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Lab Res.
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Novel technique for application of localized negative pressure in a 3-D cell culture R. Wilkes, A. McNulty, M. Schmidt, T. Feeley, C. Kauffman, A. Yanchak, J. Montalbo, K. Kieswetter, Kinetic Concepts Inc., San Antonio, TX . V.A.C. ¨ Therapy* has been shown to aid in wound healing through the application of localized negative pressure (LNP) to the wound via a specialized foam dressing. Hypothesized mechanisms for inducing cellular proliferation and matrix production include mechanical strain and fluid flow induced by the application of negative air pressure through polyurethane foam (400-600 µm pore size). An apparatus has been developed using the V.A.C.¨ GranuFoam¨ Dressing*, V.A.C. Freedom¨ System*, and a cell culture insert system (BD Falconª) to apply LNP to human dermal fibroblasts in a 3-D matrix. An engineered airtight seal between the insert and well prevents air induction into the culture that would normally desiccate or aspirate the cells and matrix when negative pressure is applied to the culture surface. Controlled flow of culture medium is provided by a peristaltic pump through an injection port at the bottom of the well. Negative pressure is applied to a port on the top of the dressing. Continuous flow of culture medium (1mL/cm2/day) through a 1.0 µm porous polyethylene terephthalate membrane in the cell culture insert creates a scalable microenvironment for studying LNP in vitro Fibroblasts were encapsulated and cultured for 3 wks in a fibrinogen (9.84 mg/mL) and thrombin (433 units/mL) matrix. LNP was applied by the V.A.C. System* continuously at 125 mmHg for 17 hrs followed by visualization with a live/dead stain to assess viability (calcein AM / ethidium homodimer-1). Results indicate most cells were viable after V.A.C. System* application, demonstrating that this testing method is appropriate for evaluating the use of the V.A.C. System* on cells *KCI, San Antonio, TX. |
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