- Start with pelleted eggs that were isolated by bleaching gravid adult worms. Ensure each step of this procedure is conducted sterilely to avoid contamination of the cultured cells. Suspend the pellet in a solution of chitinase, an enzyme that breaks down chitin-- a large polysaccharide that is a structural component of the egg shell.
After an appropriate digestion period, gently centrifuge the eggs and replace the supernatant with cell culture medium to stop the digestion. Then, pass the embryos through an 18-gauge needle to mechanically separate individual cells. Next, filter the solution to remove debris from the egg shell or unseparated cell clumps. Plate the cells in a culture dish on a glass cover slip coated with peanut agglutinin-- a lectin that helps the cells attach to the cover slip by binding cell surface carbohydrates.
In the example protocol, we will prepare embryonic cells for in vitro morphological differentiation and analysis.
- While working under a laminar flow hood to avoid introducing bacterial contamination, resuspend the pelleted eggs in one milliliter of two milligrams per milliliter chitinase and transfer them to a fresh 15 milliliter conical tube. Rock the tube for 10 to 30 minutes at room temperature, depending on the freshness of the enzyme. When approximately 80% of the egg shells are digested, centrifuge the eggs at 900 g for three minutes.
After carefully removing the supernatant, add three milliliters of L15 medium. Transfer the eggs into a six- centimeter diameter plate and begin manual dissociation using a 10-milliliter sterile syringe with an 18-gauge needle. To monitor the degree of dissociation, place a drop of suspension into a fresh Petri dish and view under a microscope. Continue until approximately 80% of the cells are dissociated.
To remove cell clumps, undigested eggs, and hatched larvae, gently filter the suspension through a five- micron filter and run an additional four to five milliliters of L15 medium through the filter to recover the cells. To culture the cells after centrifuging the filtrate at 900 g for three minutes, re-suspend the cells in complete L15 medium. Plate one milliliter per well, and store plates in a humid sealed chamber at 20 degrees Celsius in ambient air.