Biosilk 521

Biosilk 521

An optimal 3D culture environment for expansion and differentiation of human PSCs. 

Biosilk 521 is a natural biomaterial made of recombinant spider silk protein, functionalized with human recombinant laminin 521 protein (Biolaminin 521), designed for 3D cell culture. Laminin 521 is a key cell adhesion protein of the natural stem cell niche, providing the Biosilk 521 material with unique, functional properties that are ideal for integration, proliferation and subsequent lineage-specific differentiation of human pluripotent stem cells in a 3D format.



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810 µg 1 x 270 µL €240
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A natural biomaterial designed for 3D cell culture of human pluripotent stem cells 

Biosilk 521 is a natural biomaterial made of recombinant spider silk protein, functionalized with human recombinant laminin 521 protein (Biolaminin 521), designed for 3D cell culture. Laminin 521 is a key cell adhesion protein of the natural stem cell niche, providing the Biosilk 521 material with unique, functional properties that are ideal for integration, proliferation and subsequent lineage-specific differentiation of human pluripotent stem cells in a 3D format.

Biosilk 521 foam and fibers


Biosilk 521 is a biocompatible, biodegradable and non-immunogenic biomaterial, which facilitates the use in clinical applications. The extendable Biosilk 521 macrofibre material can self-assemble into several formats; foams, fibre bundles and  thin films. 

 

A MORE BIOLOGICALLY RELEVANT CULTURE ENVIRONMENT 

Compared to 2D cultures, a 3D culture is a more biologically relevant culture environment with a higher degree of architectural complexity that retains homeostasis for longer.

 

Biosilk 521 is a biorelevant cell culture system


Contrary to many other 3D scaffolds, the cells can be added to the Biosilk 521 solution before the network stabilizes, resulting in a uniform integration of the cells between the microfibers. Unlike cells cultured in hydrogel, cells seeded in the Biosilk scaffold attach along the microfibers and are in contact with other cells like in tissue. Moreover, the cells proliferate more efficiently and migrate in the Biosilk 521 scaffold and have full and even access to both the medium, matrix and neighbouring cells, compared to when encapsulated in a hydrogel.

Biosilk 521 vs hydrogel

FEATURES AND SPECIFICATIONS:

  • Flexible 30 culture system that can be assembled into different formats

  • Elastic microfibers mimicking the ECM

  • Biologically relevant hPSC culture environment

  • Biocompatible

  • Biodegradable

  • Non-immunogenic

  • Defined and animal component-free to the primary level

  • For research use only


 

 

 APPLICATION NOTE

BIOSILK 521 FOAM MORPHOLOGY

Representative pictures of the Biosilk 521 foam taken from above day 0, 1 and 3 after cell seeding. The initial small bubbles merge into bigger bubbles that disperse after a few days, resulting in a 3D scaffold with integrated cells. 

 Biosilk 521foam morphology

 

CELLS ATTACHED TO B10SILK 521 

Micrographs of human ES cells attached to Biosilk 521 day 2, 3 and 5 after seeding. When the bubbles have dispersed, the Biosilk scaffold with cells organized between the microfibers can be visualized. 

Pluripotent cells attached to Biosilk 521

EFFICIENT PROLIFERATION WITH MAINTAINED STEMNESS MARKER EXPRESSION

Human ES cells cultured in Biosilk 521 in media for culture of pluripotent cells efficiently inte-
grate, proliferate and form colonies along the microfibres with maintained expression of stern-
ness marker (NANOG; red). Scale bar 50 µm. 

Pluripotent cells cultured on Biosilk 521 

Human ES cells (HS980) and iPS cells (iPSC3) seeded at 50.000 cells/foam were cultured for 4 days in three different pluripotent cell culture media. The cells were detached with TrypLE solution and cell amount and viability was measured. 

