Epitope preservation

Abstract

The present invention provides methods of preserving epitopes presented on the surface of cells such as immune cells. The invention also provides methods of transporting cells presenting surface presented epitopes, surface presented binding proteins, surface presented binding protein-ligand complexes and / or surface presented ligands and methods of fulfilling requests and orders for cells presenting surface epitopes. Also provided is the use of a reversibly cross-linked hydrogel to preserve surface presented epitopes of cells. Cells may be in or from a whole blood sample.

Description

[0001] EPITOPE PRESERVATION

[0002] The present invention provides methods of preserving epitopes presented on the surface of cells such as immune cells. The invention also provides methods of transporting cells presenting surface presented epitopes, surface presented binding proteins, surface presented binding protein-ligand complexes and / or surface presented ligands and methods of fulfilling requests and orders for cells presenting surface epitopes. Also provided is the use of a reversibly cross-linked hydrogel to preserve surface presented epitopes of cells. Cells may be in or from a whole blood sample.

[0003] BACKGROUND

[0004] In biological and biochemical settings there is often a need to collect and preserve cells for analysis by various methods. Collected and preserved cells may be analysed for surface presented epitopes (such as antigens and / or markers) in order to characterise, quantify, select and / or separate cells in a sample (for example, in a whole blood sample). Analysis of surface epitopes may also provide diagnostic information. For example, detection of disease surface epitopes (i.e. displayed or presented as part of a disease associated antigen) may indicate the presence or absence of a disease or condition. Surface markers, such as surface epitopes, may also provide information as to the quality of cells. For example, surfaces epitopes may help determine the viability of a cell or population of cells for use in treatments or methods such as immunotherapy.

[0005] Cell surface markers or intracellular markers may be detected using flow cytometry. Flow cytometry uses fluorophore-conjugated antibodies that recognize specific epitopes on proteins to differentiate types of cells. Analysis of intracellular markers involves staining protocols which require fixation of cells. Fixation of cells is typically achieved by formaldehyde or paraformaldehyde. See for example, US Publication 2015 / 0010923.

[0006] Clinical flow cytometry utilizes antibodies to recognize epitopes exposed on the surface of the cells through extracellular or surface staining. Antibodies are added to a sample such as whole blood, allowed to bind, and then the excess unbound antibodies are removed by washing in a buffer. The red blood cells may be removed via a chemical lysing step and the remaining white blood cells may then be analysed using a flow cytometer. A fixation step is often added after the staining and lysing steps as this stabilizes the samples so that they can be tested at a later time. Fixation of the cells is always after the extracellular staining is complete as it is well known that formaldehyde alters protein structure and disrupts some epitopes that will be bound by the detection antibodies. Staining after formaldehyde fixation is generally dimmer due to decreased epitope availability. Although methods for preparing biological samples for flow cytometry analysis have been employed reliably for many years, see for example U.S. Patent No. 6,977,156 and U.S. Patent No. 6,794,152, there remains a need for continued improvements. Biological samples, such as blood, are subject to rapid deterioration. Formaldehyde is well known as a toxic substance. Some antigenic determinants can be sensitive to formaldehyde.

[0007] Alternative methods of preserving cells for future flow cytometry analysis are disclosed in EP3118623A1. EP3118623A1 and the related product Cyto-Chex® BCT provides a means to preserve whole blood samples without the use of formaldehyde. However, cells are not alive or viable after preservation with the fixation agents that are used and the levels of preserved surface epitopes can still be improved.

[0008] There is a need for improved methods of preserving surface presented epitopes of cells. There is need for improved methods of transporting cells that provide viable cells and also preserve surface presented epitopes.

[0009] BRIEF SUMMARY OF THE DISCLOSURE

[0010] The use of hydrogels to maintain and transport live cells and provide viable cells (living cells) after release from the hydrogel has been described. For example, see WO2012127224A1. However, there is no suggestion that the hydrogels and methods disclosed may be suitable for use with whole blood samples. Furthermore, there is no suggestion that the hydrogels may be suitable for preserving epitopes and antigens presented on the cell surface or preserving the expression or functionality of said epitopes.

[0011] The invention is based on the surprising finding that reversible hydrogels are not just capable of preserving the viability of cells but are also unexpectedly capable of preserving epitopes presented on a cell’s surface and / or preserving the expression of such epitopes. This finding is particularly advantageous when it is used to preserve surface epitopes of cells in biological samples such as in whole blood samples. Cells can be considered living, or viable, if they are able to perform the essential metabolic processes necessary to perform specific functions, such as proliferate. Epitope preservation is separate to this as whilst metabolic processes are needed to manufacture the proteins, these processes do not need to be active once an epitope is presented on the surface of the cell. This is demonstrated by the widely accepted practice of fixing (killing) samples before examination of epitopes e.g. by staining. The inventors have surprisingly found that the surface presentation of epitopes can be maintained using the methods of the invention.

[0012] The methods described herein may also preserve cell surface presented binding proteins (such as cell surface receptors) and / or cell surface binding proteins-ligand complexes (such as cell surface receptor-ligand complexes). That is to say that the methods described herein may preserve a cell surface binding protein in a functional state allowing future analysis and / or binding of the receptor by its cognate partner (e.g. ligand) and / or preserve a cell surface binding protein bound to a ligand in a bound state.

[0013] In addition, the methods described herein may preserve ligands presented on the surface of cells. Thus allowing a cell surface ligand to be preserved and later analysed. For example, analysed by testing binding to an extracellular binding protein or binding entity.

[0014] This may therefore provide an alternative to fixing cells or allow for surface epitopes, binding proteins, binding protein-ligand complexes and / or ligands to be preserved for a longer period of time before fixing.

[0015] The inventors have surprisingly shown that the entrapment or encapsulation of cells that include surface presented epitopes in a reversibly cross-linked hydrogel not only protects the cells but also protects the presentation of and / or expression of surface epitopes, binding proteins, binding protein-ligand complexes and / or ligands from the mechanical and environmental stresses of storage and / or transportation. Surprisingly, the entrapped or encapsulated cellular material does not require the optimum conditions (e.g. a certain temperature, oxygen and carbon dioxide level, and supporting nutrients) normally required to maintain cell morphology, structural integrity, cell viability and / or functionality of cell surface epitopes, binding proteins, binding protein-ligand complexes and / or ligands (e.g. the ability to be bound by a cognate partner of the epitope) during storage and / or transportation. The methods of the invention may be particularly useful for storing epitope-presenting cells immediately, before any deterioration has occurred. This provides flexibility to the user, as epitope-presenting cells can be safely stored until the appropriate staff are available, a GMP laboratory is accessible or until samples can be processed in bulk, without impacting endpoint performance. Accordingly, the entrapped or encapsulated cellular material can be packaged in a sealed receptacle for effective storage or delivery to its point of use, whilst maintaining the material in a condition that is fit for purpose. Furthermore, storage and / or transportation of the packaged material can effectively be undertaken at a much broader range of conditions (e.g. a broader range of temperatures, including ambient temperature) without significantly impacting cellular viability, structural integrity, morphology and / or epitope functionality.

[0016] Advantageously, the methods described herein may provide improved preservation of fresh whole blood leukocyte cell surface epitopes. Advantageously, the methods described herein may provide improved preservation of fresh whole blood leukocyte cell surface binding proteins, cell surface binding protein-ligand complexes and / or cell surface ligands.

