Biolaminin 521 CTG (CT521)

The instructions include recommendations for transfer, passaging, thawing, and cryopreservation of hPSCs on CT521.

Important notes

• “For Research Use or Non-Commercial Manufacturing of Cell, Gene, or Tissue-Based Products”.
• Biolaminin 521 CTG (CT521) has been developed and manufactured to allow customers to qualify the material for use in the manufacturing of cells for clinical research.
• CT521 is animal origin-free to the secondary level according to the ISCT guidance document ˮISCT Animal-Free Origin Survey Results-Summary”. All materials used in the production process have full traceability.
• The product has supporting documentation such as Certificate of Analysis (CoA) and Animal Origin Free Statement provided with every lot to support regulatory filings.
• 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.

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 Biolaminin 521 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 Biolaminin 521 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.