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. 2023 Nov 23;14:7660. doi: 10.1038/s41467-023-43437-2

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© The Author(s) 2023

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 2 — a Infection of high-density skin equivalents (hdSEs) with tsetse-transmitted trypanosomes. Three hdSEs were stacked (# 1, 2, 3) and exposed to tsetse flies. b Trypanosomes were detected in the skin dermis by stereo-fluorescence microscopy. Parasites (yellow) expressing the fluorescent protein tdTomato were found in multiple finger-like lesions (white arrows), probably corresponding to the bite path of the tsetse fly. Scale bar, 500 μm. c Analysis of the injection depth of tsetse-transmitted trypanosomes. Three dermal equivalents with a standardized height of 1 mm were stacked and infected with trypanosomes by tsetse flies. The numbers of parasites in each equivalent were quantified after 1 h and expressed as fractions of the total. Results are shown as median ± IQR (n = 9 independent infections). Source data are provided as a Source Data file. d Cell numbers and population doubling times (PDTs) of trypanosomes in skin equivalents at various times post-infection (hpi, dpi) over a 7-day timecourse. Numbers of monomorphic bloodstream form (BSF) parasites 7 days post syringe injection into skin equivalents are shown as comparison. Data represent means ± SD (n = 9 infected hdSEs examined per time point over three independent experiments). Source data are provided as a Source Data file. e Flow cytometry of parasite viability by Calcein-AM staining of infected hdSEs. Results are means ± SD (n = 9 independent measurements per time point). The gating strategy is described in Supplementary Fig. 5d. Source data are provided as a Source Data file. f Scanning electron microscopy of hdSEs at 4 dpi. (I) Overview showing an intact epidermal layer (yellow) and the presence of trypanosomes (purple) in the connective tissue of the dermis (yellow arrow). Scale bar, 30 µm. (II + III) Entanglement of trypanosomes with collagen fibers. (IV + V) Parasites were found in close contact with dermal fibroblasts (blue). (V + VI) Proliferation was evidenced by the double flagella of trypanosomes (orange arrows). Scale bar, 2 µm (II, III, IV, V, VI). Scanning electron microscopy was conducted on three independently infected hdSEs and representative images are shown.