[0017] Advantageously, the methods described herein may provide improved whole blood leukocyte samples for improved characterisation, quantification, selection, and / or separation of cells in a sample after storage, preservation and / or transportation. Advantageously, the methods described herein may provide improved whole blood leukocyte samples for improved detection of clinically signification epitopes and / or disease markers. The methods may provide for improved samples for diagnostic analysis and improved methods of diagnosis.

[0018] Advantageously, the methods described herein may provide improved whole blood leukocyte samples for improved determination of cell quality. For example, they may provide improved determination of quality of cells for further use in immunotherapy applications by analysis of relevant surface markers (e.g., surface presented epitopes, surface presented binding proteins, surface presented binding protein-ligand complexes and / or surface presented ligands).

[0019] Furthermore, the inventors surprisingly identified that when a mixed leukocyte population is stored in a reversibly cross-linked hydrogel for a period of time, they can be enriched for a target leukocyte.

[0020] Throughout the description the term “surface presented entity”, “surface presented entities”, “surface entities”, “cell surface entities”, and any linguistic variations thereof is used to refer to any one or more of surface presented epitopes, surface presented binding proteins, surface presented binding protein-ligand complexes and / or surface presented ligands.

[0021] The invention provides a method of preserving surface presented entities of one or more cells, the method comprising

[0022] (a) contacting the one or more cells with a hydrogel-forming polymer;

[0023] (b) polymerising the polymer to form a reversibly cross-linked cell-containing hydrogel wherein at least one or more cells is entrapped or encapsulated in the hydrogel.

[0024] Suitably, the method may further comprise: packaging and sealing the cell-containing hydrogel in a water tight or air tight receptacle.

[0025] Suitably, the method may further comprise: storing the packaged cell-containing hydrogel for a period of time; optionally wherein the period of time is at least 24 hours; and / or transporting the packaged cell-containing hydrogel from a first location to a second location.

[0026] The invention provides a method for transporting one or more cells comprising surface presented entities, wherein surface presented entities of the one or more cells are preserved, comprising: (a) preparing the one or more cells for transportation and preserving the surface presented entities by

[0027] (i) contacting the one or more cells with a hydrogel-forming polymer;

[0028] (ii) polymerising the polymer to form a reversibly cross-linked cellcontaining hydrogel wherein at least one or more cells is entrapped or encapsulated in the hydrogel;

[0029] (iii) packaging and sealing the cell-containing hydrogel in a water tight or air tight receptacle; and

[0030] (b) transporting the packaged cell-containing hydrogel of step (a) from a first location to a second location.

[0031] The invention provides a method for fulfilling an order or request for one or more cells comprising surface presented entities, the method comprising:

[0032] (a) preparing the one or more cells for transportation and preserving the surface presented entities by

[0033] (i) contacting the one or more cells with a hydrogel-forming polymer;

[0034] (ii) polymerising the polymer to form a reversibly cross-linked cellcontaining hydrogel wherein at least one or more cells is entrapped or encapsulated in the hydrogel;

[0035] (iii) packaging and sealing the cell-containing hydrogel in a water tight or air tight receptacle; and

[0036] (b) dispatching the packaged cell-containing hydrogel of step (a) for transportation; or transporting the cell-containing hydrogel of step (a) to a location specified in the order or request.

[0037] Suitably, the method may further comprise: releasing the one or more cells from the hydrogel optionally at a or the second location or a or the specified location.

[0038] Suitably, the one or more cells may comprise a population of cells individually dispersed in the hydrogel.

[0039] Suitably, the one or more cells may be comprised in or obtained from a whole blood sample.

[0040] Suitably, the one or more cells may comprise immune cells; optionally wherein the one or more cells are human cells.

[0041] Suitably, the immune cells may be selected from one or more of: lymphocytes, monocytes and / or granulocytes. Suitably, the surface presented entities may comprise one or more of : cell surface epitopes; cell surface binding proteins, optionally wherein the cell surface binding proteins comprise cell surface receptors; cell surface ligands; and / or cell surface binding protein-ligand complexes.

[0042] Suitably, the cell surface epitopes may be comprised within one or more surface antigens and wherein the surface antigens comprise one or more cell associated antigens; optionally wherein the cell associated antigen is one or more of: CD278, CD46, CD2, B2 microglobulin, CD10, CD100, CD156c, CD16, CD162, CD170, CD1172a b, CD11a, CD11 b, CD126, CD13, CD179a, CD18, CD182, CD191 , CD194, CD14, CD141 , CD218a, CD244, CD25, CD27, CD127, CD29, CD41 , CD42b, CD5, CD55, CD58, CD298, CD3, CD31 , CD317, CD33, CD61 , CD62L, CD62P, CD63, CD81 , CD82, CD87, CD35, CD354, CD367, CD28, CD4, CD9, CD93, CD95, CD97, CD99, HLA-E, Siglec-7, CD102, CD184, CD197, CD217, CD282, CD36, CD369, CD371 , CD45RO, CD7, CD84, CD88, HLA-A, HLA-B, HLA-C, HLA-DR, CD20, CD22, CD45RA, CD45RB, CD49f, Integrin b7, CX3CR1 , CCR10, CD15, CD114, CD183, CD19, CD137L, CD56, CD314, CD34, CD8, GRP56, MERTK, Siglec-8, CD24, and / or CD243.

[0043] Suitably, the cell surface epitopes may be comprised within one or more surface antigens and wherein: the one or more cells comprise lymphocytes and the surface antigens comprise one or more of CD62L, CD317, CD82, CD367 and / or Integrin b7; the one or more cells comprise monocytes and the surface antigens comprise one or more of CD62L, CD20, CD10, CD127, CD87 and / or Integrin b7; the one or more cells comprise granulocytes and surface antigens comprise one or more of CD62L, CD317, CD87, CD369, CD367 and / or CD282.

[0044] Suitably, the surface epitopes may be comprised within one or more surface antigens and wherein the surface antigens comprise one or more of: a tumour antigen; a pathogen associated antigen; an allergenic antigen; a disease associated antigen; and / or an autoantigen. Suitably, preserving may comprise: maintaining the surface presented entities of the one or more cells in a functional state at the surface of the one or more cells; and / or maintaining a level of expression of each or all surface presented entities by the one or more cells; and / or maintaining presentation of each or all of the surface presented entities by the one or more cells.

[0045] Suitably, the hydrogel may comprise cross-linked alginate, optionally wherein the hydrogel comprises cross-linked calcium-alginate, strontium alginate, barium-alginate, magnesiumalginate and / or sodium-alginate or combinations thereof.

[0046] Suitably, the cross-linked alginate may comprise from about 0.1 % (w / v) to about 5.0% (w / v) calcium alginate.

[0047] Suitably, polymerisation may be induced by a chemical agent; optionally wherein the chemical polymerisation agent comprises calcium chloride and strontium chloride.

[0048] Suitably, the sealed receptacle may be a sealed storage vial or transport tube, or wherein the receptacle is a cell culture vessel; optionally wherein the cell culture vessel is selected from a cell culture tube, blood collection tube, a cell culture flask, a cell culture dish or a cell culture plate comprising a plurality of wells; further optionally wherein the cell culture plate comprising a plurality of wells is selected from a 4-, 6-, 8-, 12-, 24-, 48-, 96-, 384-, 1536- well cell culture plate.

[0049] Suitably, the surface epitopes may be preserved for at least 24 hours.

[0050] Suitably, the one or more cells may be viable after release.

[0051] The invention provides for the use of a reversibly cross-linked hydrogel to preserve surface presented entities of one or more cells.

[0052] Suitably, the reversibly cross-linked hydrogel may comprise a hydrogel of the invention.

