3D Bioprinting

Before starting experiment

  • I reviewed the protocol from the paper (Materials fabrication from Bombyx mori silk fibroin) in order to check process one by one with my instructor.
  • I briefly rehearsed all of the process. This is because my instructor mentioned to me that she made mistakes last year.

Concern

Precautionary statements-(Prevention)
- Obtain special instructions before use.
- Do not handle until all safety precautions have been read and understood.
- Use personal protective equipment as required.
- Wash face, hands and any exposed skin thoroughly after handling.
- Do not eat, drink or smoke when using this product.
- Do not breathe dust/fume/gas/mist/vapors/spray.

Precautionary statements-(Response)
- IF exposed or concerned: Get medical advice/attention.
- IF SWALLOWED: Call a POISON CENTER or doctor/physician if you feel unwell.
- Rinse mouth.

Precautionary statements-(Storage)
- Store locked up.

Precautionary statements-(Disposal)
- Dispose of contents/container to an approved waste disposal plant.
  • And from another aspect, LiBr is categorized based on the following fire diamond which shows the "NFPA 704: Standard System for the Identification of the Hazards of Materials for Emergency Response" established by the National Fire Protection Association that is based in the US and Canada.
    • Health Hazard (Blue): Level 2 - Can cause temporary incapacitation(一時的能力障害) or residual injury (後遺障害)
    • Flammability Hazard (Red) : Level 2 - Must be heated or high ambient temperature to burn
    • Instability hazard (Yellow) : Level 0 - Stable
LiBr Rating (Japan) Rating Guide
Image from here

Assignment

1. Regeneration of silk fibroin into an aqueous suspension (please, refer to Step 1-23 in Rockwood et al. Nature Protocols, 6, 1612–1631 (2011) in background reading).

1. Fibroin extraction - Day 1-

【Equipment & materials】

Equipment Materials

【Process】

  1. Prepare Stainless steel pot filled with 2 liters of ultrapure water, cover it with a pot lid and heat until boiling.
  2. Meanwhile, cut cocoons with scissors into small pieces around 2cm.
  3. Measure 5g of the cocoon pieces on a scale.
  4. Measure 4.24 g of sodium carbonate (炭酸ナトリウム) on a scale.
  5. Add gradually the measured sodium carbonate to the water and let it completely dissolve. Note : Boiling the water while preparing the reagents (process 2 -> 5) is important, because I did 2->3->4->5->1 and had to wait for the water to boil which was a waste of time.
  6. Add the cocoon pieces once the water starts to boil and continue boiling for 30 min. (Boil for exactly 30 min. Increasing the boiling time will degrade the fibroin.) Occasionally, stir the silk with a spoon. While we waited for 30min, we poured some water into a 300ml beaker which will be used for cooling the silk later on.
  7. Remove the silk fibroin and cool it in the 300ml beaker. And discard the sodium carbonate solution in the sink.
  8. After squeezing excess water out of the silk, place it in a 1-liter beaker filled with 1 liter of ultrapure water and a stir bar. Rinse the fibroin in water for 20 min while gently stirring on a stir plate. Repeat this step for a total of three rinses. We noticed that the water color that was once cloudy turned transparent in each successive rinsing.
  9. After the third wash, remove the silk, squeeze it well and then spread it out on a clean piece of aluminum foil.
  10. Dry the silk fibroin overnight in a clean bench.
  11. Once the silk is dry, keep it in a clean plastic bag. (This can be stored indefinitely at room temperature.)
Cut silk and measure sodium carbonate Boil and stir Rinse Dry 
- MEMO : New realization through the experiment
 - Before placing the beaker on the stir plate, the stir bar should not be dropped
 but gently placed into the beaker. Because the stir bar can possibly break the beaker
 due to high speed falling and the stir plate's magnetic field can cause the stir bar to rattle.
 - It might be common sense for scientists but it was a new realization for me.

2. Dissolve silk fibroin in liBr - Day 2 -

【Equipment & materials】

12."Calculate the amount of 9.3M lithium bromide needed to prepare a 20% (wt/vol) solution based on the amount of dried fibroin available. As 20% of the solution will be silk, 80% will be LiBr. That is, a ratio of 1:4 (1 g to 4 ml) silk to LiBr. Therefore, multiply the amount of the dried silk fibroin by 4 to obtain the total volume of 9.3 M LiBr needed (X)."

  • Prepare a 9.3 M LiBr solution.
    • Total volume of silk = 3.544g
    • Required LiBr solution = 3.544 x 4 = 14.176ml
    • Required volume of LiBr = 86.85g/mol (molecular weight of LiBr) x 9.3mol/L x 1L/1000ml x 14.176 = 11.45g
  • Wear gloves for handling LiBr.
  • Place powder paper and use a spoon to measure 11.45g of LiBr on the electronic scale. Pour the LiBr into a 50ml beaker.
  • Using a pipette, add pure water to the LiBr until the total amount is 14.176ml.
  • At first I tried to stir the solution with a small stir bar and stir plate, but LiBr was too stiff for stir plate. Therefore I stirred by hand with a spoon.

