Production of cultured human mast cells and basophils for high throughput small molecule drug discovery

Abstract

Provided are methods for producing and screening proliferated populations of CD34-negative progenitor cells, mucosal mast cells, connective tissue-type mast cells and basophil cells. The methods generate uniform proliferated populations of cells. The proliferated populations contain a uniform population of a size suitable for use in high throughput screening methods, for example, screening for agents that alter exocytosis. The invention includes screening the proliferated populations with at least one candidate bioactive agent, and evaluating the cells to detect a cell with an altered phenotype. The invention also includes isolating a candidate bioactive agent that causes the altered phenotype. Additionally, cells formed according to the described methods are also encompassed by the invention.

Description

CROSS REFERENCE TO RELATED APPLICATIONS [0001] This application is a continuation of U.S. application Ser. No. 10 / 053,355, filed Nov. 8, 2001, issued as U.S. Pat. No. 7,070,996, incorporated herein by reference, which claims the benefit under 35 U.S.C. § 119(e) of U.S. provisional application Ser. No. 60 / 316,723, filed Aug. 31, 2001.FIELD OF THE INVENTION [0002] The invention relates to methods and compositions for producing a large uniform population of cultured mast cells and basophils cells, and mast and basophil progenitor cell populations, preferably human. The cell populations are suitable for use in high throughput screening methods. BACKGROUND OF THE INVENTION [0003] At least two types of mast cells are known. These include mast cells of the airway or lung and intestinal mucosa (“mucosal,” or MCT), and mast cells of the connective tissue including skin, lymph nodes and intestinal submucosa (MCTC). The majority of airway mast cells contain tryptase but lack chymase and carbox...

AI Technical Summary

Benefits of technology

The present invention provides methods for producing a population of CD34-negative cells that can be contacted with cytokines and growth factors to produce mucosal mast cells, connective tissue-type mast cells, or basophil cells. The methods involve contacting CD34-positive cells with a flt-3 ligand and stem cell factor to generate CD34-negative progenitor cells, which are then contacted with stem cell factor and IL-6, IL-4, or IL-3 to form the desired mast cell populations. The CD34-positive cells can be human cells, and the cytokines and growth factors can also be human. The invention also includes methods for screening the mast cells with a library of candidate bioactive agents to identify the agents responsible for an altered phenotype. The technical effects of the invention include the ability to produce and screen for mast cells with specific phenotypes, which can be useful in various applications such as research and treatment of mast cell-related diseases.

Problems solved by technology

Therapy for allergy remains limited to blocking the mediators released by mast cells (anti-histamines), non-specific anti-inflammatory agents such as steroids and mast cell stabilizers which are only marginally effective at limiting the symtomatology of allergy.

Historically, high throughput screening for small molecule agents that effect mast cell activation has been limited by the inability of researchers to reproducibly generate or obtain large enough numbers of a single uniform population of mast cells necessary for conducting large scale screens.

It has been very difficult to establish large numbers of these cells in culture due to their terminally differentiated character and the fact that they are frequently localized in specialized deep-tissue microenvironments.

As such, purification of mature mast cells from a single donor is labor intensive and typically yields numbers of cells which are far too low for large scale small-molecule drug discovery or for traditional genetic screening.

These methods also suffered from poor reproducibility by independent groups.

In general, reports from these groups indicated that prior methods have produced variable results and have yielded relatively small mast cell populations unsuitable for high throughput screening.

However, the expanded cells of this report were not characterized phenotypically, and fully differentiated, functional mucosal mast cells were not described.

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