title: Differential Effects of Processing Time and Duration of Collagenase Digestion on Human and Murine Fat Grafts.
abstract: BACKGROUND
  Autologous fat graft retention is unpredictable, and mechanisms of optimization are poorly understood. Attempts at improving retention use collagenase experimentally and clinically to isolate the stromal vascular fraction to "enhance" fat grafts. However, no standardized duration for collagenase digestion or time following fat graft harvest has been established. This study investigates the effect of (1) time after fat graft harvest and (2) collagenase digestion time on interstitial cell and adipocyte viability in murine fat and human lipoaspirate.
  
  
  METHODS
  Murine fat and human lipoaspirate were incubated ex vivo after harvest at room temperature for 120 minutes. Additional groups were incubated with collagenase for increasing 5-minute intervals from 30 to 60 minutes. Samples from each group were stained with BODIPY to quantify intact adipocytes and the LIVE/DEAD kit to quantify interstitial cell viability.
  
  
  RESULTS
  With increased time after harvest, the number of intact adipocytes in murine fat and human lipoaspirate remained unchanged. Human interstitial cells were resistant to the effect of increased time ex vivo, whereas murine interstitial cells decreased in viability. In both populations, increased collagenase digestion time significantly decreased the number of viable adipocytes (murine, p ≤ 0.001; human, p ≤ 0.001) and interstitial cells (murine, p ≤ 0.001; human, p ≤ 0.001).
  
  
  CONCLUSIONS
  Human and murine adipocytes and human interstitial cells appear resistant to deleterious effects of increasing time following harvest. However, murine interstitial cells are sensitive to increased time and prolonged collagenase digestion. These studies highlight the complex cellular components of fat grafts and how they respond differentially to time and collagenase digestion.
authors.affiliation: Charlottesville, Va; and San Antonio, Texas From the Departments of Biomedical Engineering and Plastic Surgery, University of Virginia; and the Hand Center of San Antonio.
authors.forename: Scott A
authors.initials: SA
authors.lastname: Seaman
authors.forename: Shruti Chudasama
authors.initials: SC
authors.lastname: Tannan
authors.forename: Yiqi
authors.initials: Y
authors.lastname: Cao
authors.forename: Shayn M
authors.initials: SM
authors.lastname: Peirce
authors.forename: Kant Y
authors.initials: KY
authors.lastname: Lin
mesh_terms: D017667:Adipocytes / Q000378:metabolism* / Q000502:physiology; D000273:Adipose Tissue / Q000637:transplantation*; D000818:Animals; D002470:Cell Survival / Q000502:physiology*; D002478:Cells, Cultured; D017364:Collagenases / Q000378:metabolism*; D006085:Graft Survival; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D018613:Microscopy, Confocal; D023421:Models, Animal; D018570:Risk Assessment; D013048:Specimen Handling; D013997:Time Factors; D020858:Tissue and Organ Harvesting
chemical_list: D017364:Collagenases