1Bio-Acoustic-MEMS Laboratory in Medicine (BAMM), HST-Center for Bioengineering, Brigham and Women’s,Harvard Medical School, 2Bio-Acoustic-MEMS Laboratory in Medicine (BAMM), HST-Center for Bioengineering,Brigham and Women’s Hospital, 3Brigham and Women’s Hospital,Harvard Medical School, 4Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology; Center for Biomedical Engineering, Department of Medicine,Brigham and Women’s Hospital
Protocol
NIH3T3 cells preparation:
A. Cells for Ejection
- Trypsinize cells, then dilute 1:1 with cell media, and transfer from a T75 flask to a 15 mL Falcon tube
- Spin down cells into a pellet by centrifuging, aspirate supernatant and wash cells with DPBS
- Spin down cells into a pellet again, and aspirate supernatant
- Resuspend cells in media
- Determine cell density with hemocytometer (~200 X 104 cells/mL per T75 flask)
- Centrifuge cell solution, aspirate supernatant, and resuspend in appropriate amount of media for varying cell concentrations
B. Cell ejection
- Vortex cells before using for ejection
- Transfer 200 µL of cell solution into syringe
- Set appropriate mode on pulse generator
- For ejecting single droplets and multiple droplets (bursts), set pulse generator to “E. BUR” mode
- For continuous droplet ejection, set pulse generator to “NORM” mode
- Change signal settings
- Set high level and low level output voltage: HIL to 5 V and LOL to 0 V and make sure the”LIM”LED is on
- Set signal as a square pulse
- Change the amount of time the solenoid valve is open for droplet ejection by changing the value for “WID” or changing duty cycle (“DUTY”)
- Change the frequency of ejection by changing the value for “PER”
- Change the number of droplets ejected in a burst by changing the value for “BUR”
- Eject cell solution onto prepared substrate for imaging with microscope
C. Staining
- Make up dye solution with 0.5 µL calcein-AM and 2 µL ethidium homodimer per mL of DPBS
- Immerse prepared substrate in dye solution
- Allow sample to incubate for 10 minutes at 37°C before imaging
Experiment Validation
- On a Nikon Eclipse TE-2000 U Fluorescent Microscope
- Spot advanced software (Diagnostics, Inc.)
- Live/Dead Assay
Disclosures
The authors have nothing to disclose.
Materials
| Material Name | Type | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| Fluorescent Microscope | Nikon | Eclipse TE-2000 U | ||
| Solenoid valve cell ejector | Operation frequency: 1 KHz, 30psi, 50nl~0.5ul. 12V Valve Driver: 2.5 Amp drive current | |||
| 5-Gallon Portable air tank | Coleman | Powermate CT5 | Pressurized air: 30 PSI | |
| Pressure regulators | Marsh Bellofram | |||
| Pulse Generator | HP8112A | Actuation frequency generation: 50 MHz | ||
| XYZ-stage | Newmark Systems | With Stepping motor: 6inch travel xy-stage(NLS4-6-25), 2.5inch travel z-stage(NLS4-2.5-25), 3axis controller(NSC-G3), 0.1um resolution | ||
| NIH 3T3 | Cell-line | fibroblasts | ||
| Trypsin | 0.05% solution | |||
| NIH 3T3 cell medium | ||||
| DPBS | Buffer | |||
| T75 | Tissue culture flasks | |||
| Plastic conical tubes | 15 ml, for tissue culture |
Cite This Article
Moon, S., Lin, P., Keles, H. O., Yoo, S., Demirci, U. Title Cell Encapsulation by Droplets. J. Vis. Exp. (8), e316, doi:10.3791/316 (2007).