Development of a skin-on-a-chip platform for the study of wound healing

Talar, Kamil

Abstract

Wound healing abnormalities and non-healing chronic wounds are a major clinical problem, primarily affecting diabetic and elderly patients. Wound management associated costs resulted in a £5.3 billion financial burden to the NHS between 2012 and 2013. These serious medical states are being recognised as mortal disease, with the fatality rates often higher than those of common cancers. Half of all patients undergoing chronic-wound associated amputation related to diabetes are expected to die within 5 years. The current chronic wound treatments are inadequate, and more sophisticated models are needed to advance this field, leading to better therapies. The aim of this work was to design, manufacture, and test a microfluidic device that would address the need for a physiologically accurate model of wound healing with improved assay lifespan, when compared to the classical static models. Here we present a new microfluidic device optimised for maintaining human skin samples for prolonged period of time, and wound healing analysis. Briefly, full thickness human skin explant samples were cultured on custom-made microfluidic devices, and in static culture. The skin samples were collected at the end of the culture period, fixed, sectioned and either stained using fluorescent TUNEL assay to analyse the tissue apoptosis, or using IHC, for K6 and K14 to study tissue architecture. Wound healing outcome was measured using wound samples wholemount-stained for K1 and K14. FACS analysis was performed on digested samples to study the immunological profile in the cultured samples. The final version of the skin-on-a-chip device (V4.0) was found to be successful at prolonging tissue survival. After the seven day culture period, four-fold decrease in the epidermal apoptotic cell death (1.4% apoptosis for the on-chip sample vs. 6.1% for the static control), and two-fold decrease in the dermal apoptosis (4.1% cell death in the flow-chip samples vs. 8.3% in the static controls) were observed. Day 7 samples maintained on the V4.0 device significantly outperformed the static control samples (p = 0.007). Furthermore, the average dermal cell death for the control samples collected on day 14 was 38.1% whilst the on-chip samples exhibited dermal cell death averaging 8.3%. V4.0 samples contain significantly less apoptotic cells in the dermal section when compared to the static controls on day 14 (p = 0.0433675). The improved tissue viability makes the model more suitable for prolonged culture experiments.
Next, it was observed that the wound area is reducing in size over the period of seven days, in both cases of the V4.0 samples and the control samples. The on-chip samples yield reduced wound perimeter when compared to the static controls from the same day. the culture method has a very significant influence on the wound size (f(1) = 75.684, p=5.61x10-6). Interestingly, the same analysis showed that the culture method does have a greater impact on the wound closure that the assay day (f(2) = 24.615, p = 0.012). The on-chip samples produced a significantly different smaller wounds on day seven of the assay than the control static culture samples (p = 0.012). In addition, the overall theme seen from the FACS data demonstrated that the wounded skin samples cultured on the V4.0 microfluidic devices yielded higher levels of immune cells than the static control samples collected on the corresponding days.
Overall, The V4.0 device allowed for an increased number of cells to be collected on day seven in every single marker group, apart from in the CD56+ CD3+ group, where the levels dropped more in the V4.0 samples than in the control samples. The same relationship was noted on day three. This indicates that maintaining samples on the V4.0 device helps to improve the immune cell retention, making the microfluidic model’s immune microenvironment more comparable to the real human skin microenvironment.

Citation

Talar, K. Development of a skin-on-a-chip platform for the study of wound healing. (Thesis). University of Hull. https://hull-repository.worktribe.com/output/4508234

• Thesis Type: Thesis
• Deposit Date: Jan 9, 2024
• Publicly Available Date: Jan 9, 2024
• Keywords: Chemistry
• Public URL: https://hull-repository.worktribe.com/output/4508234
• Additional Information: Department of Chemistry; University of Hull
• Award Date: Jun 1, 2022