This project focuses on the proof-of-concept development of a separation construct to isolate antigen-specific CD4 T cells using a buoyant lipid microparticle (microbubble) reagent attaching major histocompatibility complex class II (MHCII) molecules. These cells are implicated in the progression of cardiovascular disease. The project relied critically on preexisting reagents produced by collaborators: microbubbles and biotinylated recombinant MHCII monomers.
Current cell separation methods rely primarily on either magnetic separation or flow-cytometry based sorting. The method presented here provides the basis for a new avenue of investigation using a principally different separation scheme based on buoyancy. It is intended to be extensible to the separation of antigen-specific T cells in other contexts and was originally intended to be a removable separation reagent (unsuccessful). The ultimate goal is the development of a separation system which may be used to further clinical work or research relying on cell isolation.
Here, the buoyancy-based method is first used to perform bulk B and CD4 T cell separations. These are common lab procedures for which many commercial kits are available, which made them an ideal testbed for initial study. Flow cytometric analysis and confocal microscopy were performed to confirm successful cell enrichment.
The separation system was then further developed to separate antigen-specific CD4 T cells reactive to the endogenous murine peptide P6 (TGAYSNASSTESASY), relevant in the study of atherosclerosis. Flow cytometric and microscopic confirmation of successful separation was performed in this section as well.
Several devices for centrifugation were designed and constructed in the course of this project. These attempted to create a pendant droplet between the buoyant and non-buoyant fractions in a single column of liquid formed by centrifugation, allowing for these layers to be completely sequestered from one another. Though these devices were unsuccessful, they merit further study. Another device, a centrifuge adapter, was created as an all-purpose experimental aid to hold and centrifuge all combinations of reagent tubes used in the cell separation protocol. Computer graphics animations and diagrams of various facets of the project were also made, including molecular-scale models of separation constructs derived from Protein Data Bank (PDB) structural data.