The protocol provide a easy-to-handle and widely compatible primary cell culture method for marine invertebrates. This technique gives researchers a way to culture the marine invertebrate cells for comparatively long time. A culture of cells should be provided with consistent genetic background for different further biological experimental applications in marine biology research.
To the new research to this field, we believe that the indicated simplified protocol should be the good choice to exercise. After anesthetization, dissect and collect the anterior intestines, then section the tissue samples vertically and remove the inner contents. Wash the tissue samples in PBS twice and disinfect them by immersion in a 75%ethanol solution for no longer than two seconds.
Then wash the tissue samples in PBS three times to remove the ethanol, and transfer about 100 milligrams of tissue sample into a two milliliter sterile micro centrifuge tube. Add 1.5 milliliters of pre-optimized culture medium to the sea cucumber intestinal tissue block and mince the block with sterilized surgical scissors until the solution is cloudy. Add 400 microliters of 0.25%trypsin.
Mix the solution by inversion and incubate it for five minutes at room temperature. Then filter the solution using a 100 micron cell strainer and collect the filtrate in a new sterile two milliliter micro centrifuge tube. For optional simplified protocol, tissue blocks cut in cultured media can be directly transferred into dishes and subsequently incubated.
Centrifuge the tube at 1700xG for three minutes, discard the supernatant, then re-suspend the pellet in culture medium with antibiotics, and wash it with culture medium twice. Pre-set the incubator for cell culture, and run it in advance for at least 24 hours with the temperature of 18 degrees Celsius and saturated humidity. Then re-suspend the cells using 200 microliters of indicated medium, and transfer them into four centimeter dishes.
Culture the cells in an incubator. After six hours, take them out of the incubator and add two milliliters of indicated medium to the cell culturing dishes. Every 12 hours, change half of the medium.
After five to seven days, the cells start to divide. Reduce the adverse effects of antibiotics by reducing the concentration of indicated antibiotics, penicillin, streptomycin, and gentamicin in the culture medium by half. Every two to three days, replace the cell culture medium.
When the primary cell density reaches 60%conduct cell passaging. First, wash the cultured cells twice using PBS at room temperature. Add 200 microliters of 0.25%trypsin solution to each dish, and manually agitate the dishes, ensuring the whole bottom is covered.
Discard the trypsin solution, and incubate the cells for five minutes at room temperature. Wash the cells with one milliliter of fresh culture medium by pipetting and re-suspending the cells. Transfer 0.5 milliliters of cell suspension to a new dish.
Add 1.5 milliliters of fresh medium, and incubate the cells at 18 degrees Celsius. After 12 hours, change the medium, and observe the cells under a microscope to evaluate the conditions. Prepare three experimental groups, including a control, and two groups of solutions, with one micromolar and 100 micromolar deximethazone.
After incubation with or without deximethazone for zero, 24, or 48 hours, wash the cells three times with PBS. After turning off the light, add 300 microliters of hoechst 33258 solution per well to a 12 well plate, and gently agitate the plate to cover all cells, ensuring their staining. Incubate at 18 degrees Celsius for 30 minutes.
Then remove the hoechst staining solution and fix the cells by adding 300 microliters of four percent paraformaldehyde solution in PBS to each well. Gently agitate for 15 minutes. After that, wash the fixed cells three times in PBS.
Keep the PBS after the last washing to keep the cells covered. Turn on the fluorescent microscope hardware, including the mercury lamp power, the fluorescent light power, and the PC.Log into the operating system account, launch the software, and check its configuration. Place the prepared plate on the microscope stage.
Position the sample over the objective lens using the stage controller. Find the cells of interest under the light microscope. Switch to fluorescent microscopy and adjust the parameters of the excitation and emission to approximately 352 and 461 nanometers, respectively, for nuclei DNA observation.
Capture the images. In this study, primary intestinal cell culture of sea cucumber was established and passaged. Round cells in different stages of culturing are shown here.
Here are cells attached to the bottom of dishes on the third day, after being seeded. Here is cell proliferation on the seventh day. Here are cells attached to the bottom of the dishes after passaging.
And cells that lost activity and were dying after 10 passages. The EDU stating assays provide direct evidence to reveal the proliferative activity of these round cells in later stages. The stimulation with one micromolar deximethazone increased the apoptotic cell rate most rapidly, compared with the control and the 100 micromolar deximethazone stimulation.
The cultured cells were treated with deximethazone for different time periods, followed by hoechst 33258 staining for apoptotic cell detection. Cell treatments before incubating is critical for longterm control. It is a reminder to try the optional simplified protocol when the cell don't grow well.
Paraformaldehyde is moderately toxic by skin contact or inhalation, and it is documented as a probably human carcinogen.
