CN114621979A - Method and device for cell mechanics transfection based on flexible variable cross-section microchannel - Google Patents
Method and device for cell mechanics transfection based on flexible variable cross-section microchannel Download PDFInfo
- Publication number
- CN114621979A CN114621979A CN202210139490.8A CN202210139490A CN114621979A CN 114621979 A CN114621979 A CN 114621979A CN 202210139490 A CN202210139490 A CN 202210139490A CN 114621979 A CN114621979 A CN 114621979A
- Authority
- CN
- China
- Prior art keywords
- cell
- cells
- liquid
- transfection
- 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.)
- Granted
Links
- 238000001890 transfection Methods 0.000 title claims abstract description 27
- 238000000034 method Methods 0.000 title claims abstract description 19
- 210000004027 cell Anatomy 0.000 claims abstract description 213
- 239000007788 liquid Substances 0.000 claims abstract description 102
- 238000001125 extrusion Methods 0.000 claims abstract description 46
- 239000000126 substance Substances 0.000 claims abstract description 42
- 239000010409 thin film Substances 0.000 claims abstract description 25
- 239000012528 membrane Substances 0.000 claims abstract description 21
- 239000010408 film Substances 0.000 claims abstract description 18
- 210000000170 cell membrane Anatomy 0.000 claims abstract description 16
- 238000003151 transfection method Methods 0.000 claims abstract description 16
- 239000003814 drug Substances 0.000 claims abstract description 7
- 239000000872 buffer Substances 0.000 claims description 13
- 239000000463 material Substances 0.000 claims description 10
- 239000002953 phosphate buffered saline Substances 0.000 claims description 9
- 108090000623 proteins and genes Proteins 0.000 claims description 8
- 102000004169 proteins and genes Human genes 0.000 claims description 7
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 6
- 229940079593 drug Drugs 0.000 claims description 6
- 239000000725 suspension Substances 0.000 claims description 6
- 241000700605 Viruses Species 0.000 claims description 5
- 230000001464 adherent effect Effects 0.000 claims description 5
- 229940098773 bovine serum albumin Drugs 0.000 claims description 5
- 239000002086 nanomaterial Substances 0.000 claims description 5
- 239000013612 plasmid Substances 0.000 claims description 5
- 229920001983 poloxamer Polymers 0.000 claims description 5
- 210000004102 animal cell Anatomy 0.000 claims description 4
- 230000001580 bacterial effect Effects 0.000 claims description 4
- 210000005260 human cell Anatomy 0.000 claims description 4
- 229920001169 thermoplastic Polymers 0.000 claims description 4
- 239000004416 thermosoftening plastic Substances 0.000 claims description 4
- 150000001413 amino acids Chemical class 0.000 claims 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 claims description 3
- 239000013013 elastic material Substances 0.000 claims description 3
- 239000003102 growth factor Substances 0.000 claims description 3
- 238000002347 injection Methods 0.000 claims description 3
- 239000007924 injection Substances 0.000 claims description 3
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 3
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 3
- 230000008859 change Effects 0.000 claims description 2
- 238000011068 loading method Methods 0.000 claims description 2
- 239000004033 plastic Substances 0.000 claims description 2
- 230000006378 damage Effects 0.000 abstract description 6
- 238000004519 manufacturing process Methods 0.000 abstract description 2
- 238000002659 cell therapy Methods 0.000 abstract 1
- 230000001172 regenerating effect Effects 0.000 abstract 1
- 239000007789 gas Substances 0.000 description 26
- 230000000694 effects Effects 0.000 description 19
- 239000004205 dimethyl polysiloxane Substances 0.000 description 11
- 235000013870 dimethyl polysiloxane Nutrition 0.000 description 11
- 229920000435 poly(dimethylsiloxane) Polymers 0.000 description 11
- CXQXSVUQTKDNFP-UHFFFAOYSA-N octamethyltrisiloxane Chemical compound C[Si](C)(C)O[Si](C)(C)O[Si](C)(C)C CXQXSVUQTKDNFP-UHFFFAOYSA-N 0.000 description 10
- 238000004987 plasma desorption mass spectroscopy Methods 0.000 description 10
- 229920002307 Dextran Polymers 0.000 description 9
- 239000002609 medium Substances 0.000 description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 8
- XUIMIQQOPSSXEZ-UHFFFAOYSA-N Silicon Chemical compound [Si] XUIMIQQOPSSXEZ-UHFFFAOYSA-N 0.000 description 7
- 238000005516 engineering process Methods 0.000 description 7
- 239000011148 porous material Substances 0.000 description 7
- 229910052710 silicon Inorganic materials 0.000 description 7
- 239000010703 silicon Substances 0.000 description 7
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 6
- 210000001744 T-lymphocyte Anatomy 0.000 description 5
- 230000006835 compression Effects 0.000 description 5
- 238000007906 compression Methods 0.000 description 5
- 238000010586 diagram Methods 0.000 description 5
- 238000005119 centrifugation Methods 0.000 description 4
- 238000013461 design Methods 0.000 description 4
- 239000012091 fetal bovine serum Substances 0.000 description 4
- 239000012530 fluid Substances 0.000 description 4
- 230000003834 intracellular effect Effects 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 230000001052 transient effect Effects 0.000 description 4
- 238000005406 washing Methods 0.000 description 4
- 108020004414 DNA Proteins 0.000 description 3
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 230000021164 cell adhesion Effects 0.000 description 3
- 238000004140 cleaning Methods 0.000 description 3
- 210000000805 cytoplasm Anatomy 0.000 description 3
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 3
- 210000004940 nucleus Anatomy 0.000 description 3
- 230000032258 transport Effects 0.000 description 3
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 108010002350 Interleukin-2 Proteins 0.000 description 2
- 208000027418 Wounds and injury Diseases 0.000 description 2
- 230000004913 activation Effects 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- -1 cationic lipid Chemical class 0.000 description 2
- 230000005779 cell damage Effects 0.000 description 2
- 208000037887 cell injury Diseases 0.000 description 2
- 239000006285 cell suspension Substances 0.000 description 2
- 230000001413 cellular effect Effects 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- 230000012202 endocytosis Effects 0.000 description 2
- 230000001159 endocytotic effect Effects 0.000 description 2
- 108010048367 enhanced green fluorescent protein Proteins 0.000 description 2
- 238000005530 etching Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 239000011259 mixed solution Substances 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 108020004707 nucleic acids Proteins 0.000 description 2
- 102000039446 nucleic acids Human genes 0.000 description 2
- 150000007523 nucleic acids Chemical class 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 230000008672 reprogramming Effects 0.000 description 2
- 238000004659 sterilization and disinfection Methods 0.000 description 2
