Isolation And Primary Culture Of Vascular Smooth Muscle Cells From Spiral Modiolar Artery

1. ObjectiveThis study aimed to develop a novel method for establishing primary cultures of vascular smooth muscle cells (VSMCs) from the spiral modiolar artery (SMA) by combining two common cultural methods in vitro.2.Methods2.1 VSMCs were isolated from SMAs of guinea pigs. The arterial tissues were minced and enzymatically digested at 37°C for 20 min using a trypsin solution (0.1%). After digestion, the fragments were explanted in a 35-mm culture dish.2.2 Using this protocol, we found that the most frequently observed non-SMC cell type was the fibroblast because the unremoved adventitia layer..C The fibroblasts were removed from the cell suspension because they attach faster to the surface of the culture dish than to SMCs. To do this, the fresh cell suspension (5.7×105 cells/mL in serum-free culture medium) was added to a 35-mm tissue culture treated polystyrene dish. After 15 min of incubation at 37°C, VSMCs failed to attach to the culture dishes and could be collected with the supernatant. This process was repeated twice and the liquid supernatant was collected for further culture. We obtained pure VSMCs during the third passage. The cells obtained based on this protocol were subsequently characterized by morphology, immunofluorescence and transmission electron microscopy analysis.3. Result3.1 Using the above described protocol ,we found that the tissues need 2 days to attach, and the cells migrated out from the arterial tissues in about 7–10 d. It took about 3 weeks for the cells to become confluent. Purified VSMCs were obtained from the second to the third passage. 3.2 The results demonstrated typical characteristics of VSMCs, including a "hill-and-valley" growth pattern and the expression of cell type-specific markers (α-smooth muscle actin and myosin) by morphological and immunofluorescence analysis, respectively.3.3 The TEM study revealed contained bundles of myofilaments with dense bodies. Many dense patches, which are characteristic of SMCs, were observed. A large amount of ultrastructures including rough endoplasmic reticulum, ribosomes, polysomes, and abundant mitochondria indicated active protein synthesis in these cells4. ConclusionIn conclusion, we established a protocol for the primary culture of VSMCs from SMA in vitro. The methods used might provide a technique to obtain a number of VSMCs. Such cells could be used as a model for the in vitro/ex vivo investigation of VSMCs in physiology/pathology to reveal the precise mechanisms underlying blood flow regulation. They could also be used as pharmacological and toxicological targets that would be relevant to some hearing loss diseases.