Monitoring

The online monitoring of tissue morphogenesis and organoids in perfusion bioreactor culture is limited to a small set ofparameters. They need to be described as "black boxes" during the culture period, and the readout is defined by histological endpoints. Process control is based on online-sensing of oxygen and pH, and samples of culture supernatant are periodically harvested (at 12 h and 24 h) to this purpose. New approaches of miniaturized fluorescence-based online sensing for

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pO2, pCO2 and pH can be implemented [113, 114]. In perfusion culture, the pH and defined levels of dissolved oxygen, metabolites and factors in the PCS can be adjusted by media and gas exchange.

The monitoring of cytokines provides insight into cellular immunological processes. Successful antigen presentation leads to increased TNF-a concentrations, while the induced T-cell reaction is described by IFN-y release. Shifts in T-lymphocyte populations into humoral or cellular inductors are better described by a set of cytokines (the cytokine panel). A cellular immune response (TH1-response) is detected by increased levels of IL-2, IL-12 and IFN-y or TNF-a. In contrast, IL-4, IL-5, IL-6, IL-10 and IL-13 describe a humoral response (TH2-response) [107, 108].

Tissue formation of the generated organoids can be characterized after harvest and histological preparation. Histochemical and immune-histological staining of the matrix sheets allows investigations to be made of the tissue formation and cellular composition. To prove the development of germinal centers (GC) [106] as key structures for antibody maturation in vivo, hematoxylin and eosin (H&E) staining or May-Gruenwald-Giemsa-staining can be used. The immune-specific detection of cell surface markers can identify lymphocytes (CD3, CD4, CD19, CD20), and also verify the segregation of lymphocytes into T- and B-cell areas in the matrix. Ki-67 or BrdU-incorporation is indicative of cell proliferation, while key to the scheme is the histological confirmation of secreted or membrane-bound IgM/IgG and their spatial distribution.

Living cells can be prepared following the disintegration of organoids, and also for further cultivation, cell fusion and fluorescence-activated cell sorting (FACS). DNA and RNA preparations can be obtained by using microdissection technology, while new approaches in live cell-imaging technology, multiphoton microscopy and microtomography each allow the monitoring and characterization of cell migration, homing proliferation [115, 116], and tissue formation in situ [117].

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