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- PCTMTFRHKVHKIS-BMFZQQSSSA-N (1s,3r,4e,6e,8e,10e,12e,14e,16e,18s,19r,20r,21s,25r,27r,30r,31r,33s,35r,37s,38r)-3-[(2r,3s,4s,5s,6r)-4-amino-3,5-dihydroxy-6-methyloxan-2-yl]oxy-19,25,27,30,31,33,35,37-octahydroxy-18,20,21-trimethyl-23-oxo-22,39-dioxabicyclo[33.3.1]nonatriaconta-4,6,8,10 Chemical compound C1C=C2C[C@@H](OS(O)(=O)=O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2.O[C@H]1[C@@H](N)[C@H](O)[C@@H](C)O[C@H]1O[C@H]1/C=C/C=C/C=C/C=C/C=C/C=C/C=C/[C@H](C)[C@@H](O)[C@@H](C)[C@H](C)OC(=O)C[C@H](O)C[C@H](O)CC[C@@H](O)[C@H](O)C[C@H](O)C[C@](O)(C[C@H](O)[C@H]2C(O)=O)O[C@H]2C1 PCTMTFRHKVHKIS-BMFZQQSSSA-N 0.000 description 1
- 206010006187 Breast cancer Diseases 0.000 description 1
- 208000026310 Breast neoplasm Diseases 0.000 description 1
- 229920001917 Ficoll Polymers 0.000 description 1
- 229920000209 Hexadimethrine bromide Polymers 0.000 description 1
- 108010019160 Pancreatin Proteins 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- 102000006382 Ribonucleases Human genes 0.000 description 1
- 108010083644 Ribonucleases Proteins 0.000 description 1
- 239000012190 activator Substances 0.000 description 1
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 description 1
- 229910052782 aluminium Inorganic materials 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 238000002306 biochemical method Methods 0.000 description 1
- 229960002685 biotin Drugs 0.000 description 1
- 235000020958 biotin Nutrition 0.000 description 1
- 239000011616 biotin Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 238000007664 blowing Methods 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 239000006143 cell culture medium Substances 0.000 description 1
- 230000003915 cell function Effects 0.000 description 1
- 210000003855 cell nucleus Anatomy 0.000 description 1
- 230000003833 cell viability Effects 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- FFYPMLJYZAEMQB-UHFFFAOYSA-N diethyl pyrocarbonate Chemical compound CCOC(=O)OC(=O)OCC FFYPMLJYZAEMQB-UHFFFAOYSA-N 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 239000013536 elastomeric material Substances 0.000 description 1
- 238000004520 electroporation Methods 0.000 description 1
- 210000003743 erythrocyte Anatomy 0.000 description 1
- 210000002950 fibroblast Anatomy 0.000 description 1
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 1
- 230000004907 flux Effects 0.000 description 1
- 239000003517 fume Substances 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 231100000025 genetic toxicology Toxicity 0.000 description 1
- 230000001738 genotoxic effect Effects 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 238000003306 harvesting Methods 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000002757 inflammatory effect Effects 0.000 description 1
- 230000005732 intercellular adhesion Effects 0.000 description 1
- 230000004298 light response Effects 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 238000001000 micrograph Methods 0.000 description 1
- 230000003278 mimic effect Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000002105 nanoparticle Substances 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- 229940055695 pancreatin Drugs 0.000 description 1
- 238000005192 partition Methods 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 210000005259 peripheral blood Anatomy 0.000 description 1
- 239000011886 peripheral blood Substances 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 238000001259 photo etching Methods 0.000 description 1
- 238000003825 pressing Methods 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000004080 punching Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 238000007711 solidification Methods 0.000 description 1
- 230000008023 solidification Effects 0.000 description 1
- 238000004528 spin coating Methods 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 238000010257 thawing Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 239000013603 viral vector Substances 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
-
- 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
-
- 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
- C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
- C12M35/04—Mechanical means, e.g. sonic waves, stretching forces, pressure or shear stimuli
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
- B01L2200/02—Adapting objects or devices to another
- B01L2200/026—Fluid interfacing between devices or objects, e.g. connectors, inlet details
- B01L2200/027—Fluid interfacing between devices or objects, e.g. connectors, inlet details for microfluidic devices
-
- 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
Abstract
The present disclosure provides a method for cell mechanical transfection based on flexible variable cross-section microchannel, comprising: s1, introducing liquid containing cells and exogenous substances into a liquid path layer of the microfluidic device, wherein the liquid path layer comprises a plurality of liquid microchannels; s2, applying air pressure to the air path layer of the microfluidic device, and changing the cross-sectional dimension of the liquid path microchannel at the corresponding position by deforming the thin film layer between the liquid path layer and the air path layer; when the cell passes through the liquid micro-channel at the corresponding position, the stress is applied by the flexible film layer, a minimally invasive membrane hole is instantly generated on the cell membrane, and exogenous substances enter the cell through the minimally invasive membrane hole to finish cell transfection. The present disclosure also provides a microfluidic device for mechanical transfection of cells. The method and the device provided by the disclosure can avoid damage to cells caused by rigid extrusion, can adapt to size heterogeneity cell groups with high tolerance to cell sizes, and provide a powerful and practical mechanical transfection method for application of biological manufacturing, cell therapy, regenerative medicine and the like.
Description
Technical Field
The disclosure relates to the technical field of microfluidic chips, in particular to a cell mechanics transfection method and device based on a flexible variable cross-section microchannel.
Background
Intracellular delivery is the transport of membrane impermeable molecules (e.g., DNA, RNA, proteins, drugs, or nanomaterials) across the cell membrane into the cytoplasm or nucleus. The delivery of substances to cells is an important step in understanding cell function and reprogramming cell behavior. Viral vectors are currently the most favored method of intracellular delivery. Since the virus utilizes its own infection pathway, it has excellent gene transfer efficiency and can be permanently transferred. However, development is limited by the possibility of inflammatory immune responses, genotoxicity and lower packaging capacity. As a non-viral method, cargo is transported into cells by endocytosis using cationic lipid carriers, but for suspension cells and the like, transfection is difficult, cell type dependence is high, and some substances are delivered with low efficiency. While the technique of membrane rupture is based on the application of external electrical, thermal, optical or mechanical energy to the cell to physically open the cell membrane, delivering external cargo dispersed in solution into the cell. Currently, the most common is electroporation delivery, however, electroporated cells are less viable and tend to lose cell phenotype and function.
