Biolaminin 521 MX (MX521)

The instructions include recommendations for transfer, passaging, thawing, and cryopreservation of hPSCs on MX521.

Important notes

• “For Research Use Only”
• Avoid long exposure of the protein to ambient temperatures
• MX521 is animal-origin free to the secondary level, according to the ISCT guidance document ˮISCT Animal-Free Origin Survey Results-Summary”.
• 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.
• Avoid long exposure of the protein to ambient temperatures.
• 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 totally defined and animal origin-free with your choice of culture medium and dissociation reagent.
• It is important that the cells transferred to the MX521 matrix are of high quality.
• Some hPSC lines transferred to the MX521 matrix, might require an adaptation period before they can be cultured according to the single-cell passaging protocol.
• Once adapted to the MX521 matrix, hPSCs can be routinely cultured as single cells without ROCKi.

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 passage or passage 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 MX521 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 MX521 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.

Transfer from other substrates

• We recommend to transfer the cells as single cells (or as small clumps) and always with the addition of ROCKi for the first few (3-5) passages. Once the cells are adapted to the MX521 matrix, the cells can be cultured as single cells without ROCKi. This may take up to 5 passages.
• If the cells are hard to adapt, try increasing the coating concentration to 10 ug/ml and seed at a higher cell density 50,000 – 100,000 cells/cm². Once the cells are adapted a lower coating and seeding concentration often can be used.
• It is important that the cells transferred to the MX521 matrix are of high quality. MX521 will generally also support differentiated cells so carefully select only undifferentiated cell areas for transfer. 
• MX521 works well in combination with most commercial media brands. However, it is to be expected that cell morphology will look different depending on the medium used for culture.