SCAFFOLD MATERIAL PREPARATION FOR LAB GROWN MEAT


General Notes

Testing different protocols to produce hydrogels for 3D fat constructs.

Materials:

All parts

Misc. Consumables (pipettes, falcon tubes, flasks, etc.)
Growth Medium (prepared previously)

Feeding

DM2-Induction Medium (prepared previously)
DM2-Accumulation Media (prepared previously)Large volume for flask7 small volumes for plate
Large volume for flask
7 small volumes for plate

Hydrogel Experiments

2% (w/v) Alginate Solution
5% (w/v) CaCl2 Solution (concentrated stock)
PBS
Distilled Water
Parafilm

Methods

Feeding Fat

  1. Feed fat construct (T175) with 25mL of appropriate medium
    1. Growth Medium if still <90% confluence
    2. DM2-IM for first differentiation feeding
    3. DM2-AM for subsequent differentiation feedings
  2. Feed 48-well plate with 250uL of appropriate media
  3. Continue to feed flask and plate on the Sun,Tues,Thurs cycle

Material Testing

  1. Alginate hydrogels can be formed by combining the alginate and CaCl2 solutions.
  2. The CaCl2 solution may be diluted before use. Look in literature for reference values.
  3. Determine metrics for desirability.
  4. Choose at least 3 variables to test, and set up separate experiments for deciding hydrogel protocol.
    1. Initial test may provide insight for subsequent experiments. Record full process.
    2. Instructors may provide ideas for variables, but feel free to come up with your own!
  5. For any long-term experiments (>3h), be sure to store hydrogels in liquid medium in well plate and seal with parafilm (to prevent dehydration).
  6. Have fun investigating!

References

[1] L. Q. Wan, J. Jiang, D. E. Arnold, X. E. Guo, H. H. Lu, and V. C. Mow, “Calcium Concentration Effects on the Mechanical and Biochemical Properties of Chondrocyte-Alginate Constructs,” Cell Mol Bioeng, vol. 1, no. 1, pp. 93–102, Mar. 2008, doi: 10.1007/s12195-008-0014-x.

[2] J. S. Yuen et al., “Macroscale Adipose Tissue from Cellular Aggregates: A Simplified Method of Mass Producing Cell-Cultured Fat for Food Applications.” bioRxiv, p. 2022.06.08.495192, Jun. 18, 2022. doi: 10.1101/2022.06.08.495192.

This protocol was developed for BME 174: cultivated meat laboratory course at Tufts University. The course is administered by Professor David Kaplan's laboratory.








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