Composition and Method for Assessing Regulation of Mast Cells

Abstract

The present invention relates to mast cell cultures that are derived from hematopoietic progenitors and the use thereof. The invention describes a method for generating in-vitro cultures of human mast cells with functional phenotype of connective tissue-type mast cells. By monitoring the levels of chemokines released into the medium, such mast cell cultures can be used as a cell-based assay to assess regulation of mast cell functions and pharmacological activities of tryptase inhibitors.

1 . A mast cell culture product made by the process comprising (a) treating CD34 +  progenitor cells under hypoxic condition in a medium comprising stem cell factor (SCF), interleukin-6 (IL-6) and interleukin-3 (IL-3) to generate an initial-stage cell culture; (b) treating the initial-stage cells under hypoxic condition in a medium comprising SCF, IL-6 and interleukin-9 (IL-9) to form an intermediate stage cell culture; (c) treating the intermediate-stage cells under normoxic condition in a medium comprising SCF and IL-6, thereby generating an immature mast cell culture. 
     
     
         2 . The product of  claim 1  wherein the immature mast cell is further treated in step (d), comprising treating the immature mast cells under normoxic condition in a medium comprising SCF, IL-6 and interleukin-4 (IL-4), thereby generating a mature mast cell culture. 
     
     
         3 . The product of  claims 1  or  2  wherein the CD34 +  progenitor cells are derived from human. 
     
     
         4 . The product of  claims 1  or  2  wherein the CD34 +  progenitor cells are treated under hypoxic condition for about 1 week in step (a). 
     
     
         5 . The product of  claims 1  or  2  wherein the initial-stage cell culture is treated under hypoxic condition for 0.5-2.5 weeks in step (b). 
     
     
         6 . The product of  claim 5  wherein the initial-stage cell culture is treated under hypoxic condition for 1 week in step (b). 
     
     
         7 . The product of  claims 1  or  2  wherein the intermediate-stage cell culture is treated under normoxic condition for 2.5-3.5 weeks in step (c). 
     
     
         8 . The product of  claim 7  wherein the intermediate-stage cell culture is treated under normoxic condition for 3 weeks in step (c). 
     
     
         9 . The product of  claim 2  wherein the immature mast cell culture is treated under normoxic condition for 1 week in step (d). 
     
     
         10 . The product of  claims 1  or  2  wherein the concentration of IL-3 in the medium in step (a) is 0.1-4 ng/ml. 
     
     
         11 . The product of  claim 10  wherein the concentration of IL-3 in the medium in step (a) is 1 ng/ml. 
     
     
         12 . The product of  claims 1  or  2  wherein the concentration of IL-9 in the medium in step (b) is 15 ng/ml. 
     
     
         13 . The product of  claims 1  or  2  wherein the concentration of SCF in the media in steps (a), (b) and (c) is 100-500 ng/ml. 
     
     
         14 . The product of  claim 2  wherein the concentration of SCF in the medium in step (d) is 100-500 ng/ml. 
     
     
         15 . The product of  claims 13  or  14  wherein the concentration of SCF is 200 ng/ml. 
     
     
         16 . The product of  claims 1  or  2  wherein the concentration of IL-6 in the media in steps (a), (b) and (c) is 50-500 ng/ml. 
     
     
         17 . The product of  claim 2  wherein the concentration of IL-6 in the medium in step (d) is 50-500 ng/ml. 
     
     
         18 . The product of  claims 16  or  17  wherein the concentration of IL-6 is 100 ng/ml. 
     
     
         19 . The product of  claim 2  wherein the concentration of IL4 in the medium in step (d) is 10 ng/ml. 
     
     
         20 . A method of culturing mast cells comprising the steps of (a) treating CD34 +  progenitor cells under hypoxic condition for about 1 week in a medium comprising SCF, IL-6 and IL-3 to generate an initial-stage cell culture; (b) treating the initial-stage cell culture under hypoxic condition for about 0.5-2.5 weeks in a medium comprising SCF, IL-6 and IL-9 to form an intermediate stage cell culture; (c) treating the intermediate-stage cell culture under normoxic condition for 2.5-3.5 weeks in a medium comprising SCF and IL-6 to generate an immature mast cell culture. 
     
