Directions for Use

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1. Transfer the required weight of LifeSupport™powder into a 50 mL sterile conical centrifuge tube (see Table 2 for recommended weights). Do not weigh out more than 2.1 grams per centrifuge tube.

2. Weigh out required powder into a 50 mL centrifuge tube, and then add recommended volume of COLD media as found in Table 2. Note – these volumes are tofillthe printing dish, which is not needed for all prints. For example, printing a flat scaffold may only require the dish to be partially filled.

Table 2:

LifeSupport™can be customized to fit your specific bioink.See the Table 3 below for help on choosing the right suspension media to take advantage of the unique benefits of FRESH bioprinting.

Table 3:

*Note: centrifugation speed and time may vary slightly depending on the suspension media. Serum-based growth media requires higher centrifugation speed (ie. 2500-3000G)

3. Vortex for 1 minute, ensuring that all the powder is fully resuspended and then allow to sit for 10 minutes while the powder fully rehydrates.

4. Centrifuge the support material according to table 3.

5. Support material should be compacted at the bottom of the tube.Pour off the supernatant.

6. Aseptically scoop out the compacted LifeSupport™slurry into the desired printing dish using a sterile spatula or other device. Avoid introducing large air bubbles in the slurry. The slurry should not move if the dish is held sideways.

You are ready for FRESH bioprinting.

Printing Recommendations:1. Place LifeSupport™support slurry in your 3D bioprinter.

2. Calibrate (zero) your needle near the bottom of the dish (z), and near the middle (x,y). See below.

Note: Unlike typical printing, the needle does not have to be touching or even close to the bottom of the dish. The LifeSupport™support slurry will trap your print in-place, no matter where you start. Ensure that the print container is large enough to avoid the needle hitting into the walls during printing.

3. Bioprinting can now be initiated. Ensure the container will not shift on the print bed.Note – It is possible to print multiple constructs in the same container in order to maximize the number of prints per LifeSupport™kit.

Post Print Recommendations:1. After printing, incubate the printed construct at 37°C for at least 30 minutes to allow the LifeSupport™slurry to melt and release the bioprinted construct.

2. Cellularized bioinks printed into cell media-based support material can be directly placed in a cell incubator for 24 hours before needing to exchange media (ensuring proper levels of CO2).

3. After 30 minutes of post-print incubation, the LifeSupport™slurry will be fully melted and your printed structure will be released. Large volumes may require longer times to fully melt.

4. Transfer of bioprinted construct:

4a. Carefully transfer bioprinted constructs into desired cell culture medium.

4b. As an alternative, melted LifeSupport™slurry can be serially replaced with pre-warmed cell culture medium, to avoid handling the printed construct. For example, if you printed into a 6-well plate, this can be done by carefully aspirating out 2 mL of melted LifeSupport™and adding 2 mL of pre-warmed cell culture medium. Repeat this process until all of the melted LifeSupport™slurry has been replaced by medium.

5. Continue standard media changes in accordance with cell culture protocol.

LifeSupport™is manufactured by FluidForm under license from Carnegie Mellon University Patent No. 10,150,258.

Product Bioprints

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Product References

References for FRESH Printing:

Lee, A.et al.3D bioprinting of collagen to rebuild components of the human heart.Science365,482–487 (2019).

Thomas, H. & Adam, F. 3D printing complex scaffolds using freeform reversible embedding of suspended hydrogels (FRESH).Frontiers in Bioengineering and Biotechnology4,(2016).

Isaacson, A., Swioklo, S. & Connon, C. J. 3D bioprinting of a corneal stroma equivalent.Experimental Eye Research173,188–193 (2018).

Jeon, O., Lee, Y., Hinton, T., Feinberg, A. & Alsberg, E. Cryopreserved cell-laden alginate microgel bioink for 3D bioprinting of living tissues.Materials Today Chemistry12,61–70 (2019).

Maxson, Eva L., et al. "In vivo remodeling of a 3D-Bioprinted tissue engineered heart valve scaffold."Bioprinting(2019): e00059.

Safety and Documentation

Safety Data Sheet

Certificate of Origin

Product Disclaimer

This product is for R&D use only and is not intended for human or other uses. Please consult the Material Safety Data Sheet for information regarding hazards and safe handling practices.