Yan Zhang1, Mei Yu1, Xueyong Zhao1, Minjia Dai1, Chang Chen1, Weidong Tian1. 1. a State Key Laboratory of Oral Diseases & National Clinical Research Center for Oral Diseases & National Engineering Laboratory for Oral Regenerative Medicine & Department of Oral and Maxillofacial Surgery, West China Hospital of Stomatology , Sichuan University , Chengdu , China.
Abstract
OBJECTIVES: Adherent culture which is used to collect adipose tissue extract (ATE) previously brings the problem of inhomogeneity and non-repeatability. Here we aim to extract ATE with stirred suspension culture to speed up the extraction process, stabilize the yield, and improve consistent potency metrics of ATE. MATERIALS AND METHODS: ATE was collected with adherent culture (ATE-A) and stirred suspension culture (ATE-S) separately. Protein yield and composition were detected by SDS-PAGE, while cytokines in ATE were determined with ELISA. The adipogenic and angiogenic potential of ATE were compared by western blot and qPCR. In addition, haematoxylin and eosin staining and lactate dehydrogenase (LDH) activity assays were used to analyze the cell viability of adipose tissue cultured with different methods. RESULTS: The yield of ATE-S was consistent while ATE-A varied notably. Characterization of the protein composition and exosome-like vesicles (ELVs) indicated no significant difference between ATE-S and ATE-A. The concentrations of cytokines (VEGF, bFGF, and IL-6) showed no significant difference, while IGF in ATE-S was higher than that in ATE-A. ATE-S showed upregulated adipogenic and angiogenic potential compared to ATE-A. Morever, stirred suspension culture decreased the LDH activity of ATE while increased the number of viable adipocytes and reduced adipose tissue necrosis. CONCLUSION: Compared with adherent culture, stirred suspension culture is a reliable, time- and labor-saving method to collect ATE, which might be used to improve the downstream applications of ATE.
OBJECTIVES: Adherent culture which is used to collect adipose tissue extract (ATE) previously brings the problem of inhomogeneity and non-repeatability. Here we aim to extract ATE with stirred suspension culture to speed up the extraction process, stabilize the yield, and improve consistent potency metrics of ATE. MATERIALS AND METHODS:ATE was collected with adherent culture (ATE-A) and stirred suspension culture (ATE-S) separately. Protein yield and composition were detected by SDS-PAGE, while cytokines in ATE were determined with ELISA. The adipogenic and angiogenic potential of ATE were compared by western blot and qPCR. In addition, haematoxylin and eosin staining and lactate dehydrogenase (LDH) activity assays were used to analyze the cell viability of adipose tissue cultured with different methods. RESULTS: The yield of ATE-S was consistent while ATE-A varied notably. Characterization of the protein composition and exosome-like vesicles (ELVs) indicated no significant difference between ATE-S and ATE-A. The concentrations of cytokines (VEGF, bFGF, and IL-6) showed no significant difference, while IGF in ATE-S was higher than that in ATE-A. ATE-S showed upregulated adipogenic and angiogenic potential compared to ATE-A. Morever, stirred suspension culture decreased the LDH activity of ATE while increased the number of viable adipocytes and reduced adipose tissue necrosis. CONCLUSION: Compared with adherent culture, stirred suspension culture is a reliable, time- and labor-saving method to collect ATE, which might be used to improve the downstream applications of ATE.