Cell amplification on Biosilk 521Cell viability on Biosilk 521 

PLURIPOTENT CELLS CAN DIFFERENTIATE INTO THREE LINEAGES 

Human pluripotent stem cells were cultured in the Biosilk 521 scaffold for 2-3 days before linage specific differentiation to ectoderm (7 days, NESTIN and PAX6), endoderm (3 days, FOXA2) and mesoderm (12 days, NKX2.5). DAPI was used as nuclear counterstain. 

Differentiation on Biosilk 521

PROTOCOL

Preparation of Biosilk 521 solution

  1. Thaw the Biosilk 521 solution at RT without moving the vial.
  2. Add Rock inhibitor to the thawed solution to final concentration of 10 µM. Gently pipette 3 times.

Note:
!   Do not vortex or shake the vial and be careful when mixing to avoid introduction of air bubbles.
!   It will take around 10 min for the frozen Biosilk 521 solution to thaw at ambient temperature. The thawed solution should be used as soon as possible, within1 hour at the latest. The thawed Biosilk 521 will gradually turn milky in ambient room temperature.



Preparation of a concentrated cell suspension

  1. Prepare a concentrated single cell suspension according to “Instruction For Use BL003”, roughly 10.000-15.000 cells/µL, diluted in warm media supplemented with 10 µM ROCK inhibitor.

Note:
!   The cell suspension should be prepared freshly for foam seeding (use within 20 min after detachment from plate). Cell suspension standing too long in RT will result in reduced cell amplification rate in the foam.

 

Integration and expansion of hPSC in Biosilk 521 foam

  1. Transfer 20 µL of the prepared Biosilk 521 solution from step 1 to the center of one culture well.
  2. Use a pipette with a tip for 200 µL and set at 40 µL. Push air bubbles into the droplet by quickly pipetting up and down 20 strokes, 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.
  3. Immediately add 1–5 µL (typically 30.000-60.000 hPSCs/foam) of the cell suspension from step 2 (volume ratio of cell to Biosilk 521 ≤0.25). Use the pipette set at 40 µL with a new tip and disperse the cells throughout the 3D structure by 5 additional strokes.
  4. Repeat step 1 to 3 to create the desired number of foams. One vial of Biosilk 521 (270 µL) is sufficient for 12 foams.
  5. Place the plate with the cell-containing foams in an incubator at 37°C for 20 min. During this time, the 3D structure is stabilized. 
  6. Gently add 0.7-1 mL of the pre-warmed medium containing 10 µM ROCK inhibitor per well, enough to cover the foam.
  7. Place the plate back into the incubator.
  8. Feed the cells daily with fresh culture media without ROCK inhibitor.

Note
!    For best foam stability, cell suspension for foam seeding could be prepared beforehand or during the Biosilk 521 thawing time.
!    It takes approximately 1 min to generate each cell-containing foam. It’s good to plan the timing for best cell and foam quality.

4. Differentiation

Differentiation protocol of interest can be used when desired confluency has been reached.

IMPORTANT NOTES

  • All steps must be carried out under aseptic conditions

  • Biosilk 521 should be stored at -80°C

  • Thaw the Biosilk 521 solution at RT without moving the vial

  • Do not vortex or shake the vial and be careful when mixing to avoid introduction of air bubbles

  • Thawed Biosilk 521 solution has to be used within 1 hour. Re-freezing or storage in fridge is not recommended and will result in decreased foaming efficacy

  • For research use only

 

Instruction for use 011

Product name

Biosilk 521

 

Product code

BS521-0101

 

Declaration

For research use only

 

Product description

Recombinant spider silk protein, functionalized with human recombinant laminin 521 protein (Biolaminin 521)for 3D culture applications

 


Documents

MSDS

 

Storage

-80°C

 

Stock concentration

0.1mg/ml

 

Stability

12 months 

 

Shipping condition

Dry Ice

 

Appearance

Clear, colourless

 

Product application

Human PSC expansion and differentiation  

 

Classification

Defined and animal component-free, human recombinant protein