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Science Education
>
Biology
Title Cell Encapsulation by Droplets
Instructor Prep
concepts
Student Protocol
JoVE Journal
Biology
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JoVE Journal
Biology
Title Cell Encapsulation by Droplets
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TRANSCRIPT
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 10
4
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
Title Cell Encapsulation by Droplets
Learning Objectives
List of Materials
Print Materials List
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
Lab Prep
Print Setup Guide
Procedure
Print Procedure Steps
Tags
Cell Encapsulation
Droplet Encapsulation
Droplet-based Encapsulation