[0053] Suitably: the one or more cells may comprise one or more cells as defined elsewhere herein; and / or the surface entities may comprise one or more epitopes as defined elsewhere herein. The invention also provides a reversibly cross-linked hydrogel for use in a method of preserving surface presented entities of one or more cells, wherein the method is according to the invention.

[0054] The invention provides a method of obtaining a cell population that is enriched for a target cell, the method comprising:

[0055] (a) contacting a mixed population of cells comprising the target cell with a hydrogel forming polymer;

[0056] (b) polymerising the polymer to form a reversibly cross-linked cell-containing hydrogel wherein the mixed population of cells comprising the target cell is entrapped or encapsulated in the hydrogel; and

[0057] (c) storing the reversibly cross-linked cell-containing hydrogel of (b) to obtain a cell population that is enriched for the target cell.

[0058] Suitably, the mixed population of cells may be comprised in or obtained from a whole blood sample.

[0059] Suitably, the mixed population of cells may comprise immune cells; optionally wherein the immune cells are human cells.

[0060] Suitably, the immune cells may be selected from one or more of: lymphocytes, monocytes and / or granulocytes.

[0061] Suitably, the target cells may be selected from lymphocytes and / or monocytes.

[0062] Suitably, the hydrogel may be stored in step (c) for a period of time; optionally wherein the period of time is at least 3 days; further optionally at from about 2 °C to about 8 °C.

[0063] Throughout the description and claims of this specification, the words “comprise” and “contain” and variations of them mean “including but not limited to”, and they are not intended to (and do not) exclude other moieties, additives, components, integers or steps.

[0064] Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise. Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith.

[0065] Various aspects of the invention are described in further detail below.

[0066] BRIEF DESCRIPTION OF THE FIGURES

[0067] Embodiments of the invention are further described hereinafter with reference to the accompanying drawings, in which:

[0068] Figure 1 shows preservation of whole blood leukocyte epitope expression by BloodReady™. Whole blood was stained with a panel of 93 antibodies against leukocyte surface epitopes following 3-day storage in BloodReady™ or Cyto-Chex. Epitope expression was analysed against that of fresh whole blood leukocytes and expressed as a ratio, where 0 = fresh. Leukocyte epitope expression was analysed separately for A) lymphocyte, B) monocyte, and C) granulocyte cell subpopulations. D) Data shows the percentage of total epitopes preserved by BloodReady on each respective immune cell population, and the percentage of epitopes preserved preferentially by BloodReady over CytoChex. on each respective immune cell population.

[0069] Figure 2 shows that BloodReady™ preserves several monocyte epitopes more effectively than Cyto-Chex, including CD62L, CD20, CD10, CD127, CD87 and Integrin b7. Data shows the mean of n=3 healthy donors.

[0070] Figure 3 shows that BloodReady™ preserves several lymphocyte epitopes more effectively than Cyto-Chex, including CD62L, CD317, CD82, CD367, and Integrin b7. Data shows the mean of n=3 healthy donors.

[0071] Figure 4 shows that BloodReady™ preserves several granulocyte epitopes more effectively than Cyto-Chex, including CD62L, CD317, CD87, CD369, CD367 and CD282. Data shows the mean of n=3 healthy donors.

[0072] Figure 5 shows that BloodReady™ preserves the relative proportions of major leukocyte populations of T cells, NK cells, B cells, and monocytes in the blood.

[0073] The patent, scientific and technical literature referred to herein establish knowledge that was available to those skilled in the art at the time of filing. The entire disclosures of the issued patents, published and pending patent applications, and other publications that are cited herein are hereby incorporated by reference to the same extent as if each was specifically and individually indicated to be incorporated by reference. In the case of any inconsistencies, the present disclosure will prevail. Various aspects of the invention are described in further detail below.

[0074] DETAILED DESCRIPTION

[0075] The invention relates to methods of preserving epitopes presented on the outer surface of cells (for example, on the outer surface of the cell membrane).

[0076] Sample and Cells

[0077] The methods described herein may be used to preserve surface-presented entities of any cell. In particular, the methods may be used for preservation of surface-presented entities presented on the surface of blood cells. For example, the cells may be immune cells. Immune cells include antigen presenting cells, B-cells, basophils, cytotoxic T cells, dendritic cells, eosinophils, granulocytes, helper T cells, leukocytes, lymphocytes, macrophages, mast cells, memory cells, monocytes, natural killer cells, neutrophils, phagocytes, plasma cells and T cells. In some examples the cells may be peripheral blood mononuclear cells (PBMC) comprising at least one of dendritic cells and / or leukocyte cells (i.e. white blood cells).

[0078] In some examples, the cells may be leukocytes. The terms “leukocyte” and “leukocyte cells” as used interchangeably herein refer to cells of granulocytic and lymphocytic lineage. Examples of leukocyte cells are granulocytes (neutrophils, eosinophils, and basophils), monocytes, and lymphocytes (T cells and B cells).

[0079] In some examples, the cells used in the methods described herein may be leukocytes including lymphocytes, monocytes and / or granulocytes. In some examples, the cells may be a single type of cell. In some examples, the cells may be a single type of leukocyte. In other examples, the cells may be a mixture of cells. In other examples, the leukocytes may be a mixture of leukocytes for example, a mixture of lymphocytes, monocytes and / or granulocytes.

[0080] The terms “lymphocyte cell” and “lymphocyte” as interchangeably used herein, refer to any one of a natural killer (NK) cell (usually involved in cell-mediated, cytotoxic innate immunity), a T cell (usually involved in cell-mediated, cytotoxic adaptive immunity), a B cell (usually involved in humoral, antibody-driven adaptive immunity), a plurality thereof or any combination thereof. Peripheral blood mononucleated (PBMC) cells, tumour-infiltrating-lymphocyte (TIL) cells and lymphokine-activated killer (LAK) cells (usually involved in tumour cells' killing) are also considered lymphocyte cells. Non-limiting examples of lymphocytes include cytotoxic lymphocytes (CTLs, CD8+or CD4+), NK cells (CD2+), and T helper cells (CD4+).

[0081] "Natural killer cells (NK cells)" refers to innate immune cells that selectively show cytotoxicity to virus-infected cells or cancer cells. It has been reported that natural killer cells are effective in preventing the occurrence, proliferation, metastasis and recurrence of cancer. "T cell (T cell or T lymphocyte)" refers to one of the lymphocytes responsible for antigenspecific adaptive immunity. The T cells are classified into naive T cells that have not yet encountered antigens, effector T cells that have met antigens and mature (helper T cells, cytotoxic T cells, natural killer T cells) and memory T cells.

[0082] “Monocyte” refers to a type of white blood cell that has two main functions in the immune system: (1) replenish resident macrophages and dendritic cells under normal states, and (2) in response to inflammation signals, monocytes can move quickly (approx. 8-12 hours) to sites of infection in the tissues and divide / differentiate into macrophages and dendritic cells to elicit an immune response. Half of them are stored in the spleen. Monocytes are usually identified in stained smears by their large bilobate nucleus. In addition to the expression of CD14, monocytes also show expression of one or more of the following surface markers 1251-WVH- 1 , 63D3, Adipophilin, CB12, CD11a, CD11b, CD15, CD54, Cd163, cytidine deaminase, Flt-1 , and the like. The term monocyte includes both the classical monocyte and the non-classical pro-inflammatory monocyte, which are both present in human blood.

