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Publication Number
US-9663762-B2
Publication Date
2017-05-30
Expiration Date
Abstract
Methods for recovering target cells using degradable three dimensional matrices are described.
Core Innovation
The invention provides a method for obtaining target cells from a fluid, cell-containing sample by passing the sample through a degradable matrix filter mounted as an insert in a conical tube or filter unit. The matrix filter has a three dimensional structure fabricated of an enzyme degradable biopolymer and a porosity configured to physically retain target cells based on a size of the target cells while allowing passage of smaller non-target cells, including erythrocytes, through the matrix filter.
After passing the sample, the filter is removed from the conical tube or filter unit. Target cells retained within the matrix filter are recovered by enzymatically degrading the matrix filter such that at least 50% of the three dimensional structure of the matrix filter is enzymatically degraded, such that the three dimensional structure is no longer able to physically retain the target cells. The recovered target cells are then obtained from the enzymatically degraded matrix filter by centrifugation.
The disclosed approach is further characterized by defining target cells and by using specified biological fluids or tissue aspirates as the fluid, cell-containing sample. The degradable matrix filter can include enzyme degradable biopolymer components such as collagen, hyaluronic acid, laminin, chitosan, cellulose, and alginate, and their products. The matrix filter can also be provided with capture ligands attached to the filter, including antibodies or fragments and lectins.
Claims Coverage
The document includes one independent claim describing the full method for obtaining target cells, with multiple dependent claims that refine inventive features relating to sample type, target cell type, matrix composition, degradative enzyme constraints, and capture ligands.
Enzyme-degradable matrix filter in conical tube for size-based retention
Passing a fluid, cell-containing sample through a degradable matrix filter mounted as an insert in a conical tube or filter unit, where the filter has a three dimensional structure fabricated of an enzyme degradable biopolymer and a porosity that physically retains target cells based on a size of the target cells while allowing passage of smaller non-target cells, including erythrocytes.
Enzymatic degradation releasing physically retained target cells
Removing the filter from the conical tube or filter unit and recovering target cells retained within the matrix filter by enzymatically degrading the matrix filter such that at least 50% of the three dimensional structure of the matrix filter is enzymatically degraded, such that the three dimensional structure is no longer able to physically retain the target cells.
Centrifugation recovery from enzymatically degraded matrix filter
Recovering the target cells from the enzymatically degraded matrix filter by centrifugation.
Specified target cell and sample types
Further defining the target cells and further defining the fluid, cell-containing sample as one of specified biological fluids or tissue aspirates.
Degradable matrix biopolymer composition
Employing a matrix that includes one or more of collagen, hyaluronic acid, laminin, chitosan, cellulose, alginate and their products.
Capture ligand functionalized matrix filter
Using a matrix filter that has at least one capture ligand attached to it before the sample is passed through the filter.
Named lectin capture ligands
Where at least one capture ligand is a lectin chosen from SBA, PNA, ECL, Allo A, VAA, Con A, LcH, or PSA.
Overall claim coverage centers on a method combining a degradable enzyme-degradable three dimensional matrix filter with porosity-based physical retention of target cells, followed by enzymatic degradation of at least 50% of the matrix structure to release the retained cells, with recovery by centrifugation. Dependent claims further specify target cells and sample types, matrix composition, degradative enzyme constraints, and optional capture ligands including antibody-related capture ligands and named lectins.
Stated Advantages
Physically retains target cells based on target cell size while allowing passage of smaller non-target cells, including erythrocytes, through the matrix filter.
Enzymatic degradation of the matrix filter releases retained target cells by rendering the three dimensional structure no longer able to physically retain the target cells.
Recovery of target cells from the enzymatically degraded matrix filter by centrifugation.
Documented Applications
Processing fluid, cell-containing samples that are specified biological fluids or tissue aspirates, including peripheral blood, umbilical blood, bone marrow aspirate, lymph, cerebral spinal fluid, ductal fluid, needle biopsy aspirate, and lipoaspirate.
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