     
         21 . The method of  claim 20  further comprising the step of (d) treating the immature mast cell under normoxic condition in a medium comprising SCF, IL-6 and IL-4. 
     
     
         22 . The method of  claims 20  or  21  wherein the CD34 +  progenitor cells are derived from human. 
     
     
         23 . The method of  claims 20  or  21  wherein, the initial-stage cell culture is treated under hypoxic condition for 1 week in step (b). 
     
     
         24 . The method of  claims 20  or  21  wherein the intermediate-stage cell culture is treated under normoxic condition for 3 weeks in step (c). 
     
     
         25 . The method of  claims 20  or  21  wherein the concentration of IL-3 in the medium in step (a) is 0.1-4 ng/ml. 
     
     
         26 . The method of  claim 25  wherein the concentration of IL-3 in the medium in step (a) is 1 ng/ml. 
     
     
         27 . The method of  claims 20  or  21  wherein the concentration of IL-9 in the medium in step (b) is 15 ng/ml. 
     
     
         28 . The method of  claims 20  or  21  wherein the concentration of SCF in the media in steps (a), (b) and (c) is 100-500 ng/ml. 
     
     
         29 . The method of  claim 21  where the concentration of SCF in the medium in step (d) is 100-500 ng/ml. 
     
     
         30 . The method of  claims 28  or  29  wherein the concentration of SCF is 200 ng/ml. 
     
     
         31 . The method of  claims 20  or  21  wherein the concentration of IL-6 in the media in steps (a), (b) and (c) is 50-500 ng/ml. 
     
     
         32 . The method of  claim 31  wherein the concentration of IL-6 in the medium in step (d) is 50-500 ng/ml. 
     
     
         33 . The method of  claims 31  or  32  wherein the concentration of IL-6 is 100 ng/ml. 
     
     
         34 . The method of  claim 21  wherein the concentration of IL-4 in the medium in step (d) is 10 ng/ml. 
     
     
         35 . A method of assessing regulation of mast cell functions by measuring the level of MCP-1 wherein the MCP-1 is secreted by mast cells over a period of time in a subject. 
     
     
         36 . The method of  claim 35  wherein the regulation is a dysregulatioin of mast cell functions related to an inflammation-related condition, a disease or a disorder in a subject. 
     
     
         37 . The method of  claim 35  wherein the mast cells are stimulated by an agent selected from the group consisting of anti-immunoglobulin E (IgE) and aggregated immunoglobulin G1 (IgG 1 ). 
     
     
         38 . The method of  claim 35  wherein the level of MCP-1 is measured over 6-22 hours after stimulation. 
     
     
         39 . A method of assessing tryptase-inhibitory activity of an agent comprising the steps of (a) stimulating mast cells in the presence of the agent and; (b) measuring level of MCP-1 secreted by mast cells over a period of time. 
     
     
         40 . The method of  claim 39  wherein the mast cells are stimulated by anti-IgE or aggregated IgG 1 . 
     
     
         41 . The method of  claim 39  wherein the level of MCP-1 secreted by mast cells is measured over 6-22 hours. 
     
     
         42 . A cell culture of functionally mature human mast cells generated from progenitor cells expressing the CD34 antigen comprising said human mast cells showing phenotypic and functional maturity at six weeks in culture. 
     
     
         43 . The cell culture of  claim 42  wherein the cells express both chymase and tryptase. 
     
     
         44 . The cell culture of  claim 42  wherein the cells exhibit functional activation in response to stimulation by compound 48/80 or ionophores. 
     
     
         45 . The cell culture of  claim 42  wherein the cells exhibit functional activation in response to activation of cell surface receptors on the cells. 
     
     
         46 . The cell culture of  claim 45  wherein the cell surface receptors are selected from high-affinity IgE receptors, high-affinity IgG receptors, Toll-like receptors and Substance P receptors.