[0083] The term “the classical monocyte” refers to a type of monocyte cell characterized by high level expression of the CD14 cell surface receptor (CD14++ monocyte) and the term “the non- classical pro-inflammatory monocyte”, refers to a cell with low level expression of CD14 and with additional co-expression of the CD16 receptor (CD14+CD16+ monocyte) which are characterized by producing high amounts of pro-inflammatory cytokines such as tumour necrosis factor and interleukin- 12 in response to stimulation by microbial products. These cells develop from the CD14++ monocytes.

[0084] "Macrophage" is also referred to as a phagocyte, and is one of the main cells responsible for innate immunity.

[0085] The term “granulocyte” refers to a cell with secretory granules in its cytoplasm, i.e. neutrophils, basophils, eosinophils, mast cells and keratinocytes.

[0086] “Eosinophil" refers to granulocytes filled with eosinophilic granules that mainly act on parasites and viruses.

[0087] “Basophil" refers to leukocytes having intracellular heparin and histamine-containing granules.

[0088] “Neutrophil” refers to mature white blood cells that arise from precursor cells such as myelocytes, metamyelocytes and band forms and that have a cytoplasm that is not acidophilic or basophilic like eosinophils or basophils. The term “neutrophil” includes neutrophils and megaloblastic neutrophils that are caused because the lack of vitamin B12 and / or folate deficiency. Megaloblastic neutrophils may also have a hypersegmented nucleus. Neutrophils also cover neutrophil-like cells in animals including humans. A specific example of neutrophillike cells is HL60 cells differentiated after treatment with dibutyric cyclic AMP.

[0089] The term “peripheral blood mononuclear cell” (PBMC) refers to a cell with a spherical nucleus present in the peripheral blood, which is referred to as peripheral blood monocyte or PBMC. PBMCs may include immune cells such as B cells, T cells, macrophages, dendritic cells, and NK cells.

[0090] In some examples, the cells may be cancer cells. “Cancer cells” are cells that acquire a characteristic set of functional capabilities during their development, including the ability to evade apoptosis, self-sufficiency in growth signals, insensitivity to anti-growth signals, tissue invasion / metastasis, significant growth potential, and / or sustained angiogenesis. The term “cancer cell” is meant to encompass both pre-malignant and malignant cancer cells.

[0091] In some examples, the cancer cells may be leukocyte cancer cells. In some examples, the leukocyte cancer cells may be selected from leukaemia cells, lymphoma cells, and myeloma cells. In some examples, the leukaemia cells may be selected from myeloblasts, lymphoblasts, promyelocytes, myelocytes, metamyelocytes, mature abnormal lymphocytes, mature abnormal myeloid cells, and Philadelphia chromosome-positive cells. In some examples, the lymphoma cells may be selected from Reed-Sternberg cells, malignant B lymphocytes, malignant T lymphocytes, and follicular lymphoma cells. In some examples, the myeloma cells may be malignant plasma cells and / or plasmablasts.

[0092] The cells, such as leukocytes, used in methods described herein may be obtained from or comprised in a biological sample. Accordingly, the sample may comprise one or more leukocytes. That is to say that the cells, such as leukocytes may be separated from other components of a biological sample. In other examples, the cells, such as leukocytes, may not be separated from other components of a biological sample but be encapsulated and entrapped in the hydrogel polymer directly in the biological sample. In some examples, the sample comprising one or more leukocytes may be entrapped or encapsulated in the hydrogel polymer.

[0093] As used herein, the terms "biological sample", “test sample”, "sample" and variations thereof refer to a sample obtained or derived from a subject. For the purposes described herein, the sample may be, or may comprise, a biological fluid (also referred to herein as a bodily fluid) sample. In one example, the sample may be selected from a blood sample, tissue sample (such as a tumour), urine sample, saliva sample, cerebrospinal fluid sample, semen sample, faeces sample, and bone marrow sample. The sample may comprise one or more leukocytes. In one example, the sample may be a bone marrow sample. In one example, the bone marrow sample may comprise one or more leukocytes. In another example, the sample may be a tissue sample, optionally wherein the tissue sample may be a tumour sample. In one example, the tissue sample may comprise one or more leukocytes.

[0094] As used herein, the term “biological fluid sample” encompasses a blood sample, a urine sample, a saliva sample, a cerebrospinal fluid sample, and a semen sample.

[0095] In one example, the sample may be a blood sample. In some examples, the blood sample may comprise one or more leukocytes. A blood sample may be a whole blood sample, or a processed blood sample e.g. buffy coat. Methods for obtaining biological fluid samples (e.g. whole blood) from a subject are well known in the art. For example, methods for obtaining blood samples from a subject are well known and include established techniques used in phlebotomy. The obtained blood samples may be further processed using standard techniques. Advantageously, methods for obtaining biological fluid samples from a subject are typically low-invasive or non-invasive.

[0096] A whole blood sample is defined as a blood sample drawn from the human body and from which (substantially) no constituents (such as platelets or plasma) have been removed. In other words, the relative ratio of constituents in a whole blood sample is substantially the same as the blood in the body. In this context, “substantially the same” allows for a very small change in the relative ratio of the constituents of whole blood e.g. a change of up to 5%, up to 4%, up to 3%, up to 2%, up to 1% etc. Whole blood contains both the cell and fluid portions of blood. A whole blood sample may therefore also be defined as a blood sample with (substantially) all of its cellular components in plasma, wherein the cellular components (i.e. at least comprising the requisite white blood cells, red blood cells, platelets of blood) are intact.

[0097] In some examples, the sample may be a white blood cell sample. A white blood cell sample is a sample that includes (substantially) only white blood cells. In some examples, the white blood cell sample may comprise one or more leukocytes. That is to say other components of the bodily fluid (e.g. blood) have been removed. For example, in the case of blood, a white blood cell sample may be obtained by methods such as leukocytapheresis. In some examples, a white blood cell sample may be obtained from a blood sample by methods such as centrifugation methods (such as density gradient centrifugation), filtration methods and / or red blood cell lysis. Other methods of separating white blood cells will be known to those skilled in the art.

[0098] As used herein, “provide”, "obtain" or "obtaining" can be any means whereby one comes into possession of the sample by "direct" or "indirect" means. Directly obtaining a sample means performing a process (e.g., performing a physical method such as extraction) to obtain the sample. Indirectly obtaining a sample refers to receiving the sample from another party or source (e.g., a third party laboratory that directly acquired the sample).

[0099] The hydrogels described herein can be used to store, preserve, and / or transport a plurality of individual cells that are retained therein. These cells are in general separated or dispersed within the hydrogel, i.e. the cells are not connected in the form of a tissue or organ.

[0100] In addition to preservation of surface-presented entities , the methods described herein may also provide viable or living cells after release from the hydrogel.

[0101] For example, at least substantially all of the cells are live (or viable). For example, at least 50%, 60% or 70% of the cells remain viable after storage and / or transport, more preferably at least 80%, 85%, 90% or 95% of the cells remain viable after storage and / or transport. Viability may be assessed by T rypan blue exclusion assay or other similar means. Other similar means include the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide) assay and examination of cell colony formation post- extraction.

[0102] In some examples, the cells may be or include cells that are not viable but the surface- presented entities of the cells are preserved. For example, preserved in a functional state.

[0103] In some examples, the concentration of cells which are present in the hydrogel is 1 x 103- 1 x 101° cells / ml hydrogel solution. Generally the concentration of cells is less than 5.5 x 109, preferably 2.5 x 106- 5 x 109cells / ml hydrogel. For example cell concentrations include:

[0104] - for a whole blood sample: about 2.5x106leukocytes and 2.5x109erythrocytes per mL of hydrogel

[0105] - for a Buffy coat sample: about 3.3x107leukocytes and 6.6x109erythrocytes per mL of hydrogel

[0106] - for an isolated peripheral blood mononuclear cells (PBMCs) sample: about 2.5 x 107cells per mL of hydrogel.

