Studies on oxygen availability and the creation of natural and artificial oxygen gradients in gelatin-methacryloyl hydrogel 3D cell culture

Three-dimensional (3D) cultivation platforms allow the creation of cell models, which more closely resemble in vivo-like cell behavior. Therefore, 3D cell culture platforms have started to replace conventional two-dimensional (2D) cultivation techniques in many fields. Besides the advantages of 3D culture, there are also some challenges: cultivation in 3D often results in an inhomogeneous microenvironment and therefore unique cultivation conditions for each cell inside the construct. As a result, the analysis and precise control over the singular cell state is limited in 3D. In this work, we address these challenges by exploring ways to monitor oxygen concentrations in gelatin methacryloyl (GelMA) 3D hydrogel culture at the cellular level using hypoxia reporter cells and deep within the construct using a non-invasive optical oxygen sensing spot. We could show that the appearance of oxygen limitations is more prominent in softer GelMA-hydrogels, which enable better cell spreading. Beyond demonstrating novel or space-resolved techniques of visualizing oxygen availability in hydrogel constructs, we also describe a method to create a stable and controlled oxygen gradient throughout the construct using a 3D printed flow-through chamber

Live reporting for hypoxia : Hypoxia sensor–modified mesenchymal stem cells as in vitro reporters

Natural oxygen gradients occur in tissues of biological organisms and also in the context of three-dimensional (3D) in vitro cultivation. Oxygen diffusion limitation and metabolic oxygen consumption by embedded cells produce areas of hypoxia in the tissue/matrix. However, reliable systems to detect oxygen gradients and cellular response to hypoxia in 3D cell culture systems are still missing. In this study, we developed a system for visualization of oxygen gradients in 3D using human adipose tissue–derived mesenchymal stem cells (hAD-MSCs) modified to stably express a fluorescent genetically engineered hypoxia sensor HRE-dUnaG. Modified cells retained their stem cell characteristics in terms of proliferation and differentiation capacity. The hypoxia-reporter cells were evaluated by fluorescence microscopy and flow cytometry under variable oxygen levels (2.5%, 5%, and 7.5% O2). We demonstrated that reporter hAD-MSCs output is sensitive to different oxygen levels and displays fast decay kinetics after reoxygenation. Additionally, the reporter cells were encapsulated in bulk hydrogels with a variable cell number, to investigate the sensor response in model 3D cell culture applications. The use of hypoxia-reporting cells based on MSCs represents a valuable tool for approaching the genuine in vivo cellular microenvironment and will allow a better understanding of the regenerative potential of AD-MSCs. © 2020 The Authors. Biotechnology and Bioengineering published by Wiley Periodicals LL

How to Assess the Efficacy of Interventions for Actinic Keratosis? A Review with a Focus on Long-Term Results

Actinic keratoses (AK) are common lesions of the skin caused by cumulative sun exposure. Since AK may progress to invasive cutaneous squamous cell carcinoma (cSCC), guidelines uniformly recommend early and consequent treatment. A variety of interventions are available; however, most randomized controlled trials, meta-analyses, and guidelines focus on outcomes that are usually evaluated 8–12 weeks after the end of treatment. Importantly, these assessments can capture the short-term, transient outcomes, but do not allow any conclusions about long-term results to be drawn and do not reflect the probability of transition towards cSCC. Until now, few studies have assessed the long-term results of interventions for AK. Indeed, finding the most appropriate end-point and adjunct time point for determining the long-term results of interventions for AK remains a challenge. Here, we provide an overview of the different ways of measuring the efficacy of AK treatments, such as using recurrence rates or sustained clearance rates, and discuss methodological aspects. Furthermore, we highlight the importance of evidence from post-marketing surveillance trials for the detection of efficacy values and safety signals. Additionally, we emphasize that a follow-up period of 12 months might not be sufficient to reflect the long-term results and stress the urgent need for a longer follow-up period and regular risk-stratified surveillance