Microfluidic-based delivery technology, which is not limited by cell type and the characteristics of the delivered substance, has attracted a great deal of attention as an emerging solution, microfluidic physical perforation technology making possible the delivery of substances that were previously difficult to deliver. These techniques utilize shear or contractile forces to rapidly deform the cells, thereby creating transient pores in the cell membrane. However, it is not clear how the potential mechanical properties affect the efficiency of substance delivery, particularly the effect of rigid extrusion of narrow ducts on cell damage. Meanwhile, the delivery efficiency based on the microfluidic physical perforation technology at present is different according to cell types, different extrusion parameters need to be researched to adapt to cells with different sizes, and the microfluidic physical perforation technology is difficult to adapt to cells with heterogeneous sizes. And the narrow extruded silicon etching channel of the current microfluidic device is easy to block, and the further application of the microfluidic device is influenced by the generated cell fragments and operation problems.
Disclosure of Invention
Technical problem to be solved
In order to solve the problems, the disclosure provides a cell mechanical transfection method and a cell mechanical transfection device based on a flexible variable cross-section microchannel, which are used for at least partially solving the technical problems that the traditional cell transfection method has large damage to cells, is difficult to be applied to cells with size heterogeneity, is easy to block microchannels and the like.
(II) technical scheme
The disclosure provides a cell mechanical transfection method based on a flexible variable cross-section microchannel, which comprises the following steps: s1, introducing liquid containing cells and exogenous substances into a liquid path layer of the microfluidic device, wherein the liquid path layer comprises a plurality of liquid microchannels; s2, applying air pressure to the air path layer of the microfluidic device, and changing the cross-sectional dimension of the liquid path microchannel at the corresponding position by deforming the thin film layer between the liquid path layer and the air path layer; when the cell passes through the liquid micro-channel at the corresponding position, the stress is applied by the flexible film layer, a minimally invasive membrane hole is instantly generated on the cell membrane, and exogenous substances enter the cell through the minimally invasive membrane hole to finish cell transfection.
Further, the introducing of the liquid containing the cell and the foreign substance into the liquid path layer of the microfluidic device in S1 includes: an injection pump, pressure pump, pipettor, syringe or injector is used to drive the cell flow and regulate the flow rate of the liquid.
Further, the applying the air pressure to the air path layer of the microfluidic device in S2 includes: and applying air pressure to the air path layer by using an air pressure pump, wherein the air pressure comprises applying positive pressure to extrude the liquid microchannel at the corresponding position or restoring the liquid microchannel when negative pressure is loaded.
Further, S1 further includes: the microchannel is rinsed and incubated with a delivery buffer, wherein the delivery buffer comprises a phosphate buffered saline, pluronic, and bovine serum albumin.
Further, S1 further includes: cells were cultured and resuspended using delivery buffer.
Further, the material of the microfluidic device comprises a thermoplastic or cold-molded elastomeric material.
Further, the cell includes any one or combination of human cell, animal cell, plant cell, and bacterial cell; or the cells comprise any one or the combination of suspension cells and adherent cells; or the cells comprise any one of primary cells, cell lines, or a combination thereof.
Further, the exogenous material includes any one of or a combination of plasmids, DNA, RNA, amino acids, peptides, proteins, drugs, growth factors, nanomaterials, viruses.
In another aspect, the present disclosure provides a microfluidic device for implementing the aforementioned cell mechanical transfection method based on flexible variable cross-section microchannels, including: the liquid path layer comprises a plurality of liquid microchannels and is used for introducing liquid of cells and exogenous substances; the gas path layer comprises a plurality of gas micro-channels, and at least parts of the gas micro-channels are aligned with the liquid micro-channels; the thin film layer is arranged between the liquid path layer and the air path layer, when air pressure is applied to the air path layer, the thin film layer is used for deforming and changing the section size of the liquid path micro-channel at the corresponding position, cells in the thin film layer are extruded flexibly by the thin film layer, minimally invasive membrane holes are formed in cell membranes instantaneously, and exogenous substances enter the cells through the minimally invasive membrane holes to finish cell transfection.
Further, the liquid micro-channels and the gas micro-channels are arranged in a staggered mode; the extrusion size range of the film layer is 0-25 mu m, and the elastic modulus range is 1 KPa-1 MPa.
(III) advantageous effects
According to the cell mechanics transfection method and device based on the flexible variable cross-section microchannel, pressure is applied to the gas path layer, the thin film layer deforms and flexibly extrudes the liquid microchannel, and the cell is stressed by the flexible gas film when passing through the variable cross-section microchannel, so that a minimally invasive membrane hole is instantly generated on a cell membrane to allow foreign substances to be delivered to the cell; the flexible variable-section microchannel can flexibly and adjustably realize cell delivery with different sizes in one chip, meanwhile, the design has high tolerance on cell size and is not easy to block, the device is simple to operate, and the delivery of high-flux cells can be realized.
Drawings
FIG. 1 is a schematic diagram illustrating an extrusion flow of a cell mechanical transfection method based on a flexible variable cross-section microchannel according to an embodiment of the present disclosure;
FIG. 2 schematically illustrates a high speed microscope image showing a cell extrusion process in accordance with an embodiment of the disclosure;
FIG. 3 schematically shows a graph of 3kDa dextran control delivered fluorescence and cell activity after endocytosis and microvalve extrusion according to an embodiment of the disclosure;
FIG. 4 schematically shows a schematic graph of the delivery efficiency and cellular activity for modulating barodeformation optimized MCF-7 cell delivery of 70k Da FITC-dextran, according to an embodiment of the disclosure;
FIG. 5 schematically shows a schematic diagram of the results of 70k Da FITC-dextran delivery into primary hard-to-transfect MEF cells according to an embodiment of the present disclosure;
FIG. 6 schematically shows a diagram of the results of 2000k Da FITC-dextran delivery into primary hard-to-transfect immune T cells according to an embodiment of the disclosure;
FIG. 7 schematically shows a schematic diagram of the results of EGFP-mRNA delivery to MCF-7 cells by flexible extrusion according to an embodiment of the present disclosure;
FIG. 8 schematically shows a schematic diagram of the results of delivering plasmid life Act-TagRFP to MCF-7 cells by flexible extrusion according to an embodiment of the present disclosure.
Detailed Description
For the purpose of promoting a better understanding of the objects, aspects and advantages of the present disclosure, reference is made to the following detailed description taken in conjunction with the accompanying drawings.
