En Mikrofluid platform for High-throughput Single-celle Isolering og Kultur
Summary
Here, we present a protocol for isolating and culturing single cells with a microfluidic platform, which utilizes a new microwell design concept to allow for high-efficiency single cell isolation and long-term clonal culture.
Abstract
Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet high-throughput, method to perform single-cell culture experiments. Here, we report a microfluidic chip-based strategy for high-efficiency single-cell isolation (~77%) and demonstrate its capability of performing long-term single-cell culture (up to 7 d) and cellular heterogeneity analysis using clonogenic assay. These applications were demonstrated with KT98 mouse neural stem cells, and A549 and MDA-MB-435 human cancer cells. High single-cell isolation efficiency and long-term culture capability are achieved by using different sizes of microwells on the top and bottom of the microfluidic channel. The small microwell array is designed for precisely isolating single-cells, and the large microwell array is used for single-cell clonal culture in the microfluidic chip. This microfluidic platform constitutes an attractive approach for single-cell culture applications, due to its flexibility of adjustable cell culture spaces for different culture strategies, without decreasing isolation efficiency.
Introduction
Øjeblikket placere enkelte celler individuelt i en kultur rum opnås almindeligvis ved hjælp af begrænsende fortynding eller fluorescens-aktiveret cellesortering (FACS). For mange laboratorier, begrænsende fortynding er en bekvem metode, da den kun kræver en pipette og vævskulturplader, som er let tilgængelige. I dette tilfælde bliver en cellesuspension seriefortyndes til en passende celledensitet, og derefter anbragt i dyrkningsbrønde ved anvendelse af en manuel pipette. Disse rumopdelte enkeltceller anvendes derefter til celleanalyse, såsom genetisk heterogenitet screening 1 og kolonidannelse 2. Men denne metode er lavt gennemløb og arbejdskrævende, uden anvendelse af en robotarm for assistance, fordi Poissonfordelingen karakter af begrænsende fortyndingsmetode begrænser encellede begivenheder til en maksimal sandsynlighed på 37% 3. FACS maskiner med integreret robotarm kan overvinde begrænsningen af Poisson-fordeling ved nøjagtigt placing et enkeltdosis celle i en kultur brønd på et tidspunkt 4. Det høje mekaniske forskydningsspændingen (således sænket cellelevedygtighed) 5 og maskine køb og driftsomkostninger har begrænset dens anvendelse i mange laboratorier.
For at overvinde de ovennævnte begrænsninger, er mikroskala anordninger blevet udviklet til højt effektivt indlæse enkeltceller i mikrobrønde 6. Men mikrobrøndene ikke giver tilstrækkelig plads til de fyldte celler til at proliferere, som følge af behovet for at gøre størrelsen af hver mikrobrønd tæt på den for en enkelt celle for at maksimere enkeltcelle-loading sandsynlighed. Som kultur assays er påkrævet i mange cellebaserede applikationer (f.eks klongenicitetsassayet 7), større mikrobrønde (fra 90 – 650 um i diameter eller i sidelængde) er også blevet anvendt til at muliggøre længere cellekulturer. Men ligesom den begrænsende fortyndingsmetode, de har også en lav encellede belastningseffektiviteter, i området fra 10 -. 30% 8, 9
Tidligere har vi udviklet et high-throughput mikrofluid platform at isolere enkelte celler i individuelle mikrobrønde og demonstrere dens anvendelse i klongenicitetsassayet af de isolerede celler. 10. Anordningen blev fremstillet med poly-dimethylsiloxan (PDMS) og omfatter to sæt af mikrobrønde arrays med forskellige mikrobrønde størrelser, som i vid udstrækning kan forbedre effektiviteten i indlæsning af en enkelt celle i en mikrobrønd hvis størrelse er betydeligt større end cellen. Især denne "dual-brønd" konceptet mulighed størrelsen af kulturen område justeres fleksibelt uden at påvirke den encellede capture effektivitet, hvilket gør det nemt at justere anordningens konstruktion, der passer til forskellige celletyper og anvendelser. Denne højeffektiv metode bør være nyttige for langsigtede cellekultur forsøg til celle heterogenitet undersøgelser og monoklonal cellelinje etablering.
Protocol
Representative Results
Discussion
Mikrobrønd-baseret enhed systemer 6,14 er blevet anvendt til enkelt-celle manipulation og analyse, såsom storstilet enkelt celle trapping 6 og enkelt hæmatopoietisk stamcelleproliferation 15. Selvom godt størrelse, antal, og kan tilpasses til specifikke applikationer, single-celle isolation effektivitet altid kompromitteret, når størrelsen af brønden øges. 9,15
For at overvinde denne begrænsning, Park et al. Rapporterede en mikrof…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
This work was supported by a grant from the National Health Research Institutes (03-A1 BNMP11-014).
Materials
| AutoCAD software | Autodesk | AutoCAD LT 2011 | Part No. 057C1-74A111-1001 |
| Silicon wafer | Eltech corperation | SPE0039 | |
| Conventional oven | YEONG-SHIN company | ovp45 | |
| Plasma cleaner | Nordson | AP-300 | Bench-Top Plasma Treatment System |
| SU-8 50 negative photoresist | MicroChem | Y131269 | |
| SU-8 100 negative photoresist | MicroChem | Y131273 | |
| Spin coater | Synrex Co., Ltd. | SC-HMI 2" ~ 6" | |
| Hotplate | YOTEC company | YS-300S | |
| Msak aligner | Deya Optronic CO. | A1K-5-MDA | |
| SU-8 developer | Grand Chemical Companies | GP5002-000000-72GC | Propylene glycol monomethyl ether acetate |
| Scanning laser profilometer | KEYENCE | VK-X 100 | |
| Trichlorosilane | Gelest, Inc | SIT8174.0 | TRIDECAFLUORO-1,1,2,2-TETRAHYDROOCTYL. Hazardous. Corrosive to the respiratory tract., reacts violently with water. |
| Desiccator | Bel-Art Products | F42020-0000 | SPACE SAVER VACUUM DESICCATOR 190MM WHITE BASE |
| Polydimethylsiloxane (PDMS) kit | Dow corning | Sylgard 184 | |
| Harris Uni-Core puncher | Ted Pella Inc. | 15072 | with 0.75 mm inner-diameter |
| Removable tape | 3M Company | Scotch Removable Tape 811 | |
| Stereomicroscope | Leica Microsystems | Leica E24 | |
| Bovine serum albumin (BSA) | Bersing Technology | ALB001.500 | |
| DMEM basal medium | Gibco | 12800-017 | |
| Fetal bovine serum | Thermo Hyclone | SH30071.03HI | |
| Antibiotics | Biowest | L0014-100 | Glutamine-Penicillin-Streptomycin |
| Recombinant enzyme mixture | Innovative cell technology | AM-105 | Accumax |
| DiIC12(3) cell membrane dye | BD Biosciences | 354218 | Used as a cell tracker |
| Syringe pump | Harvard Apparatus | 703007 | |
| Plastic syringe (1 mL) | BD Biosciences | 309659 | |
| 23 gauge blunt needles | Ever Sharp Technology, Inc. | TD21 | |
| Poly-tetrafluoroethene (PTFE) tubing | Ever Sharp Technology, Inc. | TFT-23T | inner diameter, 0.51 mm; outer diameter, 0.82 mm |
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