How to Break Down Cell Walls:
-Grinding: Mortar and pestle, which is often done with plants frozen in liquid nitrogen.
-Beadbeating: Cracking open cells using ceramic or glass beads, typically done in suspension and in a vortex.
-Sonification: Using ultrasound with plant matter in a solution, by cavitation shockwave. -Homogenizer: Shear force by forcing cells through tubes smaller than cells, by rotor- stator (rotating blade) or outer layer shear (French Press).
-Freezing: Cell rupture from freeze thaw process. Can take lots of time.
-High Temperature (and Pressure): Cells walls are disrupted, but denatures proteins, and heat can damage cell contents. Typically by autoclave, microwave, steam, etc.
-Enzymes: Remove cell wall by using naturally occurring enzymes.
-Chemicals: Organic solvents like ethanol (alcohol), especially for hydrophobic (doesn’t like water) molecules. Commonly used with shearing forces.
-Bacteria: EDTA, negative bacteria, to chelate cations that bore holes in cell walls.
REF: http://bitesizebio.com/13536/bringing- down-the-walls-part-ii-8-methods-to-break- down-cell-walls/
Cell Lysis Methods:
Reagent Based Methods:
-Fast, efficient, reproducible
-Can extract total protein or subcellular fractions
-Disrupts cell wall and or lipid membrane
-Larger footprint for equipment
-Not compatible with high-throughput and small volumes
-Aggregation and denaturation of protein may occur
-Cells disrupt at different times
REF: https://www.thermofisher. com/us/en/home/life-science/protein- biology/protein-biology-learning- center/protein-biology-resource-library/pierce- protein-methods/traditional-methods-cell-
Tensile Strength of Cell Walls
Cylindrical Cell Shape: 100 atm or 1,470 psi
Spherical Cell Shape: 95 atm or 1,396 psi
Spherical Cell Shape: 30 atm or 441 psi
REF: https://www.ncbi.nlm.nih. gov/pmc/articles/PMC1074911/pdf/plntphys0 0593-0165.pdf
Plant Cell Vacuoles
The central vacuole (may be 80 percent of space) is a membrane bound sac which provides cell support and helps the plant function with growth.
Turgor Pressure: Vacuoles help to maintain and control the rigidity of the cell (structure),
by compensating the osmotic pressure from within the cell and pressure exerted from outside the cell.
Cannabis sativa: The Plant of the Thousand and One Molecules
Cell Disruption Using a Microfluidizer
Using a Microfluidizer versus a French Press using the same 20,000 psi back pressure, resulted in 92 percent breakage in 8 passes, versus only 50 percent breakge for the French Press in 7 passes.
REF: https://www.microfluidicscorp.com/sites/default/files/application-note-cell- disruption-publication-summaries.pdf
Practical Use of Continuous Processing in Developing and Scaling Up Laboratory Processes
Continuous flow reactors allow for better control of exothermic processing than do batch reactions, and allow for a more efficient and safe scale-up of rapid reactions in a smaller footprint.
REF: http://pubs.acs. org/doi/abs/10.1021/op0100605? journalCode=oprdfk