The present disclosure is directed to a method and microfluidic device for introducing membrane-impermeable molecules (e.g., DNA, RNA, proteins, drugs, or nanomaterials) into the cytoplasm or nucleus of a cell by transport across the cell membrane. The method and the device can overcome some limitations of the current intracellular delivery, such as high dependence of biochemical methods such as liposome or virus on delivered cells and delivered substances. It is not clear how the potential mechanical properties affect the efficiency of substance delivery, particularly the effect of rigid extrusion of narrow channels on cell damage. Meanwhile, the delivery efficiency based on the microfluidic physical perforation technology at present is different according to cell types, different extrusion parameters need to be researched to adapt to cells with different sizes, and the microfluidic physical perforation technology is difficult to adapt to cell groups with heterogeneous sizes. In addition, the narrow extruded silicon etching channel of the current microfluidic device is easy to block, and the generated cell fragments and operation problems influence the further application of the microfluidic device.
To achieve the above objects, the present disclosure provides a cell mechanical transfection method based on flexible variable cross-section micro-channels, referring to fig. 1, including: s1, introducing liquid containing cells and exogenous substances into a liquid path layer of the microfluidic device, wherein the liquid path layer comprises a plurality of liquid microchannels; s2, applying air pressure to the air path layer of the microfluidic device, and changing the cross-sectional dimension of the liquid path microchannel at the corresponding position by deforming the thin film layer between the liquid path layer and the air path layer; when the cell passes through the liquid micro-channel at the corresponding position, the stress is applied by the flexible film layer, a minimally invasive membrane hole is instantly generated on the cell membrane, and exogenous substances enter the cell through the minimally invasive membrane hole to finish cell transfection.
The cell and the exogenous substance flow in the micro-channel of the liquid path layer along with liquid, when the cell passes through the micro-channel at the deformation position of the thin film layer, the section size of the micro-channel at the position is reduced, the cell is stressed by the flexible air film layer, and therefore minimally invasive membrane pores are generated on the cell membrane instantaneously to allow the exogenous substance to be delivered into the cell or cell nucleus. The flexible extrusion cell transfection method avoids potential damage of the existing rigid pipeline extrusion to cells, and the delivered cells still have higher activity and can keep higher delivery efficiency.
On the basis of the above embodiment, the introducing of the liquid containing the cell and the foreign substance into the liquid path layer of the microfluidic device in S1 includes: an injection pump, pressure pump, pipettor, syringe or injector is used to drive the cell flow and regulate the flow rate of the liquid.
The flow of the liquid path cells can be driven by various methods in the present disclosure, including but not limited to the above five devices, such as an automatic or semi-automatic liquid processing system, and the like, and these devices for driving the flow of the liquid path cells are airtight with the liquid path inlet of the liquid path layer and are extruded to deliver the mixed liquid. The flow rate of the liquid is controlled, for example, in increments of 50. mu.l/min, and the cell flow rate is related to the transfection efficiency and the cell expression intensity.
On the basis of the above embodiment, the applying the air pressure to the air path layer of the microfluidic device in S2 includes: and applying air pressure to the air path layer by using an air pressure pump, wherein the air pressure comprises applying positive pressure to extrude the liquid microchannel at the corresponding position or restoring the liquid microchannel when negative pressure is loaded.
The pneumatic pump is connected to the gas circuit layer through an airtight pipe, and the pneumatic pump adjusts the middle thin film layer to generate extrusion deformation by using 200mbar increment for example, so that cells flowing at high speed in the lower liquid circuit are allowed to generate flexible extrusion. The air pressure regulation may further include restoring the liquid microchannel when the positive pressure of the air path is released, and the liquid microchannel may be expanded by loading a negative pressure through the air path. Because the section of the liquid microchannel of the device is flexible and variable, the cell mechanical transfection close to zero blockage can be realized. Except that the middle flexible thin film layer applies stress to cells by adjusting the air pressure of the air path layer, the middle thin film layer can also deform by means of other external forces, including but not limited to air pressure deformation, magnetic deformation, weight application deformation, temperature response deformation and light response deformation.
On the basis of the above embodiment, S1 further includes: the microchannel is rinsed and incubated with a delivery buffer, wherein the delivery buffer comprises a phosphate buffered saline, pluronic, and bovine serum albumin.
A delivery buffer solution of bovine serum albumin is added, on one hand, the delivery buffer solution is used for slowly moistening and washing the micro-channel and fully removing bubbles in the micro-channel; on the other hand, by incubating the microchannel, the friction between the cells and the walls of the microchannel can be reduced while preventing cell adhesion.
On the basis of the above embodiment, S1 further includes: cells were cultured and resuspended using delivery buffer.
The delivery buffer is also used for resuspending cells, can effectively reduce intercellular adhesion and reduce the risk of channel blockage, and is suitable for most types of delivery cells and delivery substances.
On the basis of the above described embodiments, the material of the microfluidic device comprises a thermoplastic or cold-moulded elastic material.
Specifically, the liquid path layer, the gas path layer and the film layer can be made of PDMS materials, and of course, the materials of the liquid path layer, the gas path layer and the film layer may be the same or different.
On the basis of the above embodiments, the cell includes any one or a combination of a human cell, an animal cell, a plant cell, and a bacterial cell; or the cells comprise any one or the combination of suspension cells and adherent cells; or the cells comprise any one of primary cells, cell lines, or a combination thereof.
The flexible extrusion delivery of the present disclosure is independent of the type of cell, including and not limited to the above cells, and is not limited by cell type.
On the basis of the above embodiments, the foreign substance includes any one of or a combination of plasmids, DNA, RNA, amino acids, peptides, proteins, drugs, growth factors, nanomaterials, viruses.
The flexible extrusion delivery of the present disclosure is also independent of the type of exogenous material, including and not limited to the above cells, and is not limited by the type and molecular weight of the exogenous material.
Compared with the prior art, the flexible thin film layer is used for applying stress, so that potential damage to cells caused by extrusion of the existing rigid pipeline can be avoided, and the cells can keep ultrahigh activity while being delivered. The technology can flexibly and adjustably realize cell delivery with different sizes in one chip through the design of the variable cross-section microchannel, and has high tolerance to the cell size.
The present disclosure also provides a microfluidic device based on flexible extrusion for cell transfection, see fig. 1, comprising: the liquid path layer comprises a plurality of liquid microchannels and is used for introducing liquid of cells and exogenous substances; the gas path layer comprises a plurality of gas micro-channels, and at least parts of the gas micro-channels are aligned with the liquid micro-channels; the thin film layer is arranged between the liquid path layer and the gas path layer, when air pressure is applied to the gas path layer, the thin film layer is used for deforming and extruding the liquid micro-channel at the corresponding position, cells in the liquid micro-channel are extruded flexibly by the thin film layer, micro-wound membrane pores are generated on the cell membrane instantaneously, and exogenous substances enter the cells through the micro-wound membrane pores to complete cell transfection.
