Biosilk

How to generate a Biosilk 3D network with evenly integrated cells

A 3D foam structure can easily be generated by the gentle introduction of air bubbles into the Biosilk solution. The cell suspension is mixed into the foam, and the silk with cells assembles into a thin film around each bubble. The bubbles disperse and the foam transforms into a stabilized 3D network with uniformly integrated cells between the microfibers.

Important notes

• All steps must be carried out under aseptic conditions
• Biosilk should be stored at -80°C
• Gently thaw the Biosilk solution at RT without moving the vial
• Do not vortex or shake the vial and be careful when mixing to avoid the introduction of air bubbles
• The Biosilk products have to be used within 45-50 min after thawing
• Re-freezing or long-term storage of thawed Biosilk solution in the fridge is not possible as this will affect product stability and functionality
• 1 vial of BioSilk is enough material to generate 12-13 foams (24 well format)
• Hydrophobic surface culture plates should be used.
• The cell suspension should be prepared fresh before mixing into the Biosilk to ensure high cell quality
• For research use only

Protocol

Preparation

• Thaw the Biosilk solution at RT without moving the vial
  
• Add Rock inhibitor to the thawed solution to a final concentration of 10 µM. Gently pipette 3 times.

Note:

!   Do not vortex or shake the vial and avoid the introduction of air bubbles.

!   It will take around 12 min for the frozen Biosilk solution to thaw at ambient temperature.  For the best result, the thawed solution should be used as soon as possible, the latest within 1 hour from removal from -80 °C. The thawed Biosilk solution will gradually turn milky in ambient RT.

• Prepare a concentrated single-cell suspension according to “Instruction For Use BL003”. For a vial of Biosilk or Biosilk 521, prepare a concentrated cell suspension of a total of 7-14 x10⁵ cells in 25 μL of medium (roughly 20 000 – 60 000 cells/μL) supplemented with 10 μM ROCK inhibitor.

Note:

!   The cell suspension should be prepared freshly before mixing into the Biosilk solution to ensure the best cell viability. For best foam stability, prepare the cell suspension for the foam incorporation beforehand or during the Biosilk thawing time.

!   Optimize the cell incorporating density for best amplification in the 3D scaffold, as this is cell type-dependent and needs to be adjusted accordingly.

• Prepare a Biosilk-Cell suspension mixture cell suspension (25 μL), and 10mM ROCK inhibitor (0.27 μL) to the thawed Biosilk solution (250 μL). The final mixture will have a concentration 10 μM ROCK inhibitor. Mix by gently pipetting 3 times without introducing air bubbles. The Biosilk-cell suspension mixture should be used within 10 min to ensure high cell viability. Biosilk can be biofunctionalized with Biolaminin. Just add Biolaminin 521 or an isoform of choice to the Biosilk solution (25 μL). The final mixture will have a concentration of 10 μg/mL Biolaminin.

Note:

!   If the Biosilk 521 (BS521-0101) pre-mixed product is used, the manual addition of Biolaminin can be omitted unless a mix with an additional Biolaminin isoform is desired.

!   Be careful when mixing to avoid the introduction of air bubbles as this will cause premature fiber formation.

3D scaffold formation and maintenance

• Transfer 20 μL of the prepared Biosilk-cell suspension solution to the center of a well.
  
• Use a pipette set at 44 μL, to push air bubbles into the droplet by quick pipetting up and down 22 times, thereby creating a dense foam. Spread out the foam in circular motions with the pipette tip during pipetting to an area covering 0.7-1 cm in diameter. See the protocol described in Fig. 1.

Note: 
!   Insufficient (<22) or excessive (> 25) pipetting for the foam formation will result in an unstable scaffold or low cell viability, respectively.

!   If the cells are sensitive to pipetting, an increased cell seeding density could help to increase cell viability. Alternatively, the cells could be mixed in after the foam has been generated. In this case, add 20 uL Biosilk solution to the well and use a pipette, set at 40 uL, to push air bubbles by quickly pipetting up and down 20 times. Add 1-4 μL dense cell suspension (roughly 20 000 – 60 000 cells/μL) for each foam and mix by pipetting an additional 5 times. See the protocol described in Fig.2.

!   It takes approximately half to one minute to generate each foam. We recommend thoroughly planning the procedure for the best cell- and foam quality. 
 

• Repeat step 4.1 to 4.2 to create the desired number of foams. The solution is sufficient for creating 12 – 13 foams.
  
• Place the lidded plate with the cell-containing foams in an incubator at 37°C for 20 min. During this time, the Biosilk product polymerizes and the 3D structure is stabilized.
  
• Remove the plate from the cell incubator. Gently add 0.7-1 mL per well of the pre-warmed medium containing 10 μM ROCK inhibitor, starting dropwise around the foam before slowly filling up to cover the foam.
  
• Place the plate back into the incubator.
  
• Feed the cells daily or at an appropriate frequency with fresh culture media without ROCK inhibitor.

Biosilk organoid differentiation

• Pluripotent stem cells seeded in Biosilk foam generally need to be cultured in medium supporting pluripotency for 4-5 days with daily feeding to reach the desired confluence before switching to differentiation medium. Depending on lineage differentiation and protocol used, culture for 1 to 2 weeks in a suitable medium with appropriate feeding frequency is needed.
  
• When cells have reached the desired confluency within the microfibrillar network, manually detach the foam from the bottom of the well using a cell scraper or a pipette tip. Cut the foam structure into 2 -4 pieces (approx. 2 mm thick) using a blade or a pair of small scissors and transfer to new low-attachment culture plates for culture as free-floating entities.
  

Note:

!   Before the foam can be detached from the culture surface for free-floating organoid culture, the cells first have to be amplified in the attached foam. It usually takes 1 to 2 weeks to generate the desired confluency.

!  Embedding the organoid in Matrigel is not needed to maintain the organoid shape and cell phenotype. If embedding is preferred, a  xenofree and defined material is recommended (e.g. HyStem™ available from Merck).

!   If using a cell type other than hPSCs, culture with appropriate culture medium and feeding frequency before detaching the foam for free-floating organoid culture.

• Feed the free-floating organoid cultures at an appropriate frequency until further analysis or desired applications.