Treatment Motivations and Expectations in Patients with Actinic Keratosis: A German-Wide Multicenter, Cross-Sectional Trial

Patient-centered motives and expectations of the treatment of actinic keratoses (AK) have received little attention until now. Hence, we aimed to profile and cluster treatment motivations and expectations among patients with AK in a nationwide multicenter, cross-sectional study including patients from 14 German skin cancer centers. Patients were asked to complete a self-administered questionnaire. Treatment motives and expectations towards AK management were measured on a visual analogue scale from 1–10. Specific patient profiles were investigated with subgroup and correlation analysis. Overall, 403 patients were included. The highest motivation values were obtained for the items “avoid transition to invasive squamous cell carcinoma” (mean ± standard deviation; 8.98 ± 1.46), “AK are considered precancerous lesions” (8.72 ± 1.34) and “treating physician recommends treatment” (8.10 ± 2.37; p < 0.0001). The highest expectation values were observed for the items “effective lesion clearance” (8.36 ± 1.99), “safety” (8.20 ± 2.03) and “treatment-related costs are covered by health insurance” (8.00 ± 2.41; p < 0.0001). Patients aged ≥77 years and those with ≥7 lesions were identified at high risk of not undergoing any treatment due to intrinsic and extrinsic motivation deficits. Heat mapping of correlation analysis revealed four clusters with distinct motivation and expectation profiles. This study provides a patient-based heuristic tool for a personalized treatment decision in patients with AK

German S3 guideline "actinic keratosis and cutaneous squamous cell carcinoma" – long version of the update 2023

Actinic keratosis (AK) are common lesions in light-skinned individuals that can potentially progress to cutaneous squamous cell carcinoma (cSCC). Both conditions may be associated with significant morbidity and constitute a major disease burden, especially among the elderly. To establish an evidence-based framework for clinical decision making, the guideline “actinic keratosis and cutaneous squamous cell carcinoma” was updated and expanded by the topics cutanepus squamous cell carcinoma in situ (Bowen’s disease) and actinic cheilitis. This guideline was developed at the highest evidence level (S3) and is aimed at dermatologists, general practitioners, ear nose and throat specialists, surgeons, oncologists, radiologists and radiation oncologists in hospitals and office-based settings, as well as other medical specialties, policy makers and insurance funds involved in the diagnosis and treatment of patients with AK and cSCC

Hypoxia Onset in Mesenchymal Stem Cell Spheroids: Monitoring With Hypoxia Reporter Cells

The therapeutic and differentiation potential of human mesenchymal stems cells (hMSCs) makes these cells a promising candidate for cellular therapies and tissue engineering. On the path of a successful medical application of hMSC, the cultivation of cells in a three-dimensional (3D) environment was a landmark for the transition from simple two-dimensional (2D) testing platforms to complex systems that mimic physiological in vivo conditions and can improve hMSC curative potential as well as survival after implantation. A 3D arrangement of cells can be mediated by scaffold materials where cells get entrapped in pores, or by the fabrication of spheroids, scaffold-free self-organized cell aggregates that express their own extracellular matrix. Independently from the cultivation method, cells expanded in 3D experience an inhomogeneous microenvironment. Many gradients in nutrient supply, oxygen supply, and waste disposal from one hand mimic in vivo microenvironment, but also put every cell in the 3D construct in a different context. Since oxygen concentration in spheroids is compromised in a size-dependent manner, it is crucial to have a closer insight on the thresholds of hypoxic response in such systems. In this work, we want to improve our understanding of oxygen availability and consequensing hypoxia onset in hMSC spheroids. Therefore, we utilized human adipose tissue-derived MSCs (hAD-MSCs) modified with a genetical sensor construct to reveal (I) the influence of spheroid production methods and (II) hMSCs cell number per spheroid to detect the onset of hypoxia in aggregates. We could demonstrate that not only higher cell numbers of MSCs, but also spheroid formation method plays a critical role in onset of hypoxia