Single-cell transcriptome conservation in a comparative analysis of fresh and cryopreserved human skin tissue: pilot in localized scleroderma

Background The purpose of this study was to assess variability in cell composition and cell-specific gene expression in the skin of patients with localized scleroderma (LS) utilizing CryoStor® CS10 in comparison to RPMI to produce adequate preservation of tissue samples and cell types of interest for use in large-scale multi-institutional collaborations studying localized scleroderma and other skin disorders. Methods We performed single-cell RNA sequencing on paired skin biopsy specimens from 3 patients with LS. Each patient with one sample cryopreserved in CryoStor® CS10 and one fresh in RPMI media using 10× Genomics sequencing. Results Levels of cell viability and yield were comparable between CryoStor® CS10 (frozen) and RPMI (fresh) preserved cells. Furthermore, gene expression between preservation methods was collectively significantly correlated and conserved across all 18 identified cell cluster populations. Conclusion Comparable cell population and transcript expression yields between CryoStor® CS10 and RPMI preserved cells support the utilization of cryopreserved skin tissue in single-cell analysis. This suggests that employing standardized cryopreservation protocols for the skin tissue will help facilitate multi-site collaborations looking to identify mechanisms of disease in disorders characterized by cutaneous pathology.

Morphological features of a cold skin wound under the influence of an extract of cryopreserved skin fragments of piglets (experimental study)

The aim of the study is to identify in an experiment the effect of an extract of cryopreserved fragments of piglets on the morphological state of a cold skin wound. Materials and methods: Hairless six-month-old male rats were used in the study. They were divided into III groups: group I included 10 rats that had not been manipulated; group II was represented by 10 rats with cold wounds on the lateral surface of the thigh; group III was represented by 10 rats that were with a cold wound, followed by the injection of an extract of cryopreserved skin fragments of piglets into the abdominal cavity at a dose of 50 μg per 100 g of animal body weight (peptide concentration 100 μg/ ml) once a day for 5 days from the time of wound modeling. Animals in groups I-III were withdrawn from the experiment on the 7th, 14th and 21st days. The material for the morphological study was the fragments of intact skin with underlying soft tissues from the thigh area in group I and the fragments of skin with underlying soft tissues from the thigh area directly from the zone of cryoexposure in groups II and III. Histological, histochemical and morphometric methods were used. Microspecimens were studied using an Olympus BX-41 microscope (Japan). Statistical processing was performed using the Statistica 6.0 and Microsoft Excel 2003 software package. Nonparametric methods were used to compare numerical values (Mann-Whitney U-test, Kruskal-Wallis test). The significance of differences between the average values of the indicators was taken at the level of p<0.05. Results: The extract of cryopreserved skin fragments of piglets has an effective wound healing effect compared to the healing processes in a cold wound, which was not subjected to any therapeutic effects. It was manifested in the improved process of cleansing the wound from necrotic tissues that entered the zone of primary necrosis, as evidenced by 1,2 times decrease of the zone of primary necrosis on the 7th, 14th and 21st days; a decrease of the zone of secondary necrosis on the 7th, 14th and 21st days, respectively, – 1.2, 1.3, 1.2 times; growth and maturation of granulation tissue activation, as evidenced by an increase in the thickness of a granulation tissue layer on 7, 14, 21 days, respectively, – 1.9, 1.8, 1.2 times; activation of proliferative processes in the epithelial layer located in the marginal sections of the wound defect or covering the regenerate surface, as evidenced by more pronounced acanthotic growths in the underlying tissue and an increase in the thickness of the epithelial layer on the 7th, 14th and 21st days, respectively, – 2.1, 2.0, 2.2 times. Conclusion: The extract of cryopreserved skin fragments of piglets has an effective wound healing effect and can be recommended for further research in order to study the possibility of its use in clinical practice.

Characterization of inflammatory infiltrate of ulcerative dermatitis in C57BL/6NCrl-Tg(HMGA1P6)1Pg mice

Ulcerative dermatitis (UD) is an idiopathic, spontaneous and progressive disease typically affecting C57BL/6 aged mice with an unknown aetiopathogenesis. For this study, we evaluated 25 cases of UD in C57BL/6NCrl-Tg(HMGA1P6)1Pg mice. Formalin-fixed, paraffin-embedded skin samples were submitted to morphological investigations. Immunohistochemical analysis was performed to characterize and quantify inflammatory cells using CD3, CD45/B220, CD4, CD8 and IL-17 antibodies. Mast cell-bound IgE was investigated by immunofluorescence, whereas serum and cryopreserved skin samples were collected for molecular analysis. Student's t-test (two-tailed) was performed to assess significant differences between the two groups. Affected skin showed extensive areas of ulceration and diffuse, severe and mixed inflammatory infiltrates. No relevant changes were observed in control mice. Immunohistochemical analysis showed a predominant CD3 + CD4 + leukocyte population with fewer CD45/B220 and IL-17 immunolabelled cells and mast cell-bound IgE. Increases in TNFα, IL-1β and Il-6 mRNA expression were observed in the skin of affected animals compared to controls. Serum TNFα and IL-6 did not vary between affected and control mice. Inflammatory infiltrates and cytokine expression were consistent with both Th2/IgE and Th17 differentiation, a typical pattern of a type I hypersensitivity reaction. Overall, our data suggest an allergic-based aetiopathogenesis of UD in C57BL/6NCrl-Tg(HMGA1P6)1Pg mice.