Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Patent
1996-06-26
1998-06-09
Ketter, James
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
435 30, 4352872, 4352873, 4352974, 4353081, G01N 3353, C12Q 124
Patent
active
057631948
DESCRIPTION:
BRIEF SUMMARY
TECHNICAL FIELD
The present invention relates to a method and apparatus for separation of cells. The apparatus includes a semi-permeable substrate to which a ligand is attached to its surface which is adapted to bind a desired cell type. The invention also relates to cells that are separated by the method or apparatus of the invention.
BACKGROUND ART
Separation of specific cells from a mixed cell population is important for cell biological and immunological studies and for use in medical cell therapy. Small-scale ligand based separation techniques (less than 10.sup.9 cells) which include fluorescent activated cell droplet sorting and cell panning are unsuitable for medical therapies. Non-ligand based separation techniques such as synthetic fibre leucocyte filters and counter-flow elutriation are not selective or adaptive for separation of specific cell subtypes. Current large-scale ligand based separation techniques such as column chromatography, magnetic bead or microsphere adsorption cannot separate easily and quickly purified cell subtypes for clinical therapies.
Cell depletion techniques for clinical applications have been adequate for large-scale removal of pathogenic cell sub-types, while cell enrichment and expansion culture technologies still require further development. Cell affinity separations are based on the selective absorption of cell phenotype using antibody, lectins, or other moieties specific for cell surface markers. Affinity techniques include plate panning, column chromatography, and magnetic bead or micro-particle absorption. Depletion of cell subsets (>1,000-fold) is possible using magnetic beads. Recovery of adherence cells involves the use of mechanical agitation and may require proteolytic enzymes. High gradient magnetic cell separation (MACS) can enrich cell populations by greater than 100-fold. Magnetic microparticles (<80 nm), which remain attached to the positive cell population, do not interfere with proliferation assays or flow cytometry. Affinity methods offer the selectivity of monoclonal antibody based separations and are required for large-scale clinical applications.
Large-scale affinity separation techniques (>10.sup.9 cells) have played an important role in the development of bone marrow transplant and adoptive cellular immunotherapy. If there is homogeneous expression of tumour markers, then magnetic bead absorption can purge tumour cells from autologous grafts. Enrichment strategies are useful for the purification of autologous haemopoietic progenitor cells using the CD34 marker or collection of lymphoid subsets for adoptive immunotherapy. About 1% of a bone marrow graft is CD34 positive. Purities of CD34 positive cells exceeding 90% require greater than 900-fold enrichment factors. Work has therefore concentrated on improving the efficiency of cell enrichment using affinity cell separation.
A method for the separation of cells from a mixed population of cells has been reported by Bigalow et al 1989, Journal of Immunological Methods 117: 289-293. These authors have reported the development of a hybrid of two separation methods, cellular adhesion chromatography (AC) and field-flow fractionation (FFF) that achieves effective separation of rat mesenteric B and T lymphocytes. This method combines the selective adhesion of AC and the control displacement forces of FFF, it also yields quantitative estimates of the binding forces of B and T lymphocytes to the adhesion surface of the system. This method uses an apparatus comprising two parallel glass plates and utilises the different binding affinities to these plates by different cell types. This method has a major problem in that it utilises the inherent binding properties of cells to the particular glass surfaces and that it cannot be scaled up to separate a large number of cells.
The present inventors have developed a method for the separation of a desired cell type from a population of cells that has the ability to select any given cell type and also may be scaled up for use in clinical applications.
DIS
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Milthorpe Bruce
Nordon Robert E.
Schindhelm Klaus
Slowiaczek Peter R.
Ketter James
Unisearch Limited
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