In a recent blog post, we summarized two key papers by Rodin et al. (1, 2) on clonal derivation and single-cell passaging of human pluripotent stem (hPS) cells in a completely defined, xeno-free environment using Laminin-521 matrix and NutriStem hPSC Medium. These publications give a wealth of information for hPS cell culture in this system and provide detailed protocols for passaging as both single cells and cell aggregates.
Here we highlight some important and practical tips from the papers for culturing hPS cells in NutriStem hPSC Medium with Laminin-521 as the basement matrix:
- Thawing a frozen vial of Laminin-521 too quickly can may cause it to precipitate. To avoid this, laminins should always be thawed slowly. The best practice is to thaw a 1 mL aliquot of Laminin-521 in the refrigerator at 4°C. By keeping the vial in the fridge like this, the complete thawing process takes about 1 to 2 hours and the coating will be smooth and even.
- Before plating, make a 1:10 dilution of Laminin-521 at 100 μg/mL stock concentration. Dilute the Laminin-521 with DPBS (with calcium and magnesium) to give a final working concentration of 10 μg/mL. Coat each well of a 24-well plate with 360 μL of the working solution. If you are working with a 96-well plate, use 60 μL per well. To scale up with Laminin-521 for larger well sizes, the authors recommend plating approximately 2 μg of Laminin-521 per 1 cm2 of cell culture surface in a volume large enough to coat the entire well and prevent evaporation if stored.
- Coating plates with Laminin-521 should be done at 4°C overnight. Seal the plates with Parafilm to prevent evaporation and to keep the wells sterile. Laminin-521-coated plates can be made and stored for up to 3 weeks 4°C. Although coating at overnight 4°C is best, in a pinch the coating procedure can also be done at 37°C for 2 hours, as long as quality plastic cell culture–treated plates are used.
- Before passaging hPS cells, remove the Parafilm wrapper and transfer the Laminin-521–coated plates from 4°C to the incubator to warm for 1 hour at 37°C and 5% CO2. After the incubation, rinse the new Laminin-521–coated wells once with PBS, and add the appropriate amount of pre-warmed NutriStem hPSC Medium to the wells. Adding media right away is important to keep the Laminin-521 coating from drying out before the cells are plated.
- Since NutriStem hPSC Medium is a complete medium, it should be used within 2 weeks once thawed. To prolong the shelf-life of the medium, simply thaw a 500 mL bottle of NutriStem hPSC Medium at 4°C overnight, divide the medium into smaller aliquots (45 or 100 mL, for example), and re-freeze these at −20°C. Making aliquots like this avoids repeated freeze/thaw cycles and preserves the media; the aliquots are stable at −20°C until the original expiry date. Always protect culture medium from light.
- To passage hPS cells as single cells, use pre-warmed aliquots of recombinant Trypsin-EDTA Solution. Be sure not to over-incubate the cells with the enzyme, and pipet gently when rinsing the wells to avoid losing cells. Generate a single-cell suspension by pipetting the cells in fresh NutriStem hPSC Medium before transferring to new wells.
- If passaging as cell clusters, other dissociation reagents can be used in place of recombinant Trypsin-EDTA. Cells can be gently scraped from the Laminin-521 matrix, pipetted to generate the right size clusters, and re-plated as smaller uniform aggregates. If the wells were close to 100% confluent when passaged, a split ratio of 1:10 may be optimal.
Note: NutriStem® hESC Medium, as referenced in the Nature articles, has recently changed its name to NutriStem® hPSC Medium. There has been no change to the formulation of this medium.
Want to try NutriStem hPSC Medium, culture your cells on Laminin-521, or learn more about hES or hiPS cell culture? Our technical training team is ready to help. E-mail us @ firstname.lastname@example.org to get started!
- Rodin, S., Antonsson, L., Hovatta, O., & Tryggvason, K. (2014). Monolayer culturing and cloning of human pluripotent stem cells on laminin-521–based matrices under xeno-free and chemically defined conditions. Nature Protocols, 9, 2354–2368.
- Rodin, S., Antonsson, L., Niaudet, C., Simonson, O. E., Salmela, E., Hansson, E. M., et al. (2014). Clonal culturing of human embryonic stem cells on laminin-521/E-cadherin matrix in defined and xeno-free environment. Nature Communications, 5, 1–13.