Optimizing culturing conditions in patient derived 3D primary slice cultures of head and neck cancer

Frontiers in Oncology

Oncology

Avneet Randhawa¹  Annette Runge²  Jozsef Dudas²  Maria do Carmo Greier²  Herbert Riechelmann²  Volker Hans Schartinger²  Lukas Carpentari²  Monika Petersson³  Melissa Mayr³ 

[1] Department of Otolaryngology, Head and Neck Surgery, Rutgers New Jersey Medical School, Newark, NJ, United States;Department of Otorhinolaryngology, Head and Neck Surgery, Medical University of Innsbruck, Innsbruck, Austria;ViraTherapeutics GmbH, Rum, Austria;

关键词: head and neck cancer;    tumor microenvironment;    cultured neoplastic cells;    platelet rich fibrin;    culture media;   

DOI  :  10.3389/fonc.2023.1145817

 received in 2023-01-16, accepted in 2023-02-27,  发布年份 2023

来源: Frontiers

【 摘 要 】

BackgroundThree-dimensional primary slice cultures (SC) of head and neck squamous cell carcinomas (HNC) are realistic preclinical models. Until now, preserving structure and viability ex vivo for several days has been difficult. The aim of this study was to optimize cultivation conditions for HNC SC and analyze the added effects of platelet rich fibrin (PRF) on these conditions.MethodsSC were prepared from the tumor biopsies of 9 HNC patients. Cultures were incubated for 1 and 7 days in three different media- Keratinocyte serum-free medium (SFM), RPMI-1640i, and 1:1 mix of both, with and without addition of PRF. After culturing, SC were fixated, embedded, and stained with Hematoxylin-Eosin (HE) and cleaved caspase-3. In addition, triple immune fluorescence staining for cytokeratin, vimentin and CD45 was performed. Outcome parameters were cell count and cell density, viability and apoptosis, SC total area and proportions of keratinocytes, mesenchymal and immune cells. The effects of culture time, medium, and addition of PRF were calculated in an SPSS generalized linear model and using the Wald Chi-Squared test.ResultsNinety-four slice cultures were analyzed. Viability remained stable for 7 days in culture. After addition of PRF, cell viability increased (p=0.05). SC total area decreased (0.44 ± 0.04 mm2 on day 1 (95% CI: 0.35 to 0.56) to 0.29 ± 0.03 mm2 on day 7 (95% CI: 0.22 to 0.36), but cell density and cell proportions remained stable. Differences in cultivation media had no significant impact on outcome parameters.ConclusionHNC SC can be preserved for up to 7 days using the tested cultivation media. Cell viability was best preserved with addition of PRF. HNC SC are a versatile experimental tool to study physiology and drug actions. Autologous PRF can help simulate realistic conditions in vitro.

【 授权许可】

Unknown   
Copyright © 2023 Greier, Runge, Dudas, Carpentari, Schartinger, Randhawa, Mayr, Petersson and Riechelmann