Methods of culturing and characterizing antibody secreting cells
This disclosure relates to methods of culturing and characterizing antibody secreting cells. In certain embodiments, this disclosure relates to methods of isolating antibody secreting cells, e.g., long lived plasma cells, replicating the isolated cells in growth media disclosed herein, and determining the nucleic acids sequences in the cells that encode the produced antibodies.
1. A method of sequencing a nucleic acid that encodes an antibody that specifically bind to an antigen comprising
isolating antibody secreting cells from a sample, providing separated single antibody secreting cells in a plurality of separate areas;
mixing the separated single antibody secreting cells in the plurality of separated areas with secretions of allogeneic mesenchymal stromal/stem cells and exogenously added A-proliferation-inducing ligand (APRIL) under conditions such that separated single antibody secreting cells replicate providing replicated homogenous antibody secreting cells in separate areas;
identifying replicated homogenous antibody secreting cells that produce antibodies that specifically bind to an antigen; and
sequencing a nucleic acid that encodes the antibody in the replicated homogenous antibody secreting cells that bind the antigen.
2. The method of claim 1 wherein mixing the separated single antibody secreting cells in the plurality of areas with secretions of allogeneic mesenchymal stromal/stem cells and exogenously added A-proliferation-inducing ligand (APRIL) is under hypoxic conditions.
3. The method of claim 1 , wherein the sample is blood, bone marrow, or product derived therefrom.
4. The method of claim 1 wherein isolating antibody secreting cells is accomplished by fluorescence activated cell sorting.
5. The method of claim 1 wherein the antibody secreting cells have a cell surface profile of CD19(−), CD38(hi), and CD138(+).
6. The method of claim 1 wherein the antibody secreting cells have a cell surface profile of CD19+, CD27hi, and CD38hi.
7. The method of claim 1 , wherein sequencing is the variable region of the heavy chain of the antibody and/or the variable region of the light chain of the antibody produced by the homogenous antibody secreting cells.