This research aimed to enhance the stability of Cordyceps fumosorosea spores (JKI-BI-1496) using spray-drying with water-soluble sunlight protectants. Submerged spores (ss) of JKI-BI-1496 were tested for survival after spray-drying. Humic acid Na, humic acid K, lignin, black tea, cocoa, coffee, skimmed milk, green tea, and calcofluor white were evaluated for integration into the spray-drying process. Absorbance profiles (280 - 800 nm) were examined. Spray-drying was conducted at 65 °C inlet, 48 °C outlet, and a maximal product temperature of 35°C. Skimmed milk powder was used as a protective agent, forming a suspension with 5% skimmed milk, 5 × 106 ss mL-1 spores, and 5% protectants. Germination rates were assessed after 1 - 4 hours of simulated solar radiation. Spores coated with black tea showed the highest germination rate (48.3%) after 4 hours, compared to < 18% with other protectants. Calcofluor white-coated spores exhibited 44.9% germination after 12 weeks at 6 °C. Black tea and calcofluor white, enhance the stability of JKI-BI-1496 spores, indicating their potential for outdoor agricultural applications.

3D cell culture has been proven to be superior to 2D culture approaches in numerous applications. Some of the advantages of threedimensional culture systems comprise the ability of the cells to form cell-cell-contacts, to establish a cellular polarity, and the formation of oxygen and nutrient gradients, among others. Moreover, co-cultures in 3D are much more physiologically relevant compared to their two-dimensional counterparts. With this in mind, we developed an in vitro-model of the leukemic stem cell niche as a test platform for third generation α-CD19 chimeric antigen receptor-T-cell (CAR-T) potency with the CAR-T-cells being one member of the so-called class of living drugs. This is of clinical relevance since leukemia initiating cells (LIC) in the bone marrow hide from circulating immune cells and thus are made responsible for the primary disease and patient relapses. The in vitro-model is based on microcavity arrays that have proven to support a plethora of cells with regard to growth, differentiation, and maintenance of stem cell characteristics. In this article, we characterize the third generation CAR-T-cell response to a CD19 positive leukemic cell line (NALM-6) in co-culture with mesenchymal stromal cells as a leukemic niche surrogate. For this, confocal microscopy data were analyzed with respect to leukemic cell count prior and after CAR-T-cell infusion into a bioreactor system as a potency assessment, distribution of leukemic cells within the niche, cellular mobility, Caspase 3/7-, and Interferon ϒ-release. We could show that this 3D model of the leukemic bone marrow niche might be of use to assess CAR-T-cell potency in a bone-marrow-like environment and thus adds additional information to classical assays such as the 51Cr-release assay of suspension cells.

Keywords: Microcavity Arrays; Leukemic Niche; Living Drugs; Potency Test

Abbreviations: CAR-T cell: Chimeric Antigen Receptor T-cell; PBS+/+: Phosphate Buffered Saline solution with Calcium and Magnesium; PBS-/-: Phosphate Buffered Saline without Calcium and Magnesium; pMSC: primary Mesenchymal Stromal Cells; LIC: Leukemia Initiating Cells.