The present disclosure relates to a microfluidic device for introducing membrane-impermeable molecule transport across cell membranes into the cytoplasm or nucleus, which is removable, comprising three layers of microfluidic chips for squeeze delivery: the liquid path layer, the thin film layer and the air path layer are arranged on the lower layer, the liquid path layer is provided with a plurality of parallel liquid path channels, different air pressures are applied to the upper layer air path layer to adjust the deformation of the middle thin film layer, and therefore the section size of the liquid path micro-channel at the corresponding position is changed. Therefore, the flexible extrusion with dynamically adjustable extrusion size can be realized, cells with different sizes can safely generate transient pores on cell membranes on the basis of keeping high activity, and foreign substances are allowed to be delivered into the cells. Of course, the gas circuit layer can be arranged on the lower layer, the liquid circuit layer can be arranged on the upper layer, and the thin film layer can be arranged between the liquid circuit layer and the gas circuit layer.
On the basis of the above embodiment, the liquid microchannels and the gas microchannels are arranged in a staggered manner.
Specifically, the liquid microchannels and the gas microchannels are arranged in a staggered mode, so that partitioned extrusion can be better achieved, for example, the liquid microchannels and the gas microchannels are divided into four areas ABCD in a design mode, 20 palindromic structures and liquid pipelines are designed and processed in each area in a staggered mode, so that cells are extruded, and the requirements of the optimized extrusion times of different cells are met through different partition combination regulation. The liquid micro-channels and the gas micro-channels can be vertically arranged in a staggered mode, and can be arranged in a staggered mode at any angle of 0-90 degrees.
In addition, the variable cross-section microchannels of the present disclosure enable near zero-plugged cell delivery, and the microchannels are flexibleThe adjustable cell delivery device can realize cell delivery with different sizes in one chip without designing and processing chips with different sizes. The device can also realize the delivery of high-flux cells, for example, a parallel liquid microchannel designed to be processed can be processed for 10 minutes in 1 minute6～107And can further expand the flux by increasing parallel channels.
On the basis of the embodiment, a liquid channel with the height of 25 mu m is designed and processed, the extrusion size range is 0-25 mu m, and the extrusion device is suitable for most cell types.
Because the section of the liquid path micro-channel in the micro-fluidic device is variable, cells with different sizes can be delivered in one chip, and the extrusion size is flexible and adjustable and is not limited to 0-25 mu m. The material of the microfluidic device comprises but is not limited to PDMS or other thermoplastic and cold-plastic elastic materials, and the material is cheap and easy to process and does not need complex operation; the film layer has a thickness of, for example, 15 μm and an elastic modulus of 1KPa to 1 MPa. The micro-fluidic device has high tolerance to cells with different sizes, and the damage of rigid extrusion to the cell activity is avoided by a flexible extrusion mode.
The microfluidic device disclosed by the invention has the characteristics of variable microchannel cross section, high tolerance on cell size, capability of adapting to large and small heterogeneous cell groups, and high efficiency of minimally invasive, and is particularly suitable for flexible mechanical transfection of cells.
The present disclosure is further illustrated by the following detailed description. The above-mentioned cytomechanical transfection method and device based on flexible variable cross-section micro-channel are specifically described in the following examples. However, the following examples are merely illustrative of the present disclosure, and the scope of the present disclosure is not limited thereto.
1. Device for intracellular delivery and method of making same
The present disclosure provides a detachable device as shown in fig. 1, the device includes a three-layer microfluidic chip for extrusion delivery, the lower layer liquid path layer has a plurality of parallel liquid path channels, and different air pressures are applied through the upper layer gas path layer to adjust the deformation of the middle thin film layer, thereby realizing the dynamic adjustment of the height of the liquid microchannel at the lower layer extrusion position, and being used for the extrusion delivery of flexible cells.
The chip with the micro valve is designed and processed by utilizing CAD drawing software to design the structures of the liquid micro channel and the gas micro channel, the structures are printed into corresponding film masks, and then male molds of the liquid micro channel and the gas micro channel are carved on a silicon wafer through photoetching experiments. The chips were then reverse molded with polydimethylsiloxane (PDMS, Sylgard 184). Firstly, mixing PDMS and a curing agent according to the proportion of 10: 1, uniformly stirring, and sufficiently removing bubbles by using a vacuum pump under negative pressure. Then pouring the PDMS mixed solution onto a positive membrane with a micro-channel, sufficiently removing bubbles by using a vacuum pump under negative pressure, and then putting the membrane into an oven to be heated and cured for 2 hours at 75 ℃. After solidification, the PDMS solidified layers of the liquid path and the gas path are cut by a nicking tool and are taken off, and a No. 19 puncher is used for punching at the inlet and the outlet of the microchannel and then is placed in a clean dish for later use. The middle film layer is uniformly stirred by PDMS mixed solution with the ratio of 25: 1, then bubbles are pumped by a vacuum pump, and poured onto a clean silicon wafer, a spin coater is used for spin coating on the surface of the silicon wafer at the rotating speed of 4500rpm (depending on the thickness of the PDMS film layer), and the silicon wafer is slightly placed and then placed into an oven for heating and curing at the temperature of 75 ℃ for 2 hours. And (3) placing the silicon wafer with the PDMS film layer and the PDMS solidified layer of the air channel in a plasma cleaning machine to clean for 40 seconds at high frequency, aligning and bonding the film layer and the air channel layer, and placing in an oven to heat for 2 hours at 75 ℃. And then removing the gas circuit layer with the thin film layer, placing the liquid circuit layer in a plasma cleaning machine in the same way, cleaning for 40 seconds at high frequency, aligning and bonding, placing in an oven, and heating for 2 hours at 75 ℃ to use.
2. Methods of demonstrating cell delivery using the device prepared in step 1
2.1 micro-valve chip Disinfection
To avoid RNase contamination affecting RNA delivery, RNase-free is required for delivery chips and catheters. Diethyl pyrocarbonate (DEPC, biotin) was first added to deionized water in a fume hood to prepare 0.1% final DEPC water, which was then shaken overnight on a shaker at room temperature. Then the chip to be extinguished and the conduit are placed in an aluminum lunch box, and the prepared 0.1 percent DEPC water is added to soak for 2 hours at room temperature. And (3) placing the DEPC water-treated chip into an autoclave for sterilization for 30 minutes, and then placing the chip into an oven at 80 ℃ for 2 hours for drying for later use.
