Tissue vasculature is essential to maintaining its viability and functionality, however, there are currently no simple ways to create thick vascularized tissue. This vascularity kit enables researchers to create thick vascularized tissue casted in a hydrogel with any vascular network design. The tissue unit created with this kit can be easily connected to other tissue units and pumps.
Storage and Handling
Pluronic should be stored at 4˚C. Be sure to wear powder-free gloves when handling this material. GelMA should be stored at -80˚C.
You Will Need
- Allevi vascularization chip
- 3 mL of Allevi pluronic
- 1.5 mL 20% w/v Allevi GelMA
- 0.5 g LAP Photoinitiator
- Allevi 5 mL syringe
- 2 x 25G tapered plastic needles
- 5 x 30G plastic needles
- Syringe coupler
- Cell media
Instructions for Use
- Place your GelMA syringe in a water bath at 37˚C;
- Dissolve LAP at 60˚C in your cell media at a 0.5% w/v concentration (this will take about 20 minutes);
- Sterile filter your LAP-media solution;
- Add 2.5 mL of your solution to your cell pellet, making sure you have the correct cell number for you desired final cell concentration (final volume is 3 mL) and that your solution is at 37˚C;
- With the sterilized Allevi syringe, draw 1.5 mL of your cell solution;
- Place the sterilized coupler on your syringe and attach the GelMA syringe to it;
- Mix back and forth until homogenous (about 10 times);
- Place your syringe in the printer and bioprint!
- NOTE: If you have an Allevi 2, make sure you transfer your solution to a 10 mL syringe in order to start your print session.
- Attach the 25G plastic tapered needle to the GelMA syringe and place it in the left extruder;
- Attach the 30G plastic tip to the pluronic syringe and load the right extruder;
- NOTE: You may use lower gauges for thicker channels.
- Set the left extruder temperature to 30˚C;
- Wait for about 5 minutes until the GelMA solution reaches 30˚C;
- Use the grid on the gasket slide to calibrate the right extruder to the x, y and z coordinates that align with the gasket inlet and outlet;
- Calibrate the right extruder at these coordinates;
- Calibrate the left extruder at the bottom right corner of the gasket, with the tip right at the top surface;
- Use our straight line or convoluted channel file, which codes for crosslinking, or create your own file that:
- Extrudes GelMA at 10 PSI for 4 s, crosslinks GelMA for 60s at 10 mW/cm², prints the desired pluronic channel at 100 PSI and 5 mm/s, extrudes GelMA at 10 PSI for 4 s and crosslinks it for 60s at 10 mW/cm².
- Note: the total gasket volume is 3 mL and the dimensions are 1.6 x 1.6 x 1.1 cm.
- After crosslinking, place your gasket in 4˚C for 5 minutes;
- Connect a pump or syringe to the luer lock gasket connection and perfuse the channel with cold (4˚C) media until all of the pluronic is washed away;
- Culture your vascularized tissue as desired.
|Speed (mm/s)||Layer height (mm)||Nozzle Diam (mm)||Gauge|
|Pressure (PSI)||Crosslink (sec)||Print Temp (°C)|
Kolesky, D. B., Truby, R. L., Gladman, A. S., Busbee, T. A., Homan, K. A., & Lewis, J. A. (2014). 3D Bioprinting of Vascularized, Heterogeneous Cell-Laden Tissue Constructs. Advanced Materials, 26(19), 3124–3130. https://doi.org/10.1002/adma.201305506