Bioreactor for application of subatmospheric pressure to three-dimensional cell culture.
Vacuum-assisted closure (VAC) negative pressure wound therapy (NPWT) is a highly successful and widely used treatment modality for wound healing, although no apparatus exists to monitor the effects of subatmospheric pressure application in vitro. Such an apparatus is desirable to better understand the biological effects of this therapy and potentially improve upon them. This article describes the development and validation of a novel bioreactor that permits such study. Tissue analogues consisting of 3-dimensional fibroblast-containing fibrin clots were cultured in off-the-shelf disposable cell culture inserts and multi-well plates that were integrated into the bioreactor module. Negative pressure dressings, commercialized for wound therapy, were placed on top of the culture, and subatmospheric pressure was applied to the dressing. Cultures were perfused with media at controlled physiologic wound exudate flow rates. The design of this bioreactor permits observation of the culture using an inverted microscope in brightfield and fluorescence modes and sustained incubation of the system in a 5% carbon dioxide atmosphere. This closed-system mimics the wound micro-environment under VAC NPWT. Matrix compression occurs as the subatmospheric pressure draws the dressing material down. At the contact zone, surface undulations were clearly evident on the fibroblast-containing tissue analogues at 24 h and appeared to correspond to the dressing microstructure. The bioreactor design, consisting of sterilizable machined plastics and disposable labware, can be easily scaled to multiple units. Validation experiments show that cell survival in this system is comparable with that seen in cells grown in static tissue culture. After application of VAC NPWT, cell morphology changed, with cells appearing thicker and with an organized actin cytoskeleton. The development and validation of this new culture system establishes a stable platform for in vitro investigations of subatmospheric pressure application to tissues.
Wilkes
Robert P
RP
Kinetic Concepts Inc., San Antonio, Texas 78249, USA. wilkesr@lci1.com
McNulty
Amy K
AK
Feeley
Teri D
TD
Schmidt
Marisa A
MA
Kieswetter
Kris
K
D000818:Animals; D001274:Atmospheric Pressure; D019149:Bioreactors; D018929:Cell Culture Techniques / Q000295:instrumentation* / Q000379:methods; D002470:Cell Survival; D002478:Cells, Cultured; D004867:Equipment Design; D019544:Equipment Failure Analysis; D004912:Erythrocytes / Q000166:cytology* / Q000502:physiology*; D040542:Mechanotransduction, Cellular / Q000502:physiology*; D013552:Swine; D023822:Tissue Engineering / Q000295:instrumentation* / Q000379:methods