78 TESTING
120 100 80 60 40 20 0
Untreated control
Placebo BZK Tx100
Figure 3: Percentage of total cell viability of HVE untreated (Untreated control), or treated with a solution without (Placebo) or with BZK 0.02% (BZK) or Triton X100 0.1% (Tx100). Treatments were performed from the 11th to the 12th day at air/liquid interface, using a nylon mesh. The experiment was performed in three biological replicates. Results are expressed as percentage of viability relative to untreated control, arbitrary set to 100%. Histogram represents mean ± standard deviation
chemical threats, comforting on its use as a new tool to define the safety profile of feminine care products. The herein described 3D HVE model was
reconstructed at the air/liquid interface for up to 18 days. At that late stage of culture, histological and functional analysis revealed a disorganized morphology and loss of biological relevance, constraining to limit the use of the model between its 11th
and 14th day of culture. In the present study, HVE at day 11 challenged
topically for 24 hours with hazardous agents displayed the expected tissue response as demonstrated by an MTS viability assay and the quantification of the inflammatory cytokines IL-1β. At that stage of reconstruction, HVE also exhibited robustness during transport at room temperature. Assays performed on HVE at day 11
demonstrated that the integrity and functionality of the tissues were preserved in an agarose- based culture media, during a delivery lasting up to 48 hours (data not shown). This result comforts on the possible distribution of this new ready-to-use, non-animal alternative model to support and accelerate discoveries in the feminine care sector. As most feminine care products are in
direct contact with the surface of the vaginal epithelium, a topical application through a nylon mesh has been tested in the present study. This approach allowed a successful treatment of the HVE model without compromising its viability. Other routes of application may however
be tested in the future, including the dilution of ingredients in the culture medium of the 3D model to replicate the systemic application of orally prescribed medications, for example. The robust release of IL-1β cytokine in
response to BZK and Tx100 stimuli strongly indicates the biochemical response of HVE. It also confirms the suitability of the 3D HVE model to screen anti-inflammatory compounds. The vaginal epithelium can also release
Interleukin-1 alpha (IL-1α) in response to various PERSONAL CARE November 2025
10.0 9.0 8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0.0
Untreated control
Placebo
BZK
Tx100
Figure 4: Mean concentration of interleukin 1 beta (IL-1β) quantified by ELISA assay from the cell culture medium of HVE untreated (Untreated control), or treated with a solution without (Placebo) or with BZK 0.02% (BZK) or Triton X100 0.1% (Tx100). Treatments were performed from the 11th to the 12th day at air/liquid interface, using a nylon mesh. The experiment was performed in three biological replicates. Histogram represents mean ± standard deviation
stimuli, including irritating agents, infections from pathogens and in certain dysbiosis vaginal microbiota conditions. Secretion of Interleukin-1 alpha (IL-1a), IL-6 and IL-8 have been reported.10 The release of inflammatory cytokines is an
active process, often preceding cell death, and contributes to the host’s immune response, though the context of the release, such as the presence of tissue damage, influences its overall effect on the host’s immune system.11,12 Monitoring inflammatory cytokines in cell
culture supernatants can provide valuable insights into the anti-inflammatory potential of bioactive compounds. Quantification by ELISA assays represents a reliable and widely used method to evaluate cellular responses under stress conditions. By applying this approach to the in vitro HVE model subjected to various stressors, it becomes possible to compare and characterize the anti-inflammatory efficacy of innovative active ingredients. Such analyses may provide a valuable assay to identify novel potent anti- inflammatory molecules aiming at reducing vaginal irritation and itching issues. In conclusion, by using its strong tissue engineering skills, Straticell releases here a new reliable, animal-free 3D vaginal model not only to meet current safety regulatory expectations but also to actively participate to the comprehension and the objectivation of the efficacy of future women’s health care products.
PC
References 1. Anderson DJ, Marathe J, Pudney J. The structure of the human vaginal stratum corneum and its role in immune defense. Am J Reprod Immunol. 2014; June 71(6), 618–623
2. Ye Z, Jiang P, Zhu Q, Pei Z, Hu Y, Zhao G. Molecular stratification of the human fetal vaginal epithelium by spatial transcriptome analysis. Acta Biochim Biophys Sin. 2024; 56(10), 1521–1536
3. McCracken JM, Calderon GA, Robinson AJ, Sullivan CN, Cosgriff-Hernandez E, Hakim
JCE. Animal models and alternatives in vaginal research: a comparative review. Reprod Sci. 2021; June 28(6), 1759–1773
4. Langhans SA. Three-dimensional in vitro cell culture models in drug discovery and drug repositioning. Frontiers in Pharmacology. 2018; 9, 6
5. Chumduri C, Turco MY. Organoids of the female reproductive tract. Journal of Molecular Medicine. 2021; 99, 531–553
6. Ayehunie S, Cannon C, Lamore S, Kubilus J, Anderson DJ, Pudney J, Klausner M. Organotypic human vaginal-ectocervical tissue model for irritation studies of spermicides, microbicides, and feminine-care products. Toxicology in Vitro. 2006; 20, 689–698
7. Goldstein MH, Silva FQ, Blender N, Tran T, Vantipalli S. Ocular benzalkonium chloride exposure: problems and solutions. Eye. 2022; 36, 361–368
8. Dayeh VR, Chow SL, Schirmer K, Lynn DH, Bols NC. Evaluating the toxicity of Triton X-100 to protozoan, fish, and mammalian cells using fluorescent dyes as indicators of cell viability. Ecotoxicology and Environmental Safety. 2004; 57, 375–382
9. Dinarello C. Overview of the IL-1 family in innate inflammation and acquired immunity. Immunol Rev. 2018; Jan 281(1), 8–27
10. Fichorova RN, Bajpai M, Chandra N, Hsiu JG, Spangler M, Ratnam V, Doncel GF. Interleukin (IL)-1, IL-6, and IL-8 predict mucosal toxicity of vaginal microbicidal contraceptives. Biology of Reproduction. 2004; 71, 761–769
11. Richardson JP, Willems HME, Moyes DL, Shoaie S, Barker KS, Tan SL, Palmer GE, Hube B, Naglik JR, Peters BM. Candidalysin drives epithelial signaling, neutrophil recruitment, and immunopathology at the vaginal mucosa. Infect Immun. 2018; 86, e00645-17
12. Ayehunie S, Cannon C, LaRosa K, Pudney J, Anderson DJ, Klausner M. Development of an in vitro alternative assay method for vaginal irritation. Toxicology. 2011; 279, Issues 1–3, 130-138
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Viability (% to treated control)
Mean concentration of IL-1 beta (pg/ml)
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