Adipose tissue is derived from the embryonic mesenchyme and contains a stroma that can be easily isolated. Preliminary studies have recently identified a putative stem cell population within the adipose stromal compartment. This cell population can differentiate to the osteogenic, adipogenic, myogenic, and chondrogenic lineages. Enzymatic digestion, the commonly used method of adipose-derived stromal cells isolation, is time consuming and expensive, especially when applied to large volumes of tissue. Additionally, mechanical stress during isolation, use of bacterial-derived products and potential contamination with endotoxins and xenoantigens are other disadvantages of this method. In this study, the characteristics of the cells obtained by adipose tissue explant culture were studied. This technique can be used to reproducibly isolate mesenchymal stromal cells from fat tissue obtained by liposuction as well as surgical resection. Explant culture gave higher yield of cells than digestion method after primary culture. Therefore, explant culture can be used as an effective way to isolate adipose-derived stromal cells for tissue engineering and regenerative medicine, especially in cases of limited starting material.