C (en) | Layered microfluidic living cell array | |
| US20190376013A1 (en) | Microfluidic devices, systems, and methods for investigating three-dimensional cellular structures | |
| US11118150B2 (en) | Layered microfluidic array | |
| KR102127765B1 (en) | Rapid Cell Culture Device For Preventing Movement Of Solidified Fluid | |
| CN108300654B (en) | Chip for evaluating pharmacokinetics of movable biological living body and real-time detection system | |
| CN117286029A (en) | Method for culturing colorectal cancer organoid chip | |
| CN115595266A (en) | Biochip and method for manufacturing the same | |
| WO2023122349A2 (en) | Human-on-chip operating system | |
| Zhao | Versatile cradle-to-grave culture platform for 3D spheroid formation and analysis |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20150311 |