This is our experiment section. Here we compile important protocols for the development of TecTissue, ranging from our bacterial transformation procedures to our cell proliferation assays. We also address cell culture maintenance and protein loaded chitosan nanoparticles. Here you may find the protocol for our growth factor delivery to damaged cells and how much harm can be inflicted in vitro.

Protocols

Chitosan nanoparticles

Protein encapsulation protocol

Reactants 

• Chitosan low molecular weight from Sigma-Aldrich
• TPP from Sigma-Aldrich
• NaOH 1M
• Acetic acid 1M
• Distilled water
• Protein of interest (10 mg/mL)

  

Procedure

Stock solutions

 

1. In a 15 mL Falcon tube add 30 mg of chitosan and 10 mL of distilled water (to get a solution with a concentration of 3 mg/mL).
2. Add 10 microliters of acetic acid for each mL of chitosan solution to solubilize the chitosan. To adjust the pH acetic acid and NaOH should be used.
NOTE: the pH should be adjusted depending on your protein of interest, taking into account the isoelectric point, always maintaining the chitosan solution positively charged (pH<6.5) and the protein of interest negatively charged (preferred).
3. In another 15 mL falcon tube add 10 mg of TPP and 10 mL of distilled water (to get a concentration of 1 mg/mL).

 

Nanoparticle preparation

1. In a 20 mL beaker add 1 mL of chitosan solution and 100 uL of your protein, stir the mix at 1100 rpm with a magnetic stirrer (the size of nanoparticles is affected by rpm value; for smaller nanoparticles use higher rpm).
2. Take 1 mL of the TPP solution and add it to the mix dropwise.
3. Continue stirring for 1 hour.

 

Particle collection 

1. Transfer the mix to 2 1.5 mL Eppendorf tubes.
NOTE: If nanoparticles are to be extracted centrifuge the tubes at 20,000 rpm for 30 minutes at 4°C.
2. Eliminate the supernatant.
3. The pellet will contain your protein of interest.
4. If nanoparticles are to be used for liberation measurements or suspended in a controlled pH solution, resuspend well and store at 4 °C.

 

TEM preparation

 

To visualize chitosan nanoparticles some previous preparation steps must be carried out (this preparation protocol may vary).

1. A film of Formvar has to be previously prepared and used to coat a glass slide for the creation of an 80-120 μm thick membrane.
2. Place a copper grid on the Formvar membrane for it to be absorbed and later removed with a needle.
3. Add 20 μL of your solution of interest into the grid and let it be absorbed. Add a solution of 1% (w/v) phosphotungstic acid until the sample dries.
4. View in a transmission electron microscope.

 

NOTE: Samples were observed at 150,000x.