This co-culture method may lead towards cellular understanding of physiological dysfunction in phenotypes, such as bladder neuron dysfunctions. The main advantage of this protocol is high efficiency. The entire isolation can be completed in six hours.
Demonstrating the procedure will be Jiao Zhang, a postgraduate student from my laboratory. Begin by placing rats on a sterilized surgical towel and exposing the abdomen. Use scissors and forceps to open the abdominal cavity and reveal the bladder.
Use another set of scissors and forceps to gently lift the bladder and cut it from the bladder neck. Then quickly place the bladder in cold oxygen stable Krebs solution to improve cell survival. Add Krebs solution into three glass dishes and three glass beakers and place them in an ice bath for pre-cooling.
Number the dishes and beakers one, two, and three to prevent confusion. In glass dish one, open the bladder with ophthalmic scissors and unfold it with forceps and a Spoons nucleus divider. Rinse the bladder in glass beaker one and place it in glass dish two.
Eliminate adherent fat on the tissue surface using the forceps and ophthalmic scissors, then rinse the bladder in beaker two and place it in dish three. Gently scrape the bladder using the forceps and the Spoons nucleus divider to remove exogenous attachments. Rinse the bladder in glass beaker three and transfer it to a 15 milliliter centrifuge tube with 14 milliliters of cold Krebs solution.
Centrifuge the sample for one minute at 360 times G and four degrees Celsius. Transfer the bladder from the centrifuge tube to a two milliliter vial containing one milliliter of digestion solution. Use ophthalmic scissors to cut the bladder into small pieces.
Mix the bladder solution with nine milliliters of digestion solution one in a sterile cell culture dish. Perform the first digestion in a shaking incubator for one hour at 37 degrees Celsius and 5%carbon dioxide at 200 RPM. After the first digestion, centrifuge the solution at 300 times G and four degrees Celsius for eight minutes.
Meanwhile, place digestion solution two in a 37 degree Celsius water bath for preheating. After centrifugation, remove most of the supernatant that contains digestion solution one, leaving some supernatant to avoid cell loss. Resuspend the cells in warm digestion solution two and shake the mixture while digesting in a 37 degree Celsius water bath for five minutes.
Once digestion is complete, immediately deactivate the trypsin by adding 10 milliliters of cold rinse media. Centrifuge the cells at 360 times G and four degrees Celsius for eight minutes, then remove as much supernatant as possible to eliminate all trypsin. Gently resuspend sediment with three milliliters of neuron media, making sure to not generate air bubbles in the solution.
Filter the suspension through a 70 micrometer cell strainer into a 50 milliliter centrifuge tube. Collect cells by centrifugation at 360 times G at four degrees Celsius for eight minutes, then gently resuspend the cell pellets in one milliliter of neuron media A and add 500 microliters of cell suspension to each well of a 48-well plate. Culture cells at 37 degrees Celsius and 5%carbon dioxide.
The cells isolated from the rat bladder culture were imaged at one, three, and seven days after plating. As the neurons grew, dendrites and axons became distinct. After five to seven days of culture, the neurons reached a mature form with long projections which were ideal for imaging or function studies.
After proper culture, neurons were identified with beta-three tubulin and MAP2 immunostaining and glia were identified with GFAP immunostaining. Mature neurons developed synaptic spines, which were close to the presynaptic specializations identified by immunostaining of Synapsin-1. Meanwhile, several neuron subtypes were recognized through immunohistochemistry experiments.
Peptidergic neurons which contain various neuropeptides were immunostained with substance-P. Purinergic neurons with expressed vascular nucleotide transporters were identified via SLC17A9 staining. Nitrogenic neurons were visualized with DOIN-LL2 which connects neural NOS with motor proteins in neurons.
And cholinergic neurons were immunoreactive with choline acetyltransferase. When performing the second digestion, remember to have the cold rinse medium beside you and ready to use. In this way, you can immediately deactivate the trypsin.
When the cell culture matures, immunocytochemistry can be performed. This will reveal expression and distribution of target proteins in the cells. This method provides researchers with an in vitro model to explore diseases like bladder neuron dysfunction.
