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

• “For Research Use Only”
• All procedures should be done under sterile conditions using aseptic techniques
• Avoid long exposure of the protein to ambient temperatures
• The laminin stock solution is long-term stable when stored at -20°C to -80°C. Please refer to the product-specific CoA for shelf life details.
• Repeated freeze-thawing should be avoided. If desired, the laminin stock can be dispensed into working aliquots and stored at -20°C to -80°C. Thawed, undiluted laminin stock is stable for at least 3 months when stored at +2°C to +8°C under aseptic conditions.
• For your convenience, the coated plates can be kept for up to 4 weeks when stored aseptically at +2°C to +8°C.
• The protocols can easily be made completely defined and animal component-free with your choice of culture medium and dissociation reagent
• The cells transferred to the LN521 matrix must be of high quality.
• Some hPSC lines transferred to the LN521 matrix might require an adaptation period before they can be cultured according to the single-cell passaging protocol.
• Once adapted to the LN521 matrix, hPSCs can routinely be cultured as single cells without ROCKi.
• The LN521 matrix facilitates long-term self-renewal of hPSC without weekend feeding.

Troubleshooting

Biolaminin plate coating

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

• Too low coating concentration used. Increase the coating concentration is high enough to support even cell growth.
• Bad coating coverage/the plate has dried out. Ensure that the entire surface is covered by the laminin coating solution when preparing fresh plates. Also, do not let the plate dry out as this will inactivate the laminin coating. Too long time in the incubator or long storage without sealing could cause too much evaporation so that part of the plate dries out (often in the center).

hPSC splitting and seeding

• We recommend single-cell passaging or passaging as small clumps.
• Stem cells are sensitive and when using an enzyme, do not treat the cells too long as that will damage the cells. Cells attach tighter on laminin compared to other matrices and scraping or pipetting without first loose up cells can affect cell integrity and viability which could result in less attachment the next day. Less confluent cells need shorter treatment time whereas more confluent cells might need longer treatment time. The cells should detach easily without too much pipetting needed. Do not use too much mechanical force (extensive pipetting or scraping) as that will damage the cells. Increase the dissociation reagent incubation time rather than increasing the force. If the cells stick very hard to the LN521 surface, try to lower the coating concentration.
• After seeding: most cells should have attached after 1 hour and the cells should be evenly distributed over the entire plate. If there is a lot of cell death after seeding, the cells have most likely been treated too harshly during splitting.
• The day after seeding: the cell has migrated and should have formed small colonies and will continue to expand as a homogenous monolayer. Cells cultured on the LN521 matrix are ready to be passaged when cell culture is 60-99% confluent. Depending on the cell line, seeding density and on the medium used, cultures are usually passaged 3-6 days after seeding.