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

Abstract

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.

Cytotoxic response of tumor-infiltrating lymphocytes of head and neck cancer slice cultures under mitochondrial dysfunction

“…Cultures of patient derived tumor tissue slices provide insights into the complex interactions within their TME, reflect their high inter- and intra-individual variability and may maintain the metabolic conditions ex vivo ( 37 ). The tumor SC of patients with HNSCC remained well preserved over the experimental period of 3 days, and the cell density on day 3, at over 9000 cells/mm², was markedly higher than in a previous study ( 21 ). This may be due to the gentle cutting technique of the tumor biopsies with the Compresstome® ( 38 ).…”

“…The tissue samples were taken with biopsy forceps from a non-necrotic tumor area during diagnostic endoscopy under anesthesia and immediately brought to the laboratory for the preparation of slice cultures (SC). Six slices with a thickness of 250 μm were cut from each patient sample using the Compresstome® VF-310-0Z (Precisionary Instruments LLC, MA, USA) and then plated ( 21 ).…”

“…Compared to cell cultures, the possible experimental interventions are limited. Although SC of HNSCC remain viable, maintain their microenvironment, and respond to experimental immunologic interventions ( 21 , 22 ), they can only do so for a few days. Moreover, they are only available in limited quantities and complex procedures like transgenic interventions are hardly feasible.…”

Transcriptomic analysis-guided assessment of precision-cut tumor slices (PCTS) as an ex-vivo tool in cancer research

“…Our data showed that PCTS from HNC and Meso retained histological fidelity for up to 72 h in culture. Other studies using PCTS derived from HNCs have demonstrated a similar viability of 2-7 days 6,7,12 . This is likely tumor-type dependent, as others have reported that PCTS from other tumor types (murine tumors, colorectal, non-small cell lung cancer and liver tumors) were viable for up to 12 days 8,[13][14][15] .…”

“…This is likely tumor-type dependent, as others have reported that PCTS from other tumor types (murine tumors, colorectal, non-small cell lung cancer and liver tumors) were viable for up to 12 days 8,[13][14][15] . It may also be culture-specific since, in our experience, different culture media, with or without serum supplementation, and different culture plates can affect PCTS viability 12 . Collectively, these data demonstrate the importance of (1) assessing viability for each type of tumor studied and (2) fully optimizing the culture conditions employed.…”