Cultivating Ex Vivo Patient-Derived Glioma Organoids Using a Tissue Chopper.

Glioblastoma, IDH-wild type, CNS WHO grade 4 (GBM) is a primary brain tumor associated with poor patient survival despite aggressive treatment. Developing realistic ex vivo models remain challenging. Patient-derived 3-dimensional organoid (PDO) models offer innovative platforms that capture the phenotypic and molecular heterogeneity of GBM, while preserving key characteristics of the original tumors. However, manual dissection for PDO generation is time-consuming, expensive and can result in a number of irregular and unevenly sized PDOs. This study presents an innovative method for PDO production using an automated tissue chopper. Tumor samples from four GBM and one astrocytoma, IDH-mutant, CNS WHO grade 2 patients were processed manually as well as using the tissue chopper. In the manual approach, the tumor material was dissected using scalpels under microscopic control, while the tissue chopper was employed at three different angles. Following culture on an orbital shaker at 37 °C, morphological changes were evaluated using bright field microscopy, while proliferation (Ki67) and apoptosis (CC3) were assessed by immunofluorescence after 6 weeks. The tissue chopper method reduced almost 70% of the manufacturing time and resulted in a significantly higher PDOs mean count compared to the manually processed tissue from the second week onwards (week 2: 801 vs. 601, P = 0.018; week 3: 1105 vs. 771, P = 0.032; and week 4:1195 vs. 784, P < 0.01). Quality assessment revealed similar rates of tumor-cell apoptosis and proliferation for both manufacturing methods. Therefore, the automated tissue chopper method offers a more efficient approach in terms of time and PDO yield. This method holds promise for drug- or immunotherapy-screening of GBM patients.

Characterization and Optimization of the Tumor Microenvironment in Patient-Derived Organotypic Slices and Organoid Models of Glioblastoma.

While glioblastoma (GBM) is still challenging to treat, novel immunotherapeutic approaches have shown promising effects in preclinical settings. However, their clinical breakthrough is hampered by complex interactions of GBM with the tumor microenvironment (TME). Here, we present an analysis of TME composition in a patient-derived organoid model (PDO) as well as in organotypic slice cultures (OSC). To obtain a more realistic model for immunotherapeutic testing, we introduce an enhanced PDO model. We manufactured PDOs and OSCs from fresh tissue of GBM patients and analyzed the TME. Enhanced PDOs (ePDOs) were obtained via co-culture with PBMCs (peripheral blood mononuclear cells) and compared to normal PDOs (nPDOs) and PT (primary tissue). At first, we showed that TME was not sustained in PDOs after a short time of culture. In contrast, TME was largely maintained in OSCs. Unfortunately, OSCs can only be cultured for up to 9 days. Thus, we enhanced the TME in PDOs by co-culturing PDOs and PBMCs from healthy donors. These cellular TME patterns could be preserved until day 21. The ePDO approach could mirror the interaction of GBM, TME and immunotherapeutic agents and may consequently represent a realistic model for individual immunotherapeutic drug testing in the future.

Rapid response flow cytometric assay for the detection of antibody responses to SARS-CoV-2.

SARS-CoV-2 has emerged as a previously unknown zoonotic coronavirus that spread worldwide causing a serious pandemic. While reliable nucleic acid-based diagnostic assays were rapidly available, only a limited number of validated serological assays were available in the early phase of the pandemic. Here, we evaluated a novel flow cytometric approach to assess spike-specific antibody responses.HEK 293T cells expressing SARS-CoV-2 spike protein in its natural confirmation on the surface were used to detect specific IgG and IgM antibody responses in patient sera by flow cytometry. A soluble angiotensin-converting-enzyme 2 (ACE-2) variant was developed as external standard to quantify spike-specific antibody responses on different assay platforms. Analyses of 201 pre-COVID-19 sera proved a high assay specificity in comparison to commercially available CLIA and ELISA systems, while also revealing the highest sensitivity in specimens from PCR-confirmed SARS-CoV-2-infected patients. The external standard allowed robust quantification of antibody responses among different assay platforms. In conclusion, our newly established flow cytometric assay allows sensitive and quantitative detection of SARS-CoV-2-specific antibodies, which can be easily adopted in different laboratories and does not rely on external supply of assay kits. The flow cytometric assay also provides a blueprint for rapid development of serological tests to other emerging viral infections.