Method and device for target cell separation
The invention relates to a method for separating viable target cells from a sample comprising the steps of contacting a sample comprising a suspension of viable target cells that display a molecule on the cell surface with non-porous microparticles that have a density of about 1.45 g/cm 3 or greater, a diameter of about 10 μm to 200 μm, and a capture ligand covalently immobilized to the microparticle surface that is capable of specifically binding to said molecule; incubating said sample without substantial agitation to form a target cell/microparticle complex; separating non-bound substances in said sample from said target cell/microparticle complex by washing said non-bound substances through a filter while retaining said target cell/microparticle complex; mechanically dissociating said target cell/microparticle complex and eluting said viable target cells through said filter while retaining said microparticles with said capture ligand covalently immobilized to the microparticle surface as well as a cartridge, kit-of-parts, an apparatus configured to be used in the method and a medicament comprising viable target cells obtainable by the method.
1 . A method for separating viable target cells from a sample comprising the steps of:
a) contacting a sample comprising a suspension of viable target cells that display a molecule on the cell surface with silicon dioxide (SiO 2 ) microparticles that have a density of about 1.45 g/cm 3 or greater, a diameter of about 10 μm to 200 μm, and a capture ligand covalently immobilized to the microparticle surface that is capable of specifically binding to said molecule;
b) incubating said sample without agitation to form a target cell/microparticle complex;
c) separating non-bound substances in said sample from said target cell/microparticle complex by washing said non-bound substances through a filter at a flow rate of 0.2 to 0.5 mm/s while retaining said target cell/microparticle complex; and
d) mechanically dissociating said target cell/microparticle complex and eluting said viable target cells through said filter to yield an eluted viable target cell suspension, while retaining said microparticles with said capture ligand covalently immobilized to the microparticle surface.
2 . The method according to claim 1 , wherein the target cells are suitable for cell therapy.
3 . The method according to claim 1 , wherein the capture ligand specifically binds to said molecule at the surface of said target cell with a dissociation constant between 10-5 and 10-12.
4 . The method according to claim 1 , wherein the capture ligand is selected from the group consisting of an antibody, a FabFC 2 , a Fab, a Fv, a Fd, a F(ab′) 2 , an Fv fragment containing only the light and heavy chain variable regions, a Fab or F(ab′) 2 fragment containing the variable regions and parts of the constant regions, a single-chain antibody, a scFv, a CDR-grafted antibody, a dAb and er a nanobody.
5 . The method according to claim 1 , wherein said cell surface molecule is selected from the group consisting of human CD2, CD3, CD4, CD8, CD11a, CD11b, CD14, CD15, CD16, CD19, CD20, CD22, CD24, CD25, CD27, CD30, CD31, CD34, CD38, CD43, CD45, CD48, CD56, CD61, CD73, CD90, CD91, CD105, CD114, CD117, CD140b, CD150, CD182, CD184, CD271, CDCP1, GD2, GPR4, Sca-1, and STRO-1.
6 . The method according to claim 1 , wherein the method further comprises obtaining a medicament comprising a viable target cell suspension of the eluted viable target cells, wherein the eluted viable target cell suspension is free from capture ligand.
7 . The method according to claim 1 , wherein the target cell is selected from the group consisting of human granulocytes, T lymphocytes, monocytes, T regulatory cells, T helper cells, cytotoxic T cells, B lymphocytes, tumour infiltrating lymphocytes, thrombocytes, natural killer cells, hematopoietic stem cells, progenitor cells, mesenchymal/stromal stem cells, hair follicle stem cells, cardiac stem cells, multipotent muscle cells, neural stem cells, hepatic stem cells, dental pulp cells, periodontal ligament cells, retinal pigment epithelial cells, adipose-derived stem and progenitor cells, pluripotent stem cells, embryonic stem cells, induced pluripotent stem cells, Car-T cells, Microvascular endothelial cells (MVEC), primary epithelial cells, keratinocytes, cervical epithelial cells, bronchial epithelial cells, tracheal epithelial cells, kidney epithelial cells, retinal epithelial cells, fibroblast cells, fibroblast cells from heart, fibroblast cells from liver, fibroblast cells from kidney, fibroblast cells from colon, fibroblast cells from intestine, fibroblast cells from esophagus, fibroblast cells from stomach, fibroblast cells from neural tissue derived from the brain and spinal cord, fibroblast cells from lung, fibroblast cells from vascular tissue derived from artery, fibroblast cells from vascular tissue derived from vein, fibroblast cells from vascular tissue derived from capillary, fibroblast cells from lymphoid tissue derived from lymph gland, fibroblast cells from lymphoid tissue derived from adenoid, fibroblast cells from lymphoid tissue derived from tonsil, fibroblast cells from lymphoid tissue derived from bone marrow, fibroblast cells from lymphoid tissue derived from blood, fibroblast cells from spleen, muscle cells, pancreatic cells, cardiac cells and cells established from such primary cells.
8 . The method according to claim 1 , wherein the target cell is selected from the group consisting of monocytes, T lymphocytes, B lymphocytes, CAR-T cells and stem cells.
9 . The method according to claim 1 , wherein said sample is selected from the group consisting of human whole blood, apheresis, bone marrow aspirate, biopsy, liquified tissue, cell culture, bioreactor culture, tumour cells, and single cell suspension.
10 . The method according to claim 1 , wherein the eluted viable target cell suspension is free from capture ligand that specifically binds to a molecule that is displayed on the target cell surface.
11 . The method according to claim 1 , wherein said silicon dioxide (SiO 2 ) microparticles have a density of about 1.95 g/cm 3 or more.
12 . The method according to claim 1 , wherein said silicon dioxide (SiO 2 ) microparticles have a diameter of about 35 μm to 50 μm.