Den neddykkede Trykning af celler på en modificeret overflade ved hjælp af en kontinuerlig strøm Microspotter
Summary
This 3D microfluidic printing technology prints arrays of cells onto submerged surfaces. We describe how arrays of cells are delivered microfluidically in 3D flow cells onto submerged surfaces. By printing onto submerged surfaces, cell microarrays were produced that allow for drug screening and cytotoxicity assessment in a multitude of areas.
Abstract
The printing of cells for microarray applications possesses significant challenges including the problem of maintaining physiologically relevant cell phenotype after printing, poor organization and distribution of desired cells, and the inability to deliver drugs and/or nutrients to targeted areas in the array. Our 3D microfluidic printing technology is uniquely capable of sealing and printing arrays of cells onto submerged surfaces in an automated and multiplexed manner. The design of the microfluidic cell array (MFCA) 3D fluidics enables the printhead tip to be lowered into a liquid-filled well or dish and compressed against a surface to form a seal. The soft silicone tip of the printhead behaves like a gasket and is able to form a reversible seal by applying pressure or backing away. Other cells printing technologies such as pin or ink-jet printers are unable to print in submerged applications. Submerged surface printing is essential to maintain phenotypes of cells and to monitor these cells on a surface without disturbing the material surface characteristics. By printing onto submerged surfaces, cell microarrays are produced that allow for drug screening and cytotoxicity assessment in a multitude of areas including cancer, diabetes, inflammation, infections, and cardiovascular disease.
Introduction
Nylige fremskridt inden for den farmaceutiske industri har ført til en øget interesse for at bruge cellulære microarrays i drug discovery processen for narkotika screening og cytotoxicological analyse 1,2,3. Udviklingen af in vitro-high-throughput assays og screeningsmetoder bruger celle microarrays vil lette en hurtig og omkostningseffektiv udvikling af lægemiddelkandidater samt forhånd grundlæggende forståelse af cellen 1,4. Den traditionelle tilgang til screening med celler anvender konventionelle vel-plade platforme; men denne fremgangsmåde er begrænset på grund af de høje omkostninger, begrænset gennemløb, og begrænset evne til kvantitativ information om celle funktion 1,5. På grund af disse begrænsninger er forskning i cellulær microarray teknologi spirende for molekylærbiologisk karakterisering, tissue engineering, og narkotika screening 1,6. Fordelene ved cellulære microarrays omfatter mindre stikprøve brug, minimale effekter afcellefænotypen heterogenitet maskering information, og vigtigst evnen til at automatisere analyser til mere high-throughput applikationer 1,7,8.
Den farmaceutiske industri i øjeblikket udnytter high-throughput cellebaserede screeningsbestemmelser med 2D celle monolagskulturer til drug screening i mikrotiterbrønd plader 9. Multiplexing celler i brønde af mikrotiterplader rummer muligheder for højere gennemløb med unikke eksperimenter muligheder. Endvidere er de nuværende teknologier til cellulære microarrays lade cellerne tørre som kan dramatisk ændre fænotypen af celler fra in vivo 10,11. For at overvinde disse problemer blev MFCA manipuleret og er vist i fig. 1. Udformningen af MFCA 3D fluidik muliggør skrivehovedet spids i figur 1 til at blive sænket ned i et bad og komprimeres mod en overflade for at danne en forsegling. Den bløde silikone spidsen af skrivehovedet opfører sig som enpakning og danner en reversibel forsegling. Den MFCA teknologi er unikt egnet til at interface med neddykkede overflader, som er nødvendig for både cellekulturer og væv slice systemer, og det er vanskeligt eller umuligt med de fleste andre metoder. Stifter eller inkjet-print vil ikke fungere, og 2D mikrofluidenheder er ikke egnet til deponering eller sammenknytning med store arrays af diskrete pletter. Yderligere, ved miniaturisering og lokalisere eksperimentet – det cellulære microarray – den MFCA overvinder de store problemer i forbindelse med high-throughput cellebaserede screeningsassays.
CFM bruger 3D-kanal netværk til at cykle små volumen væskeprøver end mikroskopiske spot steder på en overflade 12,13. Ved at udskrive med flow, biomolekyler, celler og andre reagenser opbevares i et flydende miljø i hele trykprocessen, muliggør udskrivning af følsomme biomolekyler og celler uden eksponering for luft, hvilket hindrer de aktuelle celle trykteknikker. Det er også muligt at udskrive direkte fra rå materiale såsom hybridom eller supernatanter forudsat der er en fange mekanisme på array'et overflade. Formålet med dette manuskript er at forklare i detaljer den neddykkede trykning af to celletyper på en overflade.
Protocol
Representative Results
Discussion
3D mikrofluid printteknologi beskrevet her er unikt i stand til microfluidically trykning arrays af celler i en væske fyldt godt, dvs en nedsænket overflade. Ved trykning på neddykkede overflader, kan celle microarrays produceres der opretholder det fysiologisk relevante cellulære fænotype af celler såvel som evnen til at multiplekse cellerne i bunden af en enkelt brønd fig. 4. Resultaterne af denne undersøgelse viser, at microfluidically udskrivning celler resulterer i cellefæstnelse …
Disclosures
The authors have nothing to disclose.
Acknowledgements
Forfatterne vil gerne anerkende Chris Morrow for teknisk assistance. Finansieringen blev leveret af NIH SBIR (R43) giver 1R43GM101859-01 (MPI) GRANT10940803.
