Methods for antibody production
The present invention relates to methods of constructing an integrated artificial immune system that comprises appropriate in vitro cellular and tissue constructs or their equivalents to mimic the normal tissues that interact with vaccines in mammals. The artificial immune system can be used to test the efficacy of vaccine candidates in vitro and thus, is useful to accelerate vaccine development and testing drug and chemical interactions with the immune system.
1. A method for simultaneously producing a plurality of populations of antibodies having different binding specificities, the method comprising:
(a) preparing a plurality of populations of dendritic cells, wherein each population of dendritic cells is matured in a separate culture in the presence of a different exogenous antigen, comprising adding peripheral blood mononuclear cells (PBMCs) to a plurality of separate vaccination site (VS) cultures, wherein the VS cultures are organized in a first multi-well format, wherein each of the VS cultures comprises a first substantially planar matrix, a plurality of cells consisting of endothelial cells and/or epithelial cells attached to the first matrix and an exogenous antigen of interest, wherein the exogenous antigen of interest differs between each VS culture, under conditions promoting maturation of dendritic cell precursors present in the population of PBMCs to dendritic cells; and
(b) transferring each population of dendritic cells of (a) to separate three-dimensional artificial lymphoid tissue equivalent (LTE) cultures, wherein the LTE cultures are organized in a second multi-well format, wherein each of the LTE cultures comprises a second matrix and a plurality of lymphocytes attached to the second matrix, under conditions promoting production of antibodies by the lymphocytes, thereby simultaneously producing a plurality of populations of antibodies having different binding specificities.
2. The method of claim 1 , wherein said plurality of cells forms an endothelial layer, a vascular endothelial layer, an endothelium or a vascular endothelium on one side of said first matrix.
3. The method of claim 1 , wherein said plurality of cells forms an endothelial layer, a vascular endothelial layer, an endothelium or a vascular endothelium on both sides of said first matrix.
4. The method of claim 1 , wherein said plurality of cells forms an endothelial layer on one side of said first matrix and an epithelial layer on the other side of said first matrix.
5. The method of claim 1 , wherein said plurality of cells forms an endothelial layer on one side of said first matrix and an epithelium on the other side of said first matrix.
6. The method of claim 1 , wherein said plurality of cells forms an endothelium on one side of said first matrix and an epithelium on the other side of said first matrix.
7. The method of claim 1 , wherein said plurality of cells forms a vascular endothelium on one side of said first matrix and an epithelial layer on the other side of said first matrix.
8. The method of claim 1 , wherein said plurality of cells forms an endothelium on one side of said first matrix and an epithelial layer on the other side of said first matrix.
9. The method of claim 1 , wherein said plurality of cells forms a vascular endothelium on one side of said first matrix and an epithelium on the other side of said first matrix.
10. The method of claim 1 , wherein said plurality of cells forms a vascular endothelial layer on one side of said first matrix and a lymphatic endothelial layer on the other side of said first matrix.
11. The method of claim 1 , wherein said plurality of cells forms a vascular endothelium on one side of said first matrix and a lymphatic endothelium on the other side of said first matrix.
12. The method of claim 1 , wherein said first matrix comprises a natural biopolymer.
13. The method of claim 12 , wherein said natural biopolymer is selected from the group consisting of xenographic extracellular matrix (ECM) sheet, reconstituted collagen matrix, and chitosan/collagen membrane scaffolds.
14. The method of claim 1 , wherein said first matrix comprises the natural biopolymer bovine type I collagen on a nylon mesh.
15. The method of claim 1 , wherein said first matrix comprises the natural biopolymer bovine type I collagen on a polycarbonate mesh.
16. The method of claim 1 , wherein said second matrix comprises synthetic extracellular matrix (ECM) materials.
17. The method of claim 1 , wherein said second matrix comprises synthetic extracellular matrix materials selected from the group consisting of hydrogels, poly(methyl methacrylate), poly(lactide-co-glycolide), polytetrafluoroethylene, poly(ethylene glycol dimethacrylate) hydrogels (PEGDA or PEGDMA), poly(ethylene oxide), and poly(propylene fumarate-co-ethylene glycol) (PPF-PEG).
18. The method of claim 1 , wherein said second matrix comprises natural ECM material.
19. The method of claim 1 , wherein said second matrix comprises a natural ECM material selected from the group consisting of collagen, hyaluronic acid hydrogels, calf skin gelatin, fibrinogen, thrombin, and decellularized ECM.
20. The method of claim 1 , wherein said plurality of cells in said vaccination site cultures comprises human cells.
21. The method of claim 1 , wherein said plurality of cells in said vaccination site cultures comprise human vascular endothelial cells (HUVECs).
22. The method of claim 1 , wherein said plurality of cells in said vaccination site cultures comprise blood vessel endothelial cells.
23. The method of claim 1 , wherein said plurality of lymphocytes comprises T cells and B cells.
24. The method of claim 1 , wherein said plurality of lymphocytes comprises dendritic cells.
25. The method of claim 1 , wherein said plurality of lymphocytes comprises naive T cells and naive B cells.
26. The method of claim 1 , wherein said plurality of lymphocytes comprises memory T cells and memory B cells.
27. The method of claim 1 , wherein the exogenous antigen of interest is attached to the first matrix.
28. The method of claim 1 , wherein the exogenous antigen of interest is embedded in the first matrix.
29. The method of claim 1 , wherein each of said vaccination site cultures comprises:
a first substantially planar matrix comprising an ECM membrane;
a layer of epithelial cells attached to a first side of the ECM membrane; and
a layer of endothelial cells attached to a second side of the ECM membrane.
30. The method of claim 1 , further comprising isolating antibody-producing B cells from individual LTE cultures of (b) and producing hybridomas from the antibody-producing B cells.
31. The method of claim 1 , further comprising isolating antibodies produce in the separate LTE cultures.
32. The method of claim 1 , wherein the PBMCs in individual VS cultures are from the same donor.
33. The method of claim 1 , wherein the PBMCs added to the plurality of separate VS cultures are from the same donor.
34. The method of claim 1 , wherein the different exogenous antigens may be different formulations of the same antigen or different portions of the same antigen.