Abstract: Objective To optimize the extraction method of secretory factors from adipose tissue explant (SFAE)in vitro. Methods SFAE were obtained through adherent culture (SFAE-A） and suspension culture (SFAE-S） and concentrated by filtration and centrifugation. The yield of SFAE was compared using BCA protein detection kit. P3 adipose-derived stem cells (ADSCs) were induced with equal amount of SFAE for 7-13 d, before the state of adipogenesis between suspension culture and adherent culture was compared by microscope observation and oil red O staining. Results The average amount of SFAE yielded from adherent culture and suspension culture did not show significant difference. While the yield of SFAE from suspension culture was consistent at 8.7 mg per gram of adipose tissue, the adherent culture generated an inconsistent result in the four repeat experiments, ranging from 7.3 mg to 12.4 mg per gram of adipose tissue. Moreover, ten more flasks and better distribution were needed for adherent culture to acquire an equal amount of SFAE in comparison with suspension culture. SFAE from both adherent and suspension culture promoted the adipogenesis of P3 adipose-derived stem cells. No differences on the adipogenic effect were found between the two extraction methods. Conclusion Secretory factors from adherent culture and suspension culture have the same adipogenesis effect. Suspension culture can save time and labor. The most important advantage of suspension culture is its stable yield of SFAE.