Materials
| Continuous Flow Microspotter | Wasatch Microfluidics | ||
| NIH/3T3 cells | ATCC | CRL-1658 | |
| Dubbleco's Modified Eagle Medium | Invitrogen | 11965-092 | base media for cells |
| HEPES buffer | Invitrogen | 15630-080 | cell media additive (control pH) |
| Sodium pyruvate | Invitrogen | 11360-070 | cell media additive |
| Penicillin-Streptomycin | Invitrogen | cell media additive | |
| Trypan blue | Invitrogen | 15250-061 | stain cell sfor counting |
| Haemocytometer | Fisher | 267110 | cell chamber to count cells |
| Nikon Eclipse TS100 | Nikon | Used to check on cells | |
| Nikon Eclipse TE2000-U | Nikon | Used for collecting images | |
| Phosphate Buffered Saline (with calcium and magnesium) | Invitrogen | 14040-133 | rinsing cells before passaging and before staining with PI |
| TrypLE Express | Invitrogen | A12177-01 | used to remove cells from surface |
References
- Fernandes, T. G., Diogo, M. M., Clark, D. S., Dordick, J. S., Cabral, J. High-throughput cellular microarray platforms: applications in drug discovery, toxicology and stem cell research. Trends in Biotechnology. 27, 342-349 (2009).
- Michelini, E., Cevenini, L., Mezzanotte, L., Coppa, A., Roda, A. Cell-based assays: fuelling drug discovery. Anal Bioanal Chem. 398, 227-238 (2010).
- Gao, D., et al. Recent developments in microfluidic devices for in vitro cell culture for cell-biology research. TrAC Trends in Analytical Chemistry. 35, 150-164 (2012).
- Geysen, H. M., Schoenen, F., Wagner, D., Wagner, R. Combinatorial compound libraries for drug discovery: an ongoing challenge. Nature Reviews Drug Discovery. 2, 222-230 (2003).
- Xu, Y., Shi, Y., Ding, S. A chemical approach to stem-cell biology and regenerative medicine. Nature. 453, 338-344 (2008).
- Khademhosseini, A., Langer, R., Borenstein, J., Vacanti, J. P. Microscale technologies for tissue engineering and biology. Proceedings of the National Academy of Sciences of the United States of America. 103, 2480-2487 (2006).
- Bhadriraju, K., Chen, C. S. Engineering cellular microenvironments to improve cell-based drug testing. Drug Discovery Today. 7, 612-620 (2002).
- Castel, D., Pitaval, A., Debily, M. -. A., Gidrol, X. Cell microarrays in drug discovery. Drug Discov. Today. 11, 616-622 (2006).
- Tsui, J. H., Lee, W., Pun, S. H., Kim, J., Kim, D. -. H. Microfluidics-assisted in vitro drug screening and carrier production. Advanced Drug Delivery Reviews. , (2013).
- Gottwald, E., et al. A chip-based platform for the in vitro generation of tissues in three-dimensional organization. Lab on a Chip. 7, 777-785 (2007).
- Liu, R., Lee, A. P. . Integrated Biochips for DNA Analysis. , (2008).
- Natarajan, S., et al. Continuous-flow microfluidic printing of proteins for array-based applications including surface plasmon resonance imaging. Analytical Biochemistry. 373, 141-146 (2008).
- Natarajan, S., Hatch, A., Myszka, D. G., Gale, B. K. Optimal Conditions for Protein Array Deposition Using Continuous Flow. Anal. Chem. 80, 8561-8567 (2008).
- Keighley, W. The need for high throughput kinetics early in the drug discovery process. , (2011).
- Xu, F., et al. A three-dimensional in vitro ovarian cancer coculture model using a high-throughput cell patterning platform. Biotechnol J. 6, 204-212 (2011).
- Tuckwell, D. S., Weston, S. A., Humphries, M. J. Integrins: a review of their structure and mechanisms of ligand binding. Symposia of the Society for Experimental Biology. 47, 107 (1993).
- Roberts, C., et al. Using mixed self-assembled monolayers presenting RGD and (EG) 3OH groups to characterize long-term attachment of bovine capillary endothelial cells to surfaces. Journal of the American Chemical Society. 120, 6548-6555 (1998).
- Chandra, R. A., Douglas, E. S., Mathies, R. A., Bertozzi, C. R., Francis, M. B. Programmable cell adhesion encoded by DNA hybridization. Angewandte Chemie. 118, 910-915 (2006).
- Hsiao, S. C., et al. Direct cell surface modification with DNA for the capture of primary cells and the investigation of myotube formation on defined patterns. Langmuir. 25, 6985-6991 (2009).
- Black, C. B., Duensing, T. D., Trinkle, L. S., Dunlay, R. T. Cell-Based Screening Using High-Throughput Flow Cytometry. ASSAY and Drug Development Technologies. 9, 13-20 (2011).
- Derby, B. Bioprinting: inkjet printing proteins and hybrid cell-containing materials and structures. J. Mater. Chem. 18, 5717-5721 (2008).
- Onoe, H., et al. Cellular Microfabrication: Observing Intercellular Interactions Using Lithographically-Defined DNA Capture Sequences. Langmuir. 28, 8120-8126 (2012).
- Hsiao, S. C., et al. DNA-Coated AFM Cantilevers for the Investigation of Cell Adhesion and the Patterning of Live Cells. Angewandte Chemie International Edition. 47, 8473-8477 (2008).
