Modified methods for culturing myoblasts of rats: Combination of multi-enzymatic digestion and double purification

摘要

BACKGROUND: With developments of tissue engineering and genetic engineering, we aim to culture myoblasts, which are characterized by high purity, high quality and high production, for wide application in neural regeneration researches. OBJECTIVE: To modify traditional dissociation method in order to obtain myoblasts, which are characterized by high purity, high quality and high production, and explore the biological properties under in vitro culture. DESIGN: Observational study. SETTING: Basic Institute of Academy of Military Medical Sciences of Chinese PLA. MATERIALS: Four neonatal Wistar rats of 5 days old, both genders and mean body mass of 10 g were selected in this study. The main reagents and devices were detailed as follows: DMEM medium (Gibco Company), fetus bovine serum (FBS, Hycolne Company), collagenase Ⅱ(Sigma Company), trypsin (Sigma Company), dispase Ⅱ (Sigma Company), desmin antibody (Fuzhou Maixin Company), antibody Ⅱ and ABC kit (Wuhan Boster Biotechnology Company), desk centrifuge (KUBATO, Japan), and inverted phase contrast microscope (LEICA DMIRB, Germany). METHODS: The experiment was carried out in the Basic Institute of Academy of Military Medical Sciences of Chinese PLA from June to October 2006. Neonatal rats were sacrificed under sterile condition to obtain skeletal muscles of limbs, which were washed with cold PBS (containing benzylpenicillin and estreptomicina), and muscular tissue was sheared into pieces. Then, those muscular pieces were added with mixed digestive enzyme (containing 2 g/L collagenase Ⅱ + 5 g/L dispase Ⅱ + 0.28 g/L CaCl2) as twice volume as pieces, dealt with mechanical pipetting for 5 minutes and cultured in CO2 incubator for 10 minutes. The operation was done for three times and the muscular pieces were digested for 45 minutes in total. Moreover, cells were suspended again in order to obtain myoblasts from skeletal muscle of neonatal rats. In addition, myoblasts were purified with differential attachment technique and enzyme digestion so as to observe morphological characteristics and growth, draw growth curve, analyze surface structure under scanning electron microscope, and evaluate with Desmin immunohistochemical staining. MAIN OUTCOME MEASURES: Morphological characteristics and growth of myoblasts cultured in vitro. RESULTS: ① Growth of myoblasts of skeletal muscle: Primary cells had well growth, mature and differentiation. The positive rate of Desmin was 94% and purification of cells was ideal. Growth curve of cells demonstrated that myoblasts which were characterized by high purification started proliferation plentiful through transient growth lag phase (about at one or two days after inoculation). If myoblasts were not dealt with any interventions, they might become sarcotubule gradually at 3–5 days after proliferative phase. During this period, myoblasts maintained a monocaryon-bipolarity state under inverted phase contrast microscope. Furthermore, the growth of cells was the strongest and reproductive activity was the most powerful. This suggested that myotube started to form; in addition, muscle fiber of contractility might form under a well culturing condition. ② Immunocytochemical stain with desmin antibody: Interzonal fiber of desmin from myoblasts showed strongly positive reaction. Positive staining existed in cytoplasm had a high nucleus-cytoplasm ratio. However, myoblasts showed negative or mildly positive reaction. CONCLUSION: It is ideal for modified multi-enzymatic digestion and double purification method to dissociate and purify myoblasts of skeletal muscle; meanwhile, these two methods are both the effective ways to provide convenient conditions to obtain seed cells for neural regeneration researches.