[0107] The cells are embedded in the hydrogel, i.e. the cells are generally entrapped or encapsulated within the hydrogel and not merely placed on a surface of a hydrogel.

[0108] As used herein, the term “entrapped” refers to the cells being physically captured / trapped by the hydrogel, such that they are not released from the hydrogel (unless for example the crosslinking is reversed such that the hydrogel reverts to a solution). The cells may be entrapped by virtue of being completely surrounded by the hydrogel.

[0109] The term “encapsulated” refers to enclosing the cells in the hydrogel. In the context of unbound cells (i.e. cells that are not bound / adherent to a surface such as a solid surface of a receptacle (as described elsewhere herein), the cells are “encapsulated” by a hydrogel when they are each completely surrounded by the hydrogel. In other words, in this context, encapsulation refers to enclosing available surfaces of the cells in the hydrogel.

[0110] The term "subject" as used herein may refer to an animal, plant or prokaryote. A subject therefore may refer to, for example, mammals such as dogs, cats, horses, cows, pigs, guinea pigs, and the like. The subject can be a human. When the subject is a human, the subject may be referred to herein as a patient. The terms “subject”, “individual”, and “patient” are used herein interchangeably.

[0111] Surface-Presented Entities

[0112] In some examples, the surface-presented entity is endogenous to the cell. As used herein, the term “endogenous” in the context of surface-presented entities refers to surface-presented entities originating from within the cell due to natural processes as opposed to being introduced by artificial means (such as genetic engineering or recombinant DNA technology). In other words, endogenous surface-presented entities are those that would naturally be present on the surface of the cell in question (either because that cell would naturally express such surface-presented entities, or it would naturally harbour such surface-presented entities e.g. present them on the cell surface in the context of HLA). Non-limiting examples of natural processes include gene expression of endogenous genes, presentation of pathogen epitopes, and presentation of allergenic epitopes.

[0113] These endogenous surface-presented entities may comprise surface-presented entities typically expressed by the cell, such as cell specific surface-presented entities. For example, a leukocyte surface presented entity is a surface presented entity endogenous to leukocytes. Specific examples of endogenous surface presented entities of leukocytes are outlined elsewhere herein.

[0114] Conversely, the term “exogenous” as used herein in the context of surface-presented entities refers to surface-presented entities presented on the cell which have been introduced by artificial means. Typically, exogenous surface-presented entities are introduced in view of expressing an exogenous protein, such as a recombinant protein.

[0115] Epitopes

[0116] An epitope, refers to a portion of an entity which may be bound by a cognate partner. For example, an epitope may be part of a protein or a chemical moiety. In some examples, an epitope may refer to an antigenic determinant, which is the part of an antigen that is recognized by the immune system, for example by antibodies, B cells, or T cells. The epitope may be the molecular region of an antigen which is bound by the antibody, B cell or T cell. “Epitope" includes any entity capable of specific binding to a cognate partner. For example, an epitope is typically capable of specific binding to a receptor (i.e, in the case of a ligand), an immunoglobulin (in the case of an antigen) or otherwise interacting with an immune system cell or molecule (in the case of an antigen). Epitopes generally consist of chemically active surface groupings of molecules such as, metals, lipids, amino acids or carbohydrates or sugar side chains and can have specific three-dimensional structural characteristics, as well as specific charge characteristics. An epitope may be "linear" or "conformational." A “leukocyte epitope” refers to an endogenous epitope expressed by and / or presented by a leukocyte.

[0117] The epitope may be of any chemical nature, including without limitation, peptides, carbohydrates, lipids, glycopeptides and glycolipids. The epitope may be a naturally occurring epitope, or a modified form of a naturally occurring epitope. The term "linear epitope" refers to an epitope with all of the points of interaction between the protein and the interacting molecule (such as an antibody) occur linearly along the primary amino acid sequence of the protein (continuous). The term "conformational epitope" refers to an epitope in which discontinuous amino acids that come together in three-dimensional conformation. In a conformational epitope, the points of interaction occur across amino acid residues on the protein that are separated from one another. Generally, binding molecules (such as antibodies) for a particular target antigen will preferentially recognize an epitope on the target antigen in a complex mixture of proteins and / or macromolecules. Regions of a given polypeptide that include an epitope can be identified using any number of epitope mapping techniques, well known in the art. See, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66 (Glenn E. Morris, Ed., 1996) Humana Press, Totowa, New Jersey. For example, linear epitopes may be determined by concurrently synthesizing large numbers of peptides on solid supports, the peptides corresponding to portions of the protein molecule, and reacting the peptides with antibodies while the peptides are still attached to the supports. Such techniques are known in the art and described in, e.g., U.S. Patent No. 4,708,871 ; Geysen et al., (1984) Proc. Natl. Acad. Sci. USA 8:3998-4002; Geysen et al., (1985) Proc. Natl. Acad. Sci. USA 82:78-182; Geysen et al., (1986) Mol. Immunol. 23:709-715. Similarly, conformational epitopes are readily identified by determining spatial conformation of amino acids such as by, e.g., hydrogen / deuterium exchange, x-ray crystallography and two-dimensional nuclear magnetic resonance. See, e.g., Epitope Mapping Protocols, supra. Antigenic regions of proteins can also be identified using standard antigenicity and hydropathy plots, such as those calculated using, e.g., the Omiga version 1.0 software program available from the Oxford Molecular Group. This computer program employs the Hopp / Woods method, Hopp et al., (1981) Proc. Natl. Acad. Sci USA 78:3824-3828; for determining antigenicity profiles, and the Kyte-Doolittle technique, Kyte et al., (1982) J. Mol. Biol. 157:105-132; for hydropathy plots. In some examples, the epitope may be referred to by the source that it has been derived from / from which it originates. For example, the epitope may be selected from one or more of: a cell specific epitope, an allergenic epitope, a pathogen associated epitope (e.g. a viral epitope, a bacterial epitope, parasitic epitope), disease associated epitope, an auto-antigen associated epitope or a tumour associated epitope.

[0118] In this context, an “allergenic epitope” refers to any antigenic determinant / component part of (or derived from) an allergen (the antigen) that is capable of specific binding to an immunoglobulin or otherwise interacting with an immune system molecule or cell (e.g. T cell or B cell). In other words, an allergenic epitope may be a linear or conformational epitope that is a (small) part of the allergen. The epitope may be present within a longer sequence (for example, a larger part, or all of the allergen may be present).

[0119] Similarly, a “viral epitope” refers to any antigenic determinant / component part of (or derived from) a virus (the antigen) that is capable of specific binding to an immunoglobulin or otherwise interacting with an immune system molecule or cell (e.g. T cell or B cell). In other words, a viral epitope may be a linear or conformational epitope that is a (small) part of a viral protein for example. The epitope may be present within a longer sequence (for example, the a larger part, or all of a viral protein may be present).

[0120] Similarly, a “bacterial epitope” refers to any antigenic determinant / component part of (or derived from) bacteria (the antigen) that is capable of specific binding to an immunoglobulin or otherwise interacting with an immune system molecule or cell (e.g. T cell or B cell). In other words, a bacterial epitope may be a linear or conformational epitope that is a (small) part of a bacterial protein for example. The epitope may be present within a longer sequence (for example, a larger part, or all of a bacterial protein may be present).