2.2 microvalve chip in-line surface activation
Surface activation of the channels is required to avoid cell adhesion. First, a delivery buffer was prepared, 1% Pluronic (Pluronic, Sigma) and 1% bovine serum albumin (BSA, Maclin) were added to a phosphate buffer (PBS, Invitrogen), mixed uniformly in a shaker, filtered through a 0.22 μm needle filter after complete dissolution, and stored in a four-degree refrigerator. The syringe pump slowly rinses the channel at 5. mu.l/min, and after sufficient removal of the channel bubbles, incubate for 0.5h at room temperature to prevent cell adhesion.
2.3 cell culture
Adherent cells are exemplified by the human breast cancer cell line MCF-7. Taking out cells from liquid nitrogen, rapidly thawing in water bath at 37 deg.C, adding DMEM complete medium (the components of the complete medium comprise DMEM high-sugar medium added with 10% FBS fetal bovine serum and 1% penicillin/streptomycin double antibody), centrifuging at 1000rpm for 5min, and discarding supernatant. Suspending the cells in DMEM complete medium and transferring into 6cm culture dish, adding 5% CO at 37 deg.C2Culturing in an incubator, and performing subculture digestion after 2-3 days. After the cells adhere to the wall by about 80-90%, washing with PBS, digesting with pancreatin for 1-2 minutes, adding 5mL of complete culture medium to stop digestion, and fully blowing to form cell suspension. The cells were counted separately using a hemocytometer and aspirated at 1 × 106～1*107After centrifugation of the individual cells, the supernatant was resuspended in 1mL of delivery buffer and placed on ice until use.
The suspension cells are exemplified by human immune cell T cells. The collected human peripheral blood was added to RosetteSep at 20. mu.L/mLTMThe antibody Human CD8+ T Cell Enrichment Cocktail is mixed uniformly and then stands for 20 minutes at room temperature. PBS containing 2% FBS was gently mixed with blood at a ratio of 1: 1, the mixture was slowly added to the upper layer of density gradient Ficoll, centrifuged at room temperature 1200g for 20 minutes, the intermediate white membrane layer was pipetted and transferred to a new centrifuge tube. The tube was filled with PBS containing 1% FBS and washed, and after centrifugation at 600g for 10 minutes, the red blood cells were lysed and washed once if any. Adding 100U/mL IL-2 containing X-VIVO15 complete medium, and adding 20ng/mLIL-7, 1 × 10 per branch6Cells were cryopreserved. Immediately after washing activated magnetic beads (Dynabeads human T-activator CD3/CD28beads) at 25. mu.l/harvest and rinsing with medium, the T cells were prepared in 1 × 10 with X-VIVO15 complete medium containing 20ng/mL IL-26cell/mL concentration, 1mL of cell mixture per well was added to a 24-well plate for culture. Extrusion delivery is also according to 1 x 106～1*107After centrifugation, it was resuspended in 1mL of delivery buffer and placed on ice until use.
2.4 Flexible squeeze delivery of cells
The conjugate containing a delivery substance such as FITC-labeled fluorescein isothiocyanate-dextran (3k Da FITC-dextran, Sigma-Aldrich) was added at a concentration of 0.3mg/ml to the pre-formulated 1X 106～1*107The mixture was then injected into a 21 gauge flat-ended syringe and connected to the chip fluid path inlet by a gas-tight tube with an internal diameter of 0.51mm, and a syringe pump (Harvard Apparatus, PHD4400) adjusted the cell flow rate in 50 μ l/min increments, with each fluid path channel measuring 25 μm wide and 20 μm high. The gas path micro valve is connected to a pneumatic pump through an airtight pipe with the inner diameter of 0.51mm, and the pneumatic pump(s) ((
OB1, Mk3) was adjusted at 200mbar increments to adjust the deformation of the middle 15 μm thick film layer at the compression, thereby allowing the flexible compression of the cells flowing at high speed in the lower fluid layer as shown in fig. 2, and then after incubating the recovered cells at room temperature for 20 minutes (where the incubation was to reduce the disturbance to the cells, give the cells more chance of contact with the delivered substance, and sufficient cell recovery time), PBS washing and centrifugation were resuspended in cell culture medium, and cultured in an incubator, and after 24 hours, the cells delivered by flexible compression were subjected to activity and transfection efficiency identification. LIVE/DEAD for cell viabilityTMThe cell viability/cytotoxicity kit (Invitrogen, L3224) was incubated at 37 ℃ for 20min in a ratio of 1: 1000 in a medium, washed with PBS, centrifuged, placed on ice, imaged under a confocal microscope, and the staining was observed. The exogenous substance dextran can be delivered into cells with high efficiency and safety, and is capable of maintaining high delivery efficiencyMeanwhile, the air valve is extruded to cause less damage to cells, and the delivered cells have higher activity.
3. The delivery characteristics depend on various parameters of the microvalve chip
There are many parameters that may affect substance delivery including, but not limited to, squeeze size, recovery size, surface modification at the squeeze, flow rate within the channel, cell concentration, concentration of the delivered substance, etc. The micro valve flexible extrusion chip disclosed by the invention has the advantages that the flexible adjustment of the air valve is realized, so that the chips with different extrusion sizes are not required to be designed and processed, and the extrusion parameters can be optimized on the same chip. According to the invention, the film layer at the extrusion part is deformed by accurately applying pressures with different sizes and different waveforms to the air path through a pump such as an air bottle or a compressor, so that the extrusion size is flexibly changed on one chip to optimize the delivery result, as shown in FIG. 4. The atmospheric pressure of 1-2atm of MCF-7 cells is regulated, the extrusion size is reduced along with the increase of the atmospheric pressure in a certain range, the transfection efficiency and the expression strength of the cells delivered into the cells can be further improved, the flexible extrusion of the microvalve chip can well keep the activity of the cells, and the activity is almost not different from that of a control endocytotic group, so that the microvalve chip has a great application prospect for further clinical application in the future.
The present disclosure can utilize various methods to drive cell flow in the fluid layer, including but not limited to syringe pumps, pressure pumps, pipettes, syringes, etc., in this example the cell flow rate is adjusted in increments of 50 μ l/min, with increasing transfection efficiency and cell expression intensity as the cell flow rate increases, and delivering this flexible extrusion with little difference in relative activity to the control endocytosed group relative to other extrusions.