13.Pack silk fibroin tightly into a 50ml glass beaker and add the required amount of LiBr solution on top." Make sure to add the LiBr to the silk so that the solution completely covers the silk.

14.Let fibroin dissolve in an oven at 60 °C for 4 h. Once the silk fibroin is completely dissolved, it will appear amber in color and will be transparent.

- MEMO : Mistake
 - I made a mistake about total amount of solution. Total solution amount should be 14.176ml
  but I poured 14.176ml of pure water plus 11.45g of Libr. My instructor indicated it and I realized that
  it is misunderstanding. After that we could proceed next step by her advice.
 - I acknowledged that it is important to read protocol carefully.
 - I sometimes forgot to place the empty beaker on the scale and set it to  zero before measuring reagents.
  It is crazy habit of myself and I should not make such mistake again and again.
Reagents and equipment Silk LiBr LiBr solution
Silk and solution Put in the oven 4hr later

3. Dialysis (透析) and centrifugation (遠心分離) - Days 2-4 -

【Equipment & materials】

15.Hydrate dialysis cassettes in water for a few minutes"

16.With a 20ml syringe (注射器) and an 18 gauge needle, insert 12ml of the silk-LiBr solution into a 3–12-ml dialysis cassette.

  • You should not use a needle when drawing solution from beaker into the syringe because the solution is very sticky. Only use the needle when injecting the solution into the cassette.
  • Be careful not to puncture or touch the membrane of the cassette. Because holes will cause the solution to leak.
  • The solution should be kept warm, because it is easier to handle when adding it to the cassette. We kept the solution warm on a hot plate.
  • In order allow air to escape, we inserted another needle at the top of the cassette.

17.Dialyze against 1 liter of ultrapure water per 12 ml cassette. To ensure mixing, use a large stir bar and place on a magnetic stir plate. Change the water after 1 h, 4 h, that evening, the next morning and night, as well as in the morning on the following day (i.e., six changes within 48 h)."

18.Remove silk from the cassettes with another 20ml syringe and an 18gauge needle. Place silk in a 50ml conical tube. Depending on the volume, either split it between two tubes."

19."Centrifuge to remove impurities. Place in a centrifuge and spin at 9000 r.p.m. at 4℃ for 20 min." We do not have temperature control system. Therefore we used a standard centrifuge (2500 r.p.m.) at room temperature.

20."Carefully remove tubes from the centrifuge and either pour or transfer the silk solution with a 25 ml pipette into another centrifuge tube. Be sure to leave any white flocculent or brown matter behind."

21.Repeat above centrifuge steps again.

22."To determine the concentration of the silk in solution, measure the weight of scaler. Thereafter, add 0.5 ml of the silk solution to the boat and allow it to dry at 60°C. Once the silk is dry, determine the weight of the silk and divide it by 0.5 ml. This will yield the weight per volume percentage."

23."The fibroin solution (25 ml at concentration 7–8% (wt/vol)) can either be used as is or it can be lyophilized (option A) or concentrated (option B). For storage for longer than 1 month, the silk solution should be lyophilized. In this form, the silk will be stable for years at room temperature and can be reconstituted in HFIP. The concentrated solution (20–30%, wt/vol) can be used directly for preparing silk tubes."

24."The silk fibroin can now be used to prepare a number of different materials.

MEMO : Our challenge
 - Since our silk-LiBr solution was very sticky, we couldn't inject into cassette by the needle. We push syringe continuously with full power of our hands but in vain. Therefore we decided to add LiBr solution to the silk-LiBr solution.
 - We added around 10ml of LiBr solution into this sickly solution and stirred it with a spoon on the hot plate.
 - Then our solution became while color and less sickly.
 - And we could inject this solution into cassette finally.
Draw solution into a syringe on hot plate Dialysis cassette in pure water

4. Fabrication of an implantable, biodegradable diffraction grating through soft lithography

Get diffraction grating from CD-R Heat CD-R on hot plate for easy peeling
Materials Used stir machine Kakuhanter
Diffraction grating on plate Put solution on diffraction grating Heat it in oven
After heating Peel off from plate -1 Peel off from plate -2
Peel off from diffraction grating Put Solution(Left:Solution only,Right:Module+Solution)
Final-1(After drying) Final-2 
MEMO :
- We tried to get diffraction grating from DVD and Blue ray, it was fail. CD-R is the best.

Weekly Reflection: Do your activities this week raise new ethics and/or safety considerations you had not considered in week 1? Describe what activities have raised these considerations and any changes you have implemented in response.

  • The paper shows the entire protocol for the experiment. However, It was important to rehearse before doing the actual experiment. Through such a rehearsal, we can set appropriate arrangement of equipment and acknowledge the process that need extra caution. And it will make the experimental procedure smoother without any confusion.

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