[0121] Equivalent definitions for “parasitic epitope”, “disease-associated epitope”, and “tumour- associated epitope” would be clear to a person of skill in the art. In this context, a “tumour- associated epitope” can be an epitope from a tumour associated antigen (TAA).

[0122] The epitope may be recognised and bound by a B cell, immunoglobulin or antibody, for example. Epitopes that are bound by B cells are typically at least five amino acids, more often at least six amino acids, still more often at least seven or eight amino acids in length, that may be continuous (“linear”) or discontinuous (“conformational”) (the latter being formed by the folding of a protein to bring non-contiguous parts of the primary amino acid sequence into physical proximity). B-cells may also bind to carbohydrate epitopes.

[0123] The epitope may be recognised and bound by a T cell (via a T cell receptor for example). Epitopes that are recognised by T cell receptors are typically presented to the T cell receptor in the context of class I or class II MHC molecules. The class I epitopes are usually 8 to 15, more often 9-11 amino acids in length. The class II epitopes are usually 5-24 (a 24 mer is the longest peptide which can fit in the Class II groove), more often 8-24 amino acids. Carbohydrate epitopes (as small as a single sugar unit (e.g., Tn)) may also be recognised by T cells. They are preferably no larger than five sugars. Many such epitopes are known.

[0124] Cell specific epitopes

[0125] Cell specific epitope refers to an epitope that is derived from or part of a cell specific antigen. Cell specific antigens and epitopes thereof refer to antigens that are expressed and displayed on specific cells or subsets of cells. Accordingly, leukocyte specific antigens and epitopes thereof are endogenous antigens and epitopes thereof expressed by and / or presented by a leukocyte. Accordingly, cell specific epitopes may be suitable for identifying specific cells or subsets of cells, such as leukocytes. Cell specific antigens may not be ubiquitously expressed on all cell types. A cell-specific antigen may only be expressed on the surface of, for example, certain cell types defined by their anatomical origin, location, function, or disease state. For example, the cell specific antigens include, but are not limited to, CD molecules, PD-1 , Her2, PSMA, KLB, GCGR, B2 microglobulin, HLA-E, Siglec-7, HLA-A, HLA-B, HLA-C, HLA-DR, Integrin b7, GRP56, MERTK, Siglec-8 and CNTFRa.

[0126] CD molecules or antigens, also referred to as cluster of differentiation antigens, are cellsurface molecules expressed on leukocytes and other cells relevant for the immune system. CD antigens may be used for the identification and investigation of cell surface molecules providing targets for immunophenotyping of cells. CD antigen immunophenotyping may allow cells to be defined based on what molecules are present on their surface. These markers are often used to associate cells with certain immune functions. CD molecules are utilized in cell sorting using various methods, including flow cytometry.

[0127] In some examples, the cells are immune cells. Cell specific antigens that may be used to identify and / or sort immune cells include:

[0128] Granulocyte: CD45, CD11 b, CD15, CD24, CD114, and CD182;

[0129] Monocyte: CD4, CD45, CD14, CD114, CD11a, CD11b, CD91 , and CD16;

[0130] T lymphocyte: CD45, and CD3;

[0131] T helper cell: CD45, CD3, and CD4;

[0132] T regulatory cell: CD4, CD25, and FOXP3;

[0133] Cytotoxic T cell: CD45, CD3, CD8.

[0134] B lymphocyte: CD45, CD19, CD20, CD24, CD38, and CD22;

[0135] Thrombocyte: CD45, and CD61 ; and Natural killer cell: CD16, CD56, CD31 , CD30, and CD38.

[0136] In some examples, the cells are leukocytes. Cell specific antigens that may be used to identify and / or sort leukocytes include:

[0137] Granulocyte: CD45, CD11 b, CD15, CD24, CD114, and CD182;

[0138] Monocyte: CD4, CD45, CD14, CD114, CD11a, CD11b, CD91 , and CD16;

[0139] T lymphocyte: CD45, and CD3;

[0140] T helper cell: CD45, CD3, and CD4;

[0141] T regulatory cell: CD4, CD25, and FOXP3;

[0142] Cytotoxic T cell: CD45, CD3, CD8.

[0143] B lymphocyte: CD45, CD19, CD20, CD24, CD38, and CD22; and

[0144] Natural killer cell: CD16, CD56, CD31 , CD30, and CD38.

[0145] In some examples, the cell specific antigens may be selected from the group consisting of: CD278, CD46, CD2, B2 microglobulin, CD10, CD100, CD156c, CD16, CD162, CD170, CD1172a b, CD11a, CD11 b, CD126, CD13, CD179a, CD18, CD182, CD191 , CD194, CD14, CD141 , CD218a, CD244, CD25, CD27, CD127, CD29, CD41 , CD42b, CD5, CD55, CD58, CD298, CD3, CD31 , CD317, CD33, CD61 , CD62L, CD62P, CD63, CD81 , CD82, CD87, CD35, CD354, CD367, CD28, CD4, CD9, CD93, CD95, CD97, CD99, HLA-E, Siglec-7, CD102, CD184, CD197, CD217, CD282, CD36, CD369, CD371 , CD45RO, CD7, CD84, CD88, HLA-A, HLA-B, HLA-C, HLA-DR, CD20, CD22, CD45RA, CD45RB, CD49f, Integrin b7, CX3CR1 , CCR10, CD15, CD114, CD183, CD19, CD137L, CD56, CD314, CD34, CD8, GRP56, MERTK, Siglec-8, CD24, CD243, HLA DR, CD161 , IgD, CD123, TCRgd, and / or CD125.

[0146] In some examples, the cell is, or the cells include, lymphocytes and the antigen may be selected from the group consisting of: CD62L, CD317, CD82, CD367 and / or Integrin b7.

[0147] In some examples, the cell is, or the cells include, monocytes and the antigen may be selected from the group consisting of: CD62L, CD20, CD10, CD127, CD87 and / or Integrin b7.

[0148] In some examples, the cell is, or the cells include, granulocytes and the antigen may be selected from the group consisting of: CD62L, CD317, CD87, CD369, CD367 and / or CD282.

[0149] In some examples, the cell may be a leukocyte.

[0150] In some examples, the leukocyte is, or the leukocytes include, lymphocytes and the antigen may be selected from the group consisting of: CD62L, CD317, CD82, CD367 and / or Integrin b7. In some examples, the leukocytes is, or the leukocytes include, monocytes and the antigen may be selected from the group consisting of: CD62L, CD20, CD10, CD127, CD87 and / or Integrin b7.

[0151] In some examples, the leukocytes is, or the leukocytes include, granulocytes and the antigen may be selected from the group consisting of: CD62L, CD317, CD87, CD369, CD367 and / or CD282.

[0152] CD62L (also known as L-selectin) is a cell adhesion molecule found on the cell surface of leukocytes and blastocysts. It is coded for in humans by the SELL gene. L-selectin belongs to the selectin family of proteins, which recognize sialylated carbohydrate groups containing a Sialyl LewisX (sLeX) determinant. L-selectin plays an important role in both the innate and adaptive immune responses by facilitating leukocyte-endothelial cell adhesion events. CD62L plays a pivotal role in controlling the traffic of lymphocytes and neutrophils from peripheral lymph nodes to sites of inflammation. Cryopreservation induces a profound decrease of CD62L expression (see Costantini A, Mancini S, Giuliodoro S, Butini L, Regnery CM, Silvestri G, Montroni M. Effects of cryopreservation on lymphocyte immunophenotype and function. J Immunol Methods. 2003 Jul;278(1-2):145-55). CD62L also plays a role in regulating the recruitment of monocytes to tissue from the blood during inflammation.

