US-12624338-B2 - Method and device for target cell separation

US12624338B2US 12624338 B2US12624338 B2US 12624338B2US-12624338-B2

Abstract

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.

Inventors

Luka FAJS
Laura Vuga
Katarina Katić
Monika Primon
Andrea Cardelli

Assignees

BIO-RECELL LTD.

Dates

Publication Date: 20260512
Application Date: 20230706
Priority Date: 20220428

Claims (12)

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.

Description

1. TECHNICAL FIELD The present invention is in the field of cell separation technology and pertains particularly to methods, cartridges and apparatus for targeted cell capture, separation and isolation. 2. BACKGROUND OF THE INVENTION Cell separation and targeted cell isolation from complex biological matrices such as tissues, blood, and other samples are crucial in several industrial and research processes. At its core, cell separation requires selective enrichment of target cells based on their physical, chemical, or biological properties. One of the main goals of targeted cell isolation is to produce viable cells that retain their initial biological properties as seen in the original tissue or source. This is especially important in cell and gene therapies, where cell health and metabolism are crucial to the success of such therapies. Examples of such cell and gene therapies include hematopoietic stem cell transplant for the treatment of leukaemia or solid cancers. Cells that are effective in the treatment (such as nucleated cells, including hematopoietic stem cells) are separated from bone marrow or peripheral blood by removing red blood cells and then administered to patients. Umbilical cord blood banking requires cells to be cryopreserved before use, and red blood cells are removed to prevent their haemolysis, which may occur during cryopreservation. In addition, transplantation of cell fractions of bone marrow, umbilical cord blood, or peripheral blood that are rich in stem cells such as mesenchymal stem cells, hematopoietic stem cells, and endothelial progenitor cells have been used in the treatment of ischemic diseases, such as cerebral infarction, myocardial infarction, and ischemia to promote angiogenesis or nerve regeneration. Furthermore, granulocytes can cause unwanted side effects, such as inflammation, that may reduce a therapeutic effect and it may be desirable to have them removed from the cell therapy product. Cell separation is also critical in manufacturing cell therapies such as CAR-T cells, autologous and heterologous stem cell treatment, and other novel therapeutic approaches. Sometimes, further processing of the isolated cells is required, both in industrial and research areas. The success and effectiveness of these processes are directly affected by the quality and purity of the isolated cell suspensions. Residual components such as nanoparticles, antibodies, and buffers can interfere with the downstream processes (i.e. cell expansion, gene editing) and should be excluded from the end product if possible. The same is true for clinical use of isolated cells in autologous and heterogeneous transplantation treatments: isolated cells that are contaminated with residual buffers and antibodies can negatively impact the outcome of the treatment. Furthermore, clinical use of these contaminated cells is not allowed in several countries due to regulatory constraints. This limits the access of cell therapy. Current technologies that are available on the market utilize different means of cell separation and enrichment and mostly fall into the following categories: 1) Magnetic particle separation: this method uses magnetic nanoparticles, functionalized with cell-specific antibodies, that capture target cells when passed through a magnetic source. Once the cells bind to the magnetic particles, the magnetic field retains the magnetic particles, and non-target cells are washed off. Once the magnetic field is released, so are the cells. Magnetic particles are typically nanoparticles of iron oxides, such as magnetite (Fe3O4), ranging in size from 1 to 100 nanometers, which give them superparamagnetic properties. Superparamagnetic particles are different to more common ferromagnets in that they exhibit magnetic behaviour only in the presence of an external magnetic field. This property is dependent on the small size of the particles, and enables them to be separated in suspension, along with anything they are bound to. Since the nanoparticles are often smaller or have a similar diameter as the diameter of cells, it is not possible to separate the cells and particles by size, thus the cells that are bound to the nanoparticles are separated by magnets. The cells can be eluted from the nanoparticles with the use of buffers that can highly influence cell viability, and afterwards the magnetic nanoparticles are separated from the cell solution with the use of a magnetic field. One drawback of this method is that some residual magnetic nanoparticles could remain in the final cell solution. This can happen due to the loss of the magnetic properties of the particles, making it hard to ensure that the removal of magnetic particles from the solution of target cells is performed thoroughly. Furthermore, this method requires bulky and expensive equipment to ensure magnetic separation and requires significant hands-on time by the operator (see for example EP3037171A1, U51011997082, CA2854240A1, EP0819250A1, EP0760