Our protocol provides and easy and fast approach for primary fibroblast isolation with a clear focus on avoiding bacteria contamination, which can be a huge problem. Especially for beginners. The main advantage of our technique is its simplicity.
As it does not require extensive manual skills or experience and it can be easily learned by everybody within a short training period. Presenting the technique will be Manja Newe, a technician from out laboratory. Before beginning the dissection, put on two pairs of disposable gloves, one on top of the other.
And fix the mouse to a polystyrene pad. Disinfect the fur with 70%ethanol, making sure that the animal is soaked. And use ethanol sterilized surgical forceps and atraumatic scissors to cup the skin right above the urogenital tract.
Cut three to four centimeters along the midline from the initial incision to the neck, adding relief cuts at the limbs. And pin the skin to the pad to achieve optimal access to the musculature covering the abdominal cavity. Then, disinfect the abdominal musculature two times with fresh 70%ethanol.
When the ethanol has dried, remove the first pair of gloves. And use a new sterile set of forceps and scissors to incise the muscle layer to open the abdominal cavity and the thorax. To remove the organs of interest, gently grasp each organ with the surgical forceps without piercing the tissue.
And use the scissors to carefully cut the supplying blood vessels near the entry point of the organ. Place each organ into a tube of sterile, cold PBS and close the tube tightly. Placing each tube on wet ice as the organs are collected.
When all of the organs have been harvested, spray the tubes with 70%ethanol before placing them in a sterile cell culture hood. Wearing a fresh pair of gloves, use sterile forceps to transfer each organ onto one half of a sterile six centimeter Petri dish. And briefly wash the organs with fresh PBS to remove the excess blood.
Transfer the rinsed organs to the second half of each Petri dish and remove the excess PBS. Using two sterile scalpels, mince the organs into one to two millimeter fragments. And use a sterile spatula to transfer the tissue pieces into new, individual, sterile, 15 milliliter tubes.
Add two milliliters of 0.25%Trypsin solution to each tube. And place the tubes into a cell culture incubator at 37 degrees Celsius for five minutes. At the end of the incubation, vortex the tubes at about 1400 rotations per minute for ten seconds.
And arrest the Trypsin reaction with four milliliters of an appropriate culture medium supplemented with fetal calf serum per tube. Next, add the appropriate volume of collagenase blend solution to each tube. And place the tubes in a 37 degrees Celsius ultrasonic water bath for 10 minutes.
At the end of the sonication, gently vortex the tubes for 10 seconds. Before placing the tubes back into the ultrasonic water bath for 10 more minutes. At the end of the second sonication, vortex the tubes for 10 more seconds.
And disinfect the tubes with 70%ethanol. In the cell culture hood, filter the cell suspensions through individual 40 micrometer strainers into a new, sterile, 15 milliliter tube. And collect the cells by centrifugation.
Re-suspend the pellets in one milliliter of fresh medium per tube. And see the cells in suitable cell culture vessels at appropriate plating densities. Then, place the cells in the cell culture incubator overnight.
The next morning, wash each culture three times with PBS before adding fresh medium and returning the cells to the incubator. Using this protocol, viable fibroblasts can be obtained for use in subsequent experiments. Such as amino-fluorescent staining or proliferation experiments.
Adult fibroblasts are flat, spindle shaped cells with multiple cellular processes that typically grow in monolayers. Distinct morphological differences in fibroblast populations from different organs can be observed, however. For example, renal fibroblasts are smaller and grow at higher densities than cardiac, pulmonary or hepatic fibroblasts.
The isolated fibroblasts display high proliferation rates, reaching an optical confluence of greater than 90%after approximately six days. Here, a differentiated myofibroblast with distinctive alpha smooth muscle actin microfilaments can be observed. These cells are found in abundance in the heart, kidney, liver, and lung cell cultures.
While only about 20 to 30 percent of isolated and cultured cells express orderly, arranged, alpha smooth muscle actin microfilaments, virtually all of the cells are positive for Vimentin and Discoidin Domain Receptor 2 after seven days in culture. The most important thing to remember is to use the right technique throughout the procedure as bacteria contamination must be avoided. After primary fibroblast isolation, the cells can be used for all kinds of psychiatry experiments and characterization.
Such as immunocytic chemistry proliferation or wound healing essays, or molecular biological assessments.