4. Flexible squeeze delivery independent of cell and delivery substance
The present disclosure allows passive expansion or convective delivery of foreign substances into cells by the safe generation of transient pores in the cell membrane while maintaining high cellular activity through highly dynamically adjustable flexible compression of the cells, and thus the flexible compression delivery of the present disclosure is independent of the cells and the delivered substance. The substances delivered in the present disclosure include, but are not limited to, DNA, RNA, proteins, drugs, or nanomaterialsOne substance or a mixture of a plurality of substances. In this example, some were FITC-labeled dextrans of different sizes 3k Da, 70k Da, 2000k Da to mimic nucleic acids, proteins, nanoparticles, etc. The delivery of MCF-7 cells in this example all have delivery efficiencies above 90% for different FITC-labeled dextrans and have the property of flexible extrusion without significant change in cell activity relative to control endocytosed groups. Exemplary nucleic acids in some cases of the present disclosure include, but are not limited to, EGFP mRNA (TriLink, L-7601-100), MCF-7 cells were transfected at a lower concentration of 2 μ g/mL relative to normal experiments, as shown in FIG. 7, and the present disclosure enables efficient delivery of EGFP-mRNA to MCF-7 cells by flexible extrusion with higher EGFP expression after 24h and transfection efficiency of more than 90%. The disclosed DNA includes, but is not limited to, the backbone plasmid lifeAct-TagRFP (Ibidi, p)CMV-
TagRFP), as shown in FIG. 8, MCF-7 cells were delivered at a concentration of 5. mu.g/mL in this example, and 1. mu.g/mL of Polybrene (omiget), which is a transfection assisting agent, was mixed into the delivery solution, and it was found that the expression was high after 48 hours of delivery, and the delivery efficiency was also more than 90%.
The generation of transient pores by flexible extrusion of the present disclosure is also independent of the cell type delivered, including and not limited to human cells, animal cells, plant cells, bacterial cells, applicable to suspension and adherent cells, primary cells and cell lines, and is not limited by cell type. The cell suspension can be a mixed and purified cell population, and in some embodiments, cell lines MCF-7, HeLa, IEC6 and A549 with different cell sizes from 10 μm to 20 μm and the like can have the activity which is not obviously changed relative to control endocytotic cells and simultaneously have higher transfection efficiency, so that the microfluidic chip disclosed by the invention has wider adaptability to cells with different sizes. Meanwhile, for some primary cells which are difficult to transfect, such as MEF (embryonic mouse fibroblast) cells commonly used for cell reprogramming, as shown in FIG. 5, the delivery efficiency can reach more than 50% while the high activity can be maintained, and the efficiency can be further improved through the flow rate. Primary mouse and human immune T cells that were difficult to transfect were included in this example, and as shown in fig. 6, delivery efficiency was improved by over 60% while maintaining high activity by adjusting air valve extrusion size.
The above embodiments are provided to further explain the purpose, technical solutions and advantages of the present disclosure in detail, and it should be understood that the above embodiments are merely exemplary of the present disclosure and are not intended to limit the present disclosure, and any modifications, equivalents, improvements, etc. made within the spirit and principle of the present disclosure should be included in the scope of the present disclosure.
Claims (10)
1. A cell mechanical transfection method based on a flexible variable cross-section microchannel is characterized by comprising the following steps:
s1, introducing liquid containing cells and exogenous substances into a liquid path layer of the microfluidic device, wherein the liquid path layer comprises a plurality of liquid microchannels;
s2, applying air pressure to an air path layer of the micro-fluidic device, and enabling a thin film layer between the liquid path layer and the air path layer to deform so as to change the section size of a liquid path micro-channel at a corresponding position; when the cell passes through the liquid microchannel at the corresponding position, the cell is stressed by the flexible film layer, a minimally invasive membrane hole is generated on a cell membrane instantaneously, and the exogenous substance enters the cell through the minimally invasive membrane hole to finish cell transfection.
2. The method for mechanical transfection of cells based on flexible variable cross-section micro-channels according to claim 1, wherein the step of introducing the liquid containing the cells and the exogenous substances into the liquid path layer of the micro-fluidic device in S1 comprises:
an injection pump, pressure pump, pipettor, syringe or injector is used to drive the cell flow and regulate the flow rate of the liquid.
3. A cytomechanical transfection method based on flexible variable cross-section micro-channel according to claim 2 characterized in that said applying air pressure to the air path layer of the micro-fluidic device in S2 comprises:
and applying air pressure to the air path layer by using an air pressure pump, wherein the air pressure comprises applying positive pressure to extrude the liquid microchannel at the corresponding position or restoring the liquid microchannel when loading negative pressure.
4. The method for cytomechanical transfection based on flexible variable cross-section micro-channel according to claim 1, characterized in that said S1 further comprises:
rinsing and incubating the microchannel with a delivery buffer, wherein the delivery buffer comprises a phosphate buffered saline, pluronic, and bovine serum albumin.
5. The method for cytomechanical transfection based on flexible variable cross-section micro-channel according to claim 4, characterized in that said S1 further comprises:
cells were cultured and resuspended using the delivery buffer.
6. A method for cytomechanical transfection based on flexible variable cross-section microchannels, characterized in that the material of the microfluidic device comprises thermoplastic or cold-plastic elastic material.
7. The cytomechanical transfection method based on flexible variable cross-section micro-channel according to any one of claims 1 to 6,
the cell comprises any one or the combination of human cells, animal cells, plant cells and bacterial cells; or
The cells comprise any one or the combination of suspension cells and adherent cells; or
The cells comprise any one of primary cells, cell lines or a combination thereof.
8. The cytomechanical transfection method based on the flexible variable cross-section micro-channel is characterized in that the exogenous substances comprise any one or the combination of plasmids, DNA, RNA, amino acids, peptides, proteins, drugs, growth factors, nanomaterials and viruses.
9. A microfluidic device for implementing a flexible variable cross-section microchannel-based cytomechanical transfection method according to any one of claims 1 to 8, comprising:
the liquid path layer comprises a plurality of liquid microchannels and is used for introducing liquid of cells and exogenous substances;
a gas circuit layer comprising a plurality of gas microchannels, the gas microchannels at least partially aligned with the liquid microchannels;
the thin film layer is arranged between the liquid path layer and the air path layer, when air pressure is applied to the air path layer, the thin film layer is used for deforming and changing the section size of the liquid path micro-channel at the corresponding position, the cells are flexibly extruded by the thin film layer, minimally invasive membrane holes are instantly formed in cell membranes, and exogenous substances enter the cells through the minimally invasive membrane holes to finish cell transfection.
