What ECM do you recommend for culturing iCell DopaNeurons?
Because the iCell® DopaNeurons are not amenable to many extracellular matrices, we recommend that you use a freshly prepared plate with a 0.01% poly-L-ornithine (PLO) base coat and a 3.3 µg/ml laminin top coat when culturing iCell DopaNeurons as described in our User’s Guide. Fresh laminin is required to achieve the "typical" neurite outgrowth displayed by iCell DopaNeurons, and can be left in trace amounts (1 μg/mL) when plating cells. Be sure that the culture wells do not dry out before seeding iCell DopaNeurons. We do not recommend using gelatin, fibronectin, Matrigel, or collagen ECMs.
Additional ECM Recommendations:
|Plastic||Fresh PLO (0.01%). Important to remove PLO completely before adding a fresh coat of laminin (3.3 µg/ml). PLO is very toxic to cells.|
|Glass Plates (MEA)||Fresh PEI (0.05 - 0.1%) with fresh laminin (10 µg/ml) added to cell suspension at thaw. Important to remove PEI completely before adding laminin. PEI is very toxic to cells.|
|Glass Coverslips||Pre-coated PDL/laminin. iCell DopaNeurons do not adhere well to glass coverslips, so in come cases (e.g., manual patch clamp), we recommend using plastic coverslips|
|Pre-coated plates||To enable a quicker workflow without the need to manually apply ECM to the plates, you can use the following pre-coated plates after directly adding fresh laminin (10 μg/ml) into the complete iCell DopaNeurons Maintenance Medium containing cells during thaw. Pre-coated plates can be stored at 4°C for up to 1 week in laminin coating, wrapped in Parafilm to prevent evaporation, and pre-warmed to 37°C for 1 hour before use
|Matrigel/PLO Alternative||When laminin can’t be used, use a standard PLO coating protocol (1 hour + 2 washes with DPBS or water) followed by coating with Matrigel at 8.7 μg/cm2 diluted in a cold base medium (e.g., DMEM/F12) for 1 hour before aspirating the coating and immediately adding cells. Washing Matrigel coating out of plates is not necessary.|
- Cell clumping. If the laminin is allowed to dry before seeding, cell adherence can be compromised, because the amino acid base necessary for adherence is no longer available. Cells may begin to clump and form clusters on the plate, and neurites may look like spokes. To ensure typical neurite outgrowth, make sure that culture surfaces have a fresh, intact, wet coating of laminin.
- Cell death ~1 week post-thaw. If residual PLO remains in the wells at the time of plating it can cause toxicity after several days. The cells may look healthy up to day 5 or so but may begin to die at day 6 or 7, when the somas and neurites appear very transparent, almost like they are disintegrating. To prevent PLO toxicity, thoroughly remove PLO and rinse cells completely, using a third rinse especially in the smaller well format plates (96- or 384-well plates) as it is more difficult to aspirate and dispense coating. Alternately, for smaller well formats, we strongly recommend using pre-coated PDL plates following the HTS plating protocol.
- Poor cell adherence. iCell DopaNeurons do not adhere well to glass surfaces, including Matrical glass-bottom plates. While these plates are useful for imaging and great for picture quality, they were poor for cell attachment and distribution (meaning not clumping). You may need to adjust density to compensate for attrition over time (see Solution 00000251A).