Development of a lymph node system for in vitro immunization

Monoclonal antibodies significantly impact biochemistry and medicine, representing crucial tools for diagnostics, research, and therapeutic applications. Traditional monoclonal antibody production methods rely on animal immunization, which raises ethical questions and restricts the choice of antigens. In vitro immunization presents a promising alternative for monoclonal antibody production, tackling these concerns. This method replicates an organism's natural immune response within a controlled laboratory setting, bypassing the need for animal immunization and thus aligning the production process with ethical standards. Reproducing these complex processes in vitro is challenging, and researchers have developed numerous protocols over the last decades. However, due to reliability and efficiency issues, in vitro immunization has yet to be a standard technique for antibody production. The current work focused on introducing extracellular matrix and three-dimensional cell culture techniques to the in vitro immunization procedure to achieve more lifelike conditions that might result in a robust immunization process. During the work alginic acid or alginate, a polysaccharide derived from brown algae that gels upon crosslinking with divalent cations, did not facilitate adequate cell motility, which is crucial for cell interaction and a successful immune reaction. Therefore, alginate had to be rejected as a possible matrix for in vitro immunization. The second matrix material tested was Geltrex, a basal membrane protein mixture secreted by murine Engelbreth-Holm-Swarm tumor cells that forms a gel upon warming above 15 °C. Geltrex proved suitable for long-term cell culture. Secreted antibodies from Geltrex-encapsulated cells were detectable in the culture supernatant—an essential indicator for the progress of an in vitro immunization. However, residual murine immunoglobulins in the Geltrex mixture resulted in high IgG and IgM background signals during unspecific ELISA testing. Antigen-specific ELISA testing was not impaired. Unlike alginate, Geltrex allowed for excellent cell motility of dendritic cells, T cells, and B cells. Cell proliferation measurements of Geltrex-embedded T cells confirmed the interaction and activation of immune cells in the matrix. The comparison of two cell recovery methods revealed that the proprietary non-enzymatic "Cell Recovery Solution" (CRS) from Corning is favorable to the enzymatic matrix digestion using dispase, as dispase also cleaves cell surface receptors and leads to cell damage and cell death. The final part of the work involved creating a simple, efficient, and scalable approach for high-density cell cultivation using a 3D-printed compartment in a larger cell culture plate. Using this setup, several in vitro immunizations in Geltrex were performed, resulting in antigen-specific hybridoma cell lines against chicken ovalbumin and the SARS-CoV-2 nucleocapsid protein. One of the generated monoclonal IgG antibodies was purified, identified as subclass IgG2b, and tested for specificity. The successful integration of Geltrex as an extracellular matrix to in vitro immunization protocols enables more lifelike cell interactions and immune reactions. Combined with the described three-dimensional cell culture setup, an efficient and scalable alternative to conventional antibody production techniques was established to reduce animal suffering and expand the possibilities for antibody fabrication.