Effect of collagenase concentration on the isolation of small adipocytes from human buccal fat pad

Abstract

Dedifferentiated fat (DFAT) cells were isolated from mature adipocytes using the ceiling culture method. Recently, we successfully isolated DFAT cells from adipocytes with a relatively small size (<40 μm). DFAT cells have a higher osteogenic potential than that of medium adipocytes. Therefore, the objective of this study was to determine the optimal concentration of collagenase solution for isolating small adipocytes from human buccal fat pads (BFPs). Four concentrations of collagenase solution (0.01%, 0.02%, 0.1%, and 0.5%) were used, and their effectiveness was assessed by the number of small adipocytes and DFAT cells isolated. The total number of floating adipocytes that dissociated with 0.02% collagenase was 2.5 times of that dissociated with 0.1% collagenase. The number of floating adipocytes with a diameter of ≤29 μm that dissociated with 0.02% collagenase was thrice of those dissociated with 0.1% and 0.5% collagenase. The number of DFAT cells that dissociated with 0.02% collagenase was 1.5 times of that dissociated with 0.1% collagenase. In addition, DFAT cells that dissociated with 0.02% collagenase had a higher osteogenic differentiation potential than those that dissociated with 0.1% collagenase. These results suggest that 0.02% is the optimal collagenase concentration for isolating small adipocytes from BFPs.

Dedifferentiated fat cells: current applications and future directions in regenerative medicine

“…DFAT cells from small adipocytes exhibited higher osteogenic potential than those from large adipocytes [ 54 ]. Bone regeneration involves a complex interaction between DFAT cells and biological factors [ 55 ]. DFAT cells undergo osteogenic differentiation by the stimulated of retinoic acid, an analogue of retinol that interacts with bone morphogenetic proteins (BMPs) to limit adipogenesis and promote osteogenesis [ 43 ].…”

Transplantation of dedifferentiated fat cells combined with a biodegradable type I collagen-recombinant peptide scaffold for critical-size bone defects in rats

“…After about 1 week, the medium was replaced with growth media, and the flasks were inverted so that the cells were on the bottom of the culture flasks ( Fig. 1) [22][23][24][25]. The initial DFAT cells, referred to as passage 0 (P0), were passaged at 80% confluence.…”

“…Dedifferentiated fat (DFAT) cells are established by asymmetrical division of mature adipocytes from adipose tissue with a ceiling culture technique, which relies on the inherent buoyancy of adipocytes [22][23][24][25]. Unlike terminally differentiated adipocytes, DFAT cells have the potential to actively proliferate and have the characteristics of MSCs, i.e., multiple differentiation into osteoblasts [26,27], adipocytes [26,27], chondrocytes [26], skeletal myocytes [28], smooth muscle cell lineages [29,30], cardiomyocytes [31], and endothelial cells [32,33] in vitro.…”

“…Two types of adipose-derived stem cells (ASCs), identified as MSCs and DFAT cells, can be isolated from subcutaneous adipose tissues or buccal fat pads [22][23][24][25][26][27]. Several studies reported that DFAT cells and ASCs potentially contribute to regeneration of periodontal tissue in animal models [22,24,27].…”

Transplantation of Mature Adipocyte-Derived Dedifferentiated Fat Cells Facilitates Periodontal Tissue Regeneration of Class II Furcation Defects in Miniature Pigs

“…DFAT cells have the characteristics of MSCs, with the multipotent capacity to differentiate into adipocytes [20][21][22], osteoblasts [20,21,23], chondrocytes [20], skeletal myocytes [24], smooth muscle cells [25,26], cardiomyocytes [27], and endothelial cells [28,29]. ASCs and DFAT cells can be isolated from subcutaneous adipose tissues or buccal fat pads [14,20,[30][31][32][33]. Recent oral and maxillofacial research has demonstrated that DFAT cells and ASCs potentially contribute to the generation of skeletal bone and periodontal tissue in animals [14,30,32,34].…”