Biolaminin 521 LN (LN521)

Coating plates

hPSC culture on LN521

The instructions include recommendations for transfer, passaging, thawing, and cryopreservation of hPSCs on LN521.

Embryoid body formation

Important notes

Preparation and handling

• Perform all procedures under sterile conditions using aseptic techniques.
• Minimize exposure of the protein to ambient temperatures.

Storage and stability

Laminin stock solution storage

• Store the frozen laminin stock solution at -20°C to -80°C for long-term stability. Refer to the product-specific Certificate of Analysis (CoA) for detailed shelf-life information.
• For long-term storage of thawed stock solution, dispense into working aliquots and store at -30°C to -80°C.
• Avoid repeated freeze-thaw cycles. Thawed, undiluted Biolaminin stock remains stable for 3 months at +2°C to +8°C.

Coated plate storage

• Store coated plates aseptically at +2°C to +8°C for up to 4 weeks. Do not let the surface dry.

Cell culture setup

• Use appropriate culture media and dissociation reagents to create fully defined, animal component-free protocols.
• Ensure high-quality cells when transferring to the LN521 matrix.
• Some human pluripotent stem cell (hPSC) lines may require an adaptation period when transitioning to the LN521 matrix before implementing single-cell passaging protocols.
• Once adapted, hPSCs can be routinely cultured as single cells without the need for ROCK inhibitor (ROCKi).

Troubleshooting Guide

Biolaminin plate coating

An uneven cell spread is often a coating issue and could be caused by the following:

• Low coating concentration: Ensure the laminin coating concentration is high enough to support even cell growth. Increase the concentration if necessary.
• Poor coating coverage or plate drying: Confirm that the entire surface is covered with the laminin coating solution when preparing fresh plates. Avoid drying out the plate, as this will inactivate the laminin. Prolonged time in the incubator or long storage without proper sealing can cause evaporation, leading to localized drying, often in the center of the plate.

hPSC splitting and seeding

• We recommend single-cell passaging or passaging as small clumps.
• Enzymatic treatment: Stem cells are sensitive to enzymes. Avoid over-treatment, as it can damage the cells.
  • Less confluent cells need shorter treatment times.
  • More confluent cells might need longer treatment times.
  • Cells should detach easily with minimal pipetting. Avoid excessive mechanical force (like extensive pipetting or scraping); instead, increase the dissociation reagent incubation time if needed.
• Cells attach more tightly to laminin compared to some other matrices. If cells adhere too firmly, try lowering the coating concentration.

After seeding

• Most cells should attach within 1 hour and be evenly distributed across the plate. Significant cell death at this stage usually indicates the cells were handled too harshly during splitting.
• The day after seeding, cells should have migrated and begun forming small colonies, which will continue to expand into a homogenous monolayer.
• hPSCs cultured on the LN521 matrix are typically ready for passaging at 60-99% confluence. Depending on the cell line, seeding density, and medium used, cultures are generally passaged 3-6 days after seeding.