Title Cell Encapsulation by Droplets
Sangjun Moon1, 2, Pei-Ann Lin1, 2, Hasan Onur Keles1, 2, Seung-Schick Yoo3, Utkan Demirci1, 4, 2
1Bio-Acoustic-MEMS Laboratory in Medicine (BAMM), HST-Center for Bioengineering, Brigham and Women&#x, 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
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0:07 Introduction7 0:21 Subscription Lock21 0:28 Equipment28 5:22 Preparation of cells for ejection322 10:29 Ejection629 12:19 Staining and imaging739
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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
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| Fluorescent Microscope |
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Nikon |
Eclipse TE-2000 U |
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| Solenoid valve cell ejector |
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Operation frequency: 1 KHz, 30psi, 50nl~0.5ul. 12V Valve Driver: 2.5 Amp drive current
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| 5-Gallon Portable air tank |
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Coleman |
Powermate CT5 |
Pressurized air: 30 PSI |
| Pressure regulators |
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Marsh Bellofram |
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| Pulse Generator |
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HP8112A |
Actuation frequency generation: 50 MHz
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| XYZ-stage |
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Newmark Systems |
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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 |
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fibroblasts |
| Trypsin |
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0.05% solution |
| NIH 3T3 cell medium |
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| DPBS |
Buffer |
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| T75 |
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Tissue culture flasks |
| Plastic conical tubes |
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15 ml, for tissue culture |
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Moon S, Lin PA, Keles HO, Yoo SS, Demirci U (2007). Title Cell Encapsulation by Droplets. JoVE. 8. http://www.jove.com/index/details.stp?id=316, doi: 10.3791/316
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Allowed tags: i, b, u, sup, sub 
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Mailer.URL: http://www.jove.com/index/details.stp?id=316 |
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