Primary culture and identification of rat skeletal muscle-derived stem cells

Aibin LI; Linglong WANG; Huamin JIN; Jingjing LI

ABSTRACT

BACKGROUND:

Skeletal muscle-derived stem cells (MDSCs), another adult pluripotent stem cells, have become a hot topic in the field of gene therapy and cell-based tissue engineering. It has wide prospect in treating stress incontinence by periurethral injection of patients' MDSCs.

OBJECTIVE:

To investigate the method of culturing MDSCs in vitro so as to provide experimental basis for treating stress incotinence by injecting MDSCs.

DESIGN:

Repeated observation.

SETTING:

Department of Reproductive Medical Center and Department of Urology, Renmin Hospital of Wuhan University.MATERIALS This experiment was conducted at the Laboratory of Department of Urology , Renmin Hospital , wuhan University from December 2003 to May 2004. Totally 20 female SD rats , aged 4 to 6 weeks , were involved . Type- Ⅺ pancreatin, collgenase , pancreatin ,polylysine (Sigma), dispase enzyme (Gibco) , chick embryo extract (self made).

METHODS:

① Rats were anesthetized intraperitoneally with 10 g/Lpentobarbital sodium (30 g/kg). Under aspetic condition, gastroeneminus were isolated and immediately put into pre-cooled DMEM media (Gibco)containing antibiotics and then removed into the hood. After washed with D-hank's solution three times, muscle biopsies were removed of fascia,tendon, nerve and blood vessels and minced into small pieces about 1-3 mm3,and then transferred into a centrifugation tube. 0.2% collgenase-type Ⅺ(Sigma) and 0.1% pancreatin were added to the left tissue . Skeletal muscle cells of the rats were isolated with collagenase. ② Differential attachment was used to purify the skeletal muscle cells of the rats. After screened with cell screen cloth , cell suspension was transferred into a polylysine-coated flask and cultured at 37 ℃ in a humid atmosphere with 0.05 CO2 in air for 1 hour. All the cells that did not adhere to the flask were then transferred to another flask with new culture medium and cultured for approximately 1 hour at 37 ℃ .Again,the non-adhering cells were transferred to another flask and were incubated at 37 ℃ overnight.The technique was carried out for an additional 4-5 days. This operation was repeated every 24 hours until the fifth day. Those cells attached on days 5-6 were MDSCs.③ After they grew with 70% confluence , the cells were digested with trypsin and passaged at the ratio of 12. After digestion, the generative cells were inoculated to the culture plate with 6-well cover glass. Growing culture medium was added and cell slide was prepaared 24 hours later. The expression of specific protein antigen and stem cell antigen-1 of the cultured cells were identified with immunohistochemical staining.MAIN OUTCOME

MEASURES:

Morphology and identification of MDSCs.

RESULTS:

① Morphology of MDSCs Cells isolated from skeletal muscle tissue took the form of spheres and had high refraction. When subcultured,they began to adhere at the 12th hour, at that time they were still round,and complete their attachment at the 48th hour. Then they began to expand and ultimately became fusiform-shaped or spindle-shaped, having two poles and small size. As time going, they fused with each other to form mature poly-nuclear myotubes. ② Identification results of muscle-derived stem cells MDSCs were desmin and stem cell antigen-1 (Sca-1) positive specific for some stem cells. Red fluorescence effluenced from cytoplasm was found under the fluorescence microscope and the positive rate reached 90%.

CONCLUSION:

MDSC belongs to adult stem cells and has the advantages of rich source, low immunogenicity and long survival after transplantation. High-purity muscle-derived stem cells can be obtained through primary culture , and immunohistochemical technique can identify muscle-derived and specific characteristics of stem cells, so it has a broad application perspective in tissue engineering and gene therapy.