[0153] CD317 is also known as bone marrow stromal antigen 2 and tetherin. CD317 is constitutively expressed in mature B cells, plasma cells and plasmacytoid dendritic cells.

[0154] CD82 associates with CD4 or CD8 and delivers costimulatory signals for the TCR / CD3 pathway. CD82 is a membrane glycoprotein that is a member of the transmembrane 4 superfamily. Expression of this gene has been shown to be downregulated in tumor progression of human cancers and can be activated by p53 through a consensus binding sequence in the promoter. Its expression and that of p53 are strongly correlated, and the loss of expression of these two proteins is associated with poor survival for prostate cancer patients.

[0155] CD367 (also known as C-type lectin domain family 4 member A) is a C-type lectin receptor that binds carbohydrates mannose and fucose but also weakly interacts with N- acetylglucosamine (GIcNAc) in a Ca2+-dependent manner. Once triggered by antigen, it is internalized by clathrin-dependent endocytosis and delivers its antigenic cargo into the antigen presentation pathway resulting in cross-priming of CD8+ T cells. This cross-presentation and cross-priming are enhanced by TLR7 and TLR8 agonists with increased expansion of the CD8+ T cells, high production of IFNG and TNF with reduced levels of IL4, IL5 and IL13.

[0156] Integrin b7 is a member of the integrin superfamily. Members of this family are adhesion receptors that function in signalling from the extracellular matrix to the cell. Integrins are heterodimeric integral membrane proteins composed of an alpha chain and a beta chain. The encoded protein forms dimers with an alpha4 chain or an alphaE chain and plays a role in leukocyte adhesion. Dimerization with alpha4 forms a homing receptor for migration of lymphocytes to the intestinal mucosa and Peyer's patches. Dimerization with alphaE permits binding to the ligand epithelial cadherin, a calcium-dependent adhesion molecule. Integrin b7 also functions in the homing of T cells to intestinal sites

[0157] CD20 is a member of the membrane-spanning 4A gene family. CD20 is a B-lymphocyte- specific membrane protein that plays a role in the regulation of cellular calcium influx necessary for the development, differentiation, and activation of B-lymphocytes.

[0158] CD10 (also known as Neprilysin and membrane metallo-endopeptidase (MME)) is a zincdependent metalloprotease that cleaves peptides at the amino side of hydrophobic residues and inactivates several peptide hormones including glucagon, enkephalins, substance P, neurotensin, oxytocin, and bradykinin. CD10 is expressed by early B, pro-B and pre-B lymphocytes, and by lymph node germinal centers.

[0159] CD127 is a receptor for interleukin 7 (IL7). The function of this receptor requires the interleukin 2 receptor, gamma chain (IL2RG), which is a common gamma chain shared by the receptors of various cytokines, including interleukins 2, 4, 7, 9, and 15. This protein has been shown to play a critical role in V(D)J recombination during lymphocyte development.

[0160] CD87 (also known as urokinase receptor and urokinase plasminogen activator surface receptor) multidomain glycoprotein tethered to the cell membrane with a glycosylphosphotidylinositol anchor. CD87 facilitates CD11b / CD18-mediated adhesion of human monocytes.

[0161] CD369 is a member of the C-type lectin / C-type lectin-like domain (CTL / CTLD) superfamily. The encoded glycoprotein is a small type II membrane receptor with an extracellular C-type lectin-like domain fold and a cytoplasmic domain with a partial immunoreceptor tyrosine-based activation motif. It functions as a pattern-recognition receptor for a variety of p-1 ,3-linked and P-1 ,6-linked glucans from fungi and plants, and in this way plays a role in innate immune response. Expression is found on myeloid dendritic cells, monocytes, macrophages and B cells.

[0162] CD282 is a toll-like receptor (TLR) family member, which plays a fundamental role in pathogen recognition and activation of innate immunity. TLRs recognize pathogen-associated molecular patterns (PAMPs) that are expressed on infectious agents, and mediate the production of cytokines necessary for the development of effective immunity. The various TLRs exhibit different patterns of expression. The CD282 gene is expressed most abundantly in peripheral blood leukocytes, and mediates host response to Gram-positive bacteria[6] and yeast via stimulation of NF-KB.

[0163] Allergenic epitopes

[0164] In some examples, the epitope is derived from or part of an allergenic antigen. In other words, the epitope may be comprised in an allergenic antigen. In some examples, the antigen may comprise part of an allergen (which includes an allergenic epitope). In other examples, the antigen may be an allergen (which includes an allergenic epitope). In some examples, allergenic antigens may be presented by leukocytes as part of the allergy response. Accordingly, in the context of leukocytes, allergenic antigens may be endogenous.

[0165] Allergenic epitopes refer to an epitope derived from an antigen that causes an allergic reaction (i.e. and allergen). The term “allergen” encompasses "allergen extracts" and "allergenic epitopes”. Examples of allergens include but are not limited to pollens; house dust and dust mites; animal allergens; mold and fungus; insect bodies and insect venom; feathers; food; and drugs (e.g., penicillin). In some examples, allergenic epitopes may be presented by leukocytes as part of the allergy response. Accordingly, in the context of leukocytes, allergenic epitopes may be endogenous.

[0166] Viral epitopes

[0167] In some examples, the epitope is derived from or part of a viral antigen. In some examples, the antigen may comprise part of a viral protein (which includes a viral epitope). In other examples, the antigen may be a viral protein (which includes a viral epitope). In some examples, the epitope is derived from or part of a viral antigen, such as an epitope derived from an infectious virus.

[0168] Examples of infectious virus include: Retroviridae (e.g., human immunodeficiency viruses, such as HIV-1 (also referred to as HTLV-III, LAV or HTLV- lll / LAV, or HLV-III; and other isolates, such as HIV-LP; Picornaviridae (e.g., polio viruses, hepatitis A virus; enteroviruses, human coxsackie viruses, rhinoviruses, echoviruses); Calciviridae (e.g., strains that cause gastroenteritis); Togaviridae (e.g., equine encephalitis viruses, rubella viruses); Flaviridae (e.g., dengue viruses, encephalitis viruses, yellow fever viruses); Coronaviridae (e.g., coronaviruses); Rhabdoviridae (e.g., vesicular stomatitis viruses, rabies viruses); Filoviridae (e.g., ebola viruses); Paramyxoviridae (e.g., parainfluenza viruses, mumps virus, measles virus, respiratory syncytial virus); Orthomyxoviridae (e.g., influenza viruses); Bungaviridae (e.g., Hantaan viruses, bunga viruses, phleboviruses and Nairo viruses); Arena viridae (hemorrhagic fever viruses); Reoviridae (e.g., reoviruses, orbiviurses and rotaviruses); Birnaviridae', Hepadnaviridae (Hepatitis B virus); Parvoviridae (parvoviruses); Papovaviridae (papilloma viruses, polyoma viruses); Adenoviridae (most adeno viruses); Herpesviridae (herpes simplex virus (HSV) 1 and 2, varicella zoster virus, cytomegalovirus (CMV), hecpes viruses'); Poxviridae (variola viruses, vaccinia viruses, pox viruses); and Iridoviridae (e.g., African swine fever virus); and unclassified viruses (e.g., the etiological agents of Spongiform encephalopathies, the agent of delta hepatitis (thought to be a defective satellite of hepatitis B virus), the agents of non-A, non-B hepatitis (class 1 = internally transmitted; class 2 = parenterally transmitted (i.e. , Hepatitis C); Norwalk and related viruses, and astro viruses).