10. The microfluidic device of claim 9, wherein the liquid microchannel is staggered with the gas microchannel;
the extrusion size range of the film layer is 0-25 mu m, and the elastic modulus range is 1 KPa-1 MPa.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210139490.8A CN114621979B (en) | 2022-02-15 | 2022-02-15 | Cell mechanical transfection method and device based on flexible variable-section micro-channel |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210139490.8A CN114621979B (en) | 2022-02-15 | 2022-02-15 | Cell mechanical transfection method and device based on flexible variable-section micro-channel |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114621979A true CN114621979A (en) | 2022-06-14 |
| CN114621979B CN114621979B (en) | 2024-04-09 |
Family
ID=81898512
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210139490.8A Active CN114621979B (en) | 2022-02-15 | 2022-02-15 | Cell mechanical transfection method and device based on flexible variable-section micro-channel |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114621979B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115254214A (en) * | 2022-06-29 | 2022-11-01 | 中国科学院精密测量科学与技术创新研究院 | Microfluidic channel, microfluidic chip and biochemical molecule delivery method |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105132284A (en) * | 2011-05-13 | 2015-12-09 | 加利福尼亚大学董事会 | Photothermal substrates for selective transfection of cells |
| CN107922911A (en) * | 2015-07-09 | 2018-04-17 | 麻省理工学院 | Material is delivered to cytode |
| WO2019079787A1 (en) * | 2017-10-20 | 2019-04-25 | The Regents Of The University Of California | Microfluidic systems and methods for lipoplex-mediated cell transfection |
| US20190322976A1 (en) * | 2018-04-20 | 2019-10-24 | Cellix Limited | Method and device for transfecting cells |
| EP3871773A1 (en) * | 2020-02-27 | 2021-09-01 | Cellix Limited | A method of determining the transfection status of a plurality of cells |
| CN113817589A (en) * | 2021-09-03 | 2021-12-21 | 南昌大学 | Cell transfection device, cell transfection method and micro-channel manufacturing method |
-
2022
- 2022-02-15 CN CN202210139490.8A patent/CN114621979B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105132284A (en) * | 2011-05-13 | 2015-12-09 | 加利福尼亚大学董事会 | Photothermal substrates for selective transfection of cells |
| CN107922911A (en) * | 2015-07-09 | 2018-04-17 | 麻省理工学院 | Material is delivered to cytode |
| WO2019079787A1 (en) * | 2017-10-20 | 2019-04-25 | The Regents Of The University Of California | Microfluidic systems and methods for lipoplex-mediated cell transfection |
| US20190322976A1 (en) * | 2018-04-20 | 2019-10-24 | Cellix Limited | Method and device for transfecting cells |
| EP3871773A1 (en) * | 2020-02-27 | 2021-09-01 | Cellix Limited | A method of determining the transfection status of a plurality of cells |
| CN113817589A (en) * | 2021-09-03 | 2021-12-21 | 南昌大学 | Cell transfection device, cell transfection method and micro-channel manufacturing method |
Non-Patent Citations (2)
| Title |
|---|
| JONATHAN J CHOU: "A design approach for layer-by-layer surface-mediated siRNA delivery", ACTA BIOMATER * |
| 王欣: "PCM修饰脂质体的制备及心肌靶向性初步评价", 第二军医大学学报 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115254214A (en) * | 2022-06-29 | 2022-11-01 | 中国科学院精密测量科学与技术创新研究院 | Microfluidic channel, microfluidic chip and biochemical molecule delivery method |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114621979B (en) | 2024-04-09 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| AU2018201426B2 (en) | A Device for Mechanical and Hydrodynamic Microfluidic Transfection | |
| Leclerc et al. | Cell culture in 3-dimensional microfluidic structure of PDMS (polydimethylsiloxane) | |
| JP6805152B2 (en) | Method of injecting a substance into a cell to transform the cell {Method for Modifying a Cell by Putting Material into the Cell} | |
| EP3500662B1 (en) | High-throughput system and method for the temporary permeabilization of cells | |
| Khoury et al. | A microfluidic traps system supporting prolonged culture of human embryonic stem cells aggregates | |
| US20190275520A1 (en) | Flow-through microfluidic methods and devices featuring membrane-perturbing surface interactions for intracellular delivery | |
| US20080248575A1 (en) | Drug and Gene Delivery by Polymer Nanonozzle and Nanotip Cell Patch | |
| CN114621979B (en) | Cell mechanical transfection method and device based on flexible variable-section micro-channel | |
| Nejad et al. | Laterally confined microfluidic patterning of cells for engineering spatially defined vascularization | |
| CN113874488A (en) | Erythrocyte removing device, monocyte recovering device, cell culture system, cell culture method, and monocyte recovering method | |
| Ding et al. | A modular 3D printed microfluidic system: a potential solution for continuous cell harvesting in large-scale bioprocessing | |
| WO2002058847A9 (en) | Cell transformation using a single chip silicon microfabricated array incorporating integrated micro-piercing injectors | |
| Jiang et al. | Noncovalent reversible binding-enabled facile fabrication of leak-free PDMS microfluidic devices without plasma treatment for convenient cell loading and retrieval | |
| Okanojo et al. | Nuclear transplantation between allogeneic cells through topological reconnection of plasma membrane in a microfluidic system | |
| Williams et al. | Filtroporation: A simple, reliable technique for transfection and macromolecular loading of cells in suspension | |
| Piraino et al. | Polyester μ-assay chip for stem cell studies | |
| Leclerc et al. | A multi-layer PDMS microfluidic device for tissue engineering applications | |
| Huang et al. | A pneumatically-driven microfluidic system for size-tunable generation of uniform cell-encapsulating collagen microbeads with the ultrastructure similar to native collagen | |
| US11866685B2 (en) | Temperature responsive device for mechanobiological manipulation | |
| Huang et al. | Microfluidic channel with embedded monolayer nanofibers for cell culture and co-culture | |
| Dong et al. | Design and fabrication of vascular network for muscle tissue engineering | |
| Hu et al. | Controlled patterning of magnetic hydrogel microfibers under magnetic tweezers | |
| Kashani et al. | Cell-imprinted-based integrated microfluidic device for biomedical applications: Computational and experimental studies | |
| US20220306993A1 (en) | Erythrocyte removal device, mononuclear cell collector, cell culture device, cell culture system, cell culture method, and mononuclear cell collection method | |
| Bourguignon et al. | Micro and milli-chamber bioreactors for human induced pluripotent stem cell culture: Model and experimental validation |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |