Two alternative procedures for isolating adipofibroblasts from sheep skeletal muscle

Methods to isolate cells used in the study of adipocyte differentiation are lengthy, potentially damaging to the cells collected, and usually result in a mixed group of cells which are difficult to define clearly. Additionally, much work done on the differentiation of fibroblasts to preadipocytes or preadipocytes to adipocytes has relied on the use of populations of cells which are either embryonic in origin or from genetically unique animals. We therefore report two simple and rapid protocols for obtaining relatively pure populations of preadipocytes from perimuscular fat and from intramuscular fat depots of normal sheep skeletal muscle. In the first procedure, a finely minced preparation of perimuscular adipose tissue is placed directly into flasks for ceiling culture. During the second procedure, free-floating adipocytes, resulting from the enzymatic preparation of satellite cells from skeletal muscle, are placed into ceiling culture. Cells from both isolation procedures attach, grow, and are later harvested for use. These cells demonstrate proliferation and differentiation abilities of normal preadipocytes. Cell populations may be expanded for use in cloning pure populations of adipocytes or used directly in studies to identify mechanisms of adipocyte development and intercellular communication with myogenic cells.