Methods for Preserving Target Cells
Methods for obtaining and preserving target cells using degradable three dimensional matrices are described.
1 . A method for preserving freshly isolated target cells from a fluid, cell-containing sample, the method comprising:
a) providing a fluid, cell-containing sample;
b) passing the sample through a degradable, three-dimensional matrix, wherein the matrix has a porosity that selectively retains target cells while allowing undesired cells to pass through the matrix; and
c) cryopreserving the matrix and freshly isolated target cells retained by the matrix.
2 . The method of claim 1 , wherein the fluid, cell-containing sample is peripheral blood, umbilical cord blood, bone marrow aspirate, lymph, cerebral spinal fluid, ductal fluid, needle biopsy aspirate, or lipoaspirate.
3 . The method of claim 1 , wherein the matrix comprises one or more of collagen, hyaluronic acid, laminin, chitosan, cellulose, alginate and products thereof.
4 . The method of claim 1 , wherein the matrix comprises a degradable natural or synthetic polymer.
5 . The method of claim 4 , wherein the polymer is selected from one or more of: poly(glycolic acid) (PGA), poly(lactic acid) (PLA), poly(lactic acid)-poly(glycolic acid) (PLGA), polyhydroxybutyrate (PHB), polycaprolactone, polyanhydride, polyorthoester, and poly(amino acid).
6 . The method of claim 1 , wherein the matrix and freshly isolated target cells are cryopreserved with a solution comprising dimethylsulfoxide.
7 . The method of claim 6 , wherein the solution further comprises one or more reagents selected from the group consisting of glycerol, 1,2-propanediol, dextran, ethylene glycol, albumin, polyvinyl pyrolidone, hyaluronic acid, and hydroxyethyl starch.
8 . The method of claim 1 , further comprising recovering the freshly isolated target cells retained by the matrix by substantially degrading the matrix with a degradative reagent for an amount of time sufficient to substantially degrade the matrix.
9 . The method of claim 8 , wherein the degradative reagent is a collagenase, hyaluronidase, or protease.
10 . The method of claim 1 wherein the matrix comprises at least one capture ligand attached thereto, the capture ligand having affinity for a target cell in the sample.
11 . The method of claim 10 , wherein the at least one capture ligand is an antibody or an antigen binding fragment thereof having a binding affinity for CD71, CD36, CD45, glycophorin A, CD35, CD47, CD117, SSEA-4, or CD146.
12 . The method of claim 10 , wherein at least two different capture ligands are attached to the matrix.
13 . The method of claim 10 , wherein at least one capture ligand is a lectin selected from the group consisting of soybean agglutinin (SBA), peanut agglutinin (PNA), Erythrina cristagalli lectin (ECL), Allomyrina dichotoma lectin (Allo A), Viscum album agglutinin (VAA), concanavalin A (Con A), Lens culinaris lectin (LcH), and Pisum sativum agglutin (PSA).
14 . A method for preserving target cells from a fluid, cell-containing sample, the method comprising:
a) providing a fluid, cell-containing sample from a subject;
b) passing the sample through a three-dimensional matrix, the matrix comprising an inner core and an outer layer disposed around the inner core, the inner core comprising a non-degradable substrate, the outer layer composed of a degradable polymer and having a porosity that retains target cells; and
c) cryopreserving the matrix and target cells retained by the matrix.
15 . The method of claim 14 , the method further comprising recovering target cells retained by the matrix by substantially degrading the outer layer of the matrix.
16 . The method of claim 14 , further comprising concentrating the cells isolated from the matrix.
17 . The method of claim 14 wherein the outer layer comprises a capture ligand attached thereto, the capture ligand having affinity for a target cell in the sample.
18 . The method of claim 14 , wherein the inner core comprises a woven or non-woven polypropylene, nylon, or silk mesh.
19 . The method of claim 14 , wherein the fluid, cell-containing sample comprises erythrocytes, and wherein the method comprises removing erythrocytes from the fluid, cell-containing sample to produce an erythrocyte depleted sample before passing the erythrocyte depleted sample through the three-dimensional matrix.
20 . The method of claim 14 , wherein the fluid, cell-containing sample is first centrifuged to fractionate cells according to their respective specific gravity before passing one or more of the cell fractions through the three dimensional matrix.