[0169] In some examples, viral antigens may be presented by leukocytes as part of the immune response. Accordingly, in the context of leukocytes, viral antigens may be endogenous.

[0170] In some examples, viral epitopes may be presented by leukocytes as part of the immune response. Accordingly, in the context of leukocytes, viral epitopes may be endogenous.

[0171] Bacterial epitopes

[0172] In some examples, the epitope is derived from or part of a bacterial antigen. In other words, the antigen may comprise a bacterial epitope. In some examples, the antigen may comprise part of a bacterial protein (which includes a bacterial epitope). In other examples, the antigen may be a bacterial protein (which includes a bacterial epitope). In some examples, the epitope is derived from or part of a bacterial antigen, such as an epitope derived from an infectious bacteria.

[0173] Examples of infectious bacteria include: Helicobacter pyloris, Borelia burgdorferi, Legionella pneumophilia, Mycobaderia sps (e.g. M. tuberculosis, M. avium, M. intracellulare, M. kansaii, M. gordonae), Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitidis, Listeria monocytogenes, Streptococcus pyogenes (Group A Streptococcus), Streptococcus agalactiae (Group B Streptococcus), Streptococcus (viridans group), Streptococcus faecalis, Streptococcus bovis, Streptococcus (anaerobic sps.), Streptococcus pneumoniae, pathogenic Campylobacter spp., Enterococcus spp., Haemophilus influenzae, Bacillus antracis, Corynebaderium diphtheriae, Corynebacterium spp., Erysipelothrix rhusiopathiae, Clostridium perfringers, Clostridium tetani, Enterobacter aerogenes, Klebsiella pneumoniae, Pasturella multocida, Bacteroides spp., Fusobacterium nucleatum, Streptobacillus moniliformis, Treponema pallidium, Treponema pertenue, Leptospira, and Actinomyces israelii.

[0174] In some examples, bacterial antigens may be presented by leukocytes as part of the immune response. Accordingly, in the context of leukocytes, bacterial antigens may be endogenous.

[0175] In some examples, bacterial epitopes may be presented by leukocytes as part of the immune response. Accordingly, in the context of leukocytes, bacterial epitopes may be endogenous.

[0176] Parasitic epitopes In some examples, the epitope is derived from or part of a parasitic antigen. In some examples, the antigen may comprise part of a parasitic protein (which includes a parasitic epitope). In other examples, the antigen may be a parasitic protein (which includes a parasitic epitope). In some examples, the epitope is derived from or part of a parasitic antigen, such as a protein from Hookworm (Necator americanus), Scabies mite (Sarcoptes scabiei var. hominis), Roundworm (Ascaris lumbricoides), Flatworm blood fluke (Schistosoma mansoni, S. haematobium, S. japonicum), Tapeworm (Taenia solium), Pinworm (Enterobius vermicularis), Toxoplasma gondii, Giardia lamblia or Entamoeba histolytica.

[0177] In some examples, parasitic antigens may be presented by leukocytes as part of the immune response. Accordingly, in the context of leukocytes, parasitic antigens may be endogenous.

[0178] In some examples, parasitic epitopes may be presented by leukocytes as part of the immune response. Accordingly, in the context of leukocytes, parasitic epitopes may be endogenous.

[0179] Disease-associated epitope

[0180] In some examples, the epitope is a disease-associated epitope.

[0181] An epitope may be indirectly associated with a disease if the epitope is of an antigen which is specifically produced or overproduced by diseased cells of the subject, or which is specifically produced or overproduced by other cells of the subject in specific, but non-immunological, response to the disease, e.g., an angiogenic factor which is overexpressed by nearby cells as a result of regulatory substances secreted by a tumour.

[0182] In some examples, disease-associated antigens may be presented by leukocytes as part of the immune response. Accordingly, in the context of leukocytes, disease-associated antigens may be endogenous.

[0183] In some examples, disease-associated epitopes may be presented by leukocytes as part of the immune response. Accordingly, in the context of leukocytes, disease-associated epitopes may be endogenous.

[0184] Tumour-associated epitopes

[0185] An epitope may be said to be directly associated with a particular tumour if it is an epitope or antigen that is present on said tumour. It need not be present on all cells of the tumour type in question, or on all cells of a particular tumour, or throughout the entire life of the tumour. It need not be specific to the tumour in question. An epitope may be said to be “tumour- associated” in general if it is so associated with any tumour (cancer, neoplasm).

[0186] The tumour-associated epitope may be comprised in a tumour-associated antigen (TAA), or derived from a TAA. In some examples, the epitope is derived from or part of a TAA. In some examples, the antigen may comprise part of a TAA (which includes a tumour-associated epitope). In other examples, the antigen may be a TAA (which includes a tumour-associated epitope). Several TAAs are known.

[0187] In some examples, TAAs may be presented by leukocytes as part of the immune response. Accordingly, in the context of leukocytes, TAAs may be endogenous.

[0188] In some examples, TAA epitopes may be presented by leukocytes as part of the immune response. Accordingly, in the context of leukocytes, TAA epitopes may be endogenous.

[0189] Tumours may be of mesenchymal or epithelial origin. Cancers include cancers of the colon, rectum, cervix, breast, lung, stomach, uterus, skin, mouth, tung, lips, larynx, kidney, bladder, prostate, brain, and blood cells.

[0190] In the case of a “tumour-specific” epitope, the epitope is more frequently associated with that tumour that with other tumours, or with normal cells. Preferably, there should be a statistically significant (p=0.05) difference between its frequency of occurrence in association with the tumour in question, and its frequency of occurrence in association with (a) normal cells of the type from which the tumour is derived, and (b) at least one other type of tumour. An epitope may be said to be “tumour-specific” in general if it is associated more frequently with tumours (of any or all types) than with normal cells. It need not be associated with all tumours.

[0191] In general, tumour-versus-normal specificity is more important than tumour-versus-tumour specificity as (depending on the route of administration and the particular normal tissue affected) higher specificity generally leads to fewer adverse effects. Tumour-versus-tumour specificity is more important in diagnostic as opposed to therapeutic uses.

[0192] The term “specific” is not intended to connote absolute specificity, merely a clinically useful difference in probability of occurrence in association with a pathogen or tumour rather than in a matched normal subject.

[0193] Tumour-associated epitopes include, but are not limited to, peptide epitopes such as those of mutant p53, the point mutated Ras oncogene gene product, her 2 / neu, c / erb2, and the MLIC1 core protein, and carbohydrate epitopes such as sialyl Tn (STn), TF, Tn, CA 125, sialyl Lex, sialyl Leaand P97.

[0194] Carbohydrate epitopes are also of interest. For example, any of three types of tumour- associated carbohydrate epitopes which are highly expressed in common human cancers may be presented. These particularly include the lacto series type 1 and type 2 chains, cancer associated ganglio chains, and neutral glycosphingolipids. Examples of the lacto series Type 1 and Type 2 chains are as follows: Lewis a, dimeric Lewis a, Lewis b, Lewis b / Lewis a, Lewis x, Lewis, y, Lewis a / Lewis x. dimeric Lewis x, Lewis y / Lewis x, trifucosyl Lewis y, trifucosyl Lewis b, sialosyl Lewis x, sialosyl Lewis y, sialosyl dimeric Lewis x, Tn, sialosyl Tn, sialosyl TF, TF. Examples of cancer-associated ganglio chains are as follows: GM3. GD3, GM2, GM4, GD2, GM1 , GD-1a, GD-1b. Neutral sphingolipids include globotriose, globotetraose, globopentaose, isoglobotriose, isoglobotetraose, mucotriose, mucotetraose, lