Novel cell culture system for monitoring cells during continuous and variable negative-pressure wound therapy

Abstract

Background: Although the clinical efficacy of negative-pressure wound therapy (NPWT) is well known, many of its molecular biological mechanisms remain unresolved, mainly due to the difficulty and paucity of relevant in vitro studies. We attempted to develop an in vitro cell culture system capable of real-time monitoring of cells during NPWT treatment.

Materials and methods: A novel negative-pressure cell culture system was developed by combining an inverted microscope, a stage-top incubator, a sealed metal chamber for cell culture, and an NPWT treatment device. Human keratinocytes, PSVK-1, were divided into ambient pressure (AP), continuous negative-pressure (NPc), and intermittent negative-pressure (NPi) groups and cultured for 24 h with scratch assay using our real-time monitoring system and device. Pressure inside the device, medium evaporation rate, and the residual wound area were compared across the groups.

Results: Pressure in the device was maintained at almost the same value as set in all groups. Medium evaporation rate was significantly higher in the NPi group than in the other two groups; however, it had negligible effect on cell culture. Residual wound area after 9 h evaluated by the scratch assay was significantly smaller in the NPc and NPi groups than in the AP group.

Conclusion: We developed a negative-pressure cell culture device that enables negative-pressure cell culture under conditions similar to those used in clinical practice and is able to monitor cells under NPWT. Further experiments using this device would provide high-quality molecular biological evidence for NPWT.

Keywords: epithelial-to-mesenchymal transition; intermittent negative-pressure wound therapy; keratinocyte; microdeformational wound therapy; negative-pressure incubator; topical negative pressure; vacuum-assisted closure; wound healing.

FIGURE 1

We constructed a novel negative‐pressure cell culture system by combining readily available materials. (A) Appearance of the apparatus. (Left) Overall view of the apparatus. A personal computer connected to the microscope enables cell monitoring and image analysis. (Upper right) Stage‐top incubator and two metal chambers placed there. (Lower right) The chambers just fit a 35 mm dish. (B) Schematic illustration of the sealed metal chamber for airtight culture and its internal structure. The chamber is sized to just fit a 35‐mm dish. There is a hole at the bottom for microscopic observation, and a ring‐shaped silicon sheet is placed on the bottom to ensure airtightness. The lid has three holes to which up to three dedicated connectors can be connected, enabling the exchange of gases and liquids. Cells were cultured in a monolayer using 3000 μl of filtered medium. (C) Schematic illustration of the entire apparatus. Two sealed chambers were set inside a stage‐top incubator equipped with a microscope and adjusted to ambient and negative pressure, respectively. Internal pressures were recorded by a barometer connected to the very upstream of the circuit. Negative pressure was applied using a negative‐pressure treatment device. A water bottle was placed upstream of the circuit in order to maintain high humidity during the negative‐pressure treatment, as water vapor saturation in the chamber would be disturbed and the medium would evaporate. All tubing was connected in such a way that it did not interfere with the movement of the microscope stage and cell observation.

FIGURE 2

Two independent sealed culture chambers were used simultaneously in each experiment, one of which was treated under ambient pressure (AP) and the other under continuous negative‐pressure of −120 mmHg (NPc) or intermittent negative‐pressure of −120 mmHg for 5 min and −25 mmHg for 2 min (NPi). Pressure in each chamber was recorded every 5 s by a barometer, and the relative pressure in the circuit was recorded as the NPc or NPi value minus the AP value. Experiments were conducted with and without RENASYS Softport in the circuit, and results are shown in the graph. (A) Without Softport, negative pressure was maintained as set in NPc group, whereas rapid pressure increase was not obtained in NPi group, and the next high‐pressure cycle began without sufficient pressure return. (B) With Softport, negative pressure was successfully applied almost exactly as set in both the NPc and NPi groups.

FIGURE 3

Weight of the chamber was measured before and after 24‐h treatment, and the amount of medium evaporated was calculated from the reduction and specific gravity of the culture media. The value was divided by the volume of medium at the start of the experiment (3000 μl) and defined as %Medium loss; the values were compared across different conditions. AP: ambient pressure group, n = 10; NPc: continuous negative‐pressure group, n = 5; NPi: intermittent negative‐pressure group, n = 5. Values are expressed as means ± standard deviation. Statistical significance is marked with ***p < 0.005.

FIGURE 4

Scratch assay using the apparatus was conducted on human keratinocytes PSVK‐1. (A) Scratch wounds were created using a 200‐μl pipette tip on cells confluent in 35‐mm dishes. Cells were divided into three groups, namely, AP, NPc, and NPi, and incubated with the apparatus shown in Figure 1. Time‐lapse images of four coordinates in each dish were acquired every 30 min, and the remaining wound area relative to that in the beginning of the experiment (T0) was calculated as %Wound area. (B) The residual wound area was compared 3, 6, 9, and 12 h after the start of the experiment (T3, T6, T9, and T12). Both NPc and NPi groups had significantly less residual wound area than the AP group. AP: ambient pressure group, n = 10; NPc: continuous negative‐pressure group, n = 5; NPi: intermittent negative‐pressure group, n = 5. Values are expressed as means ± standard deviation. Statistical significance is marked with *p < 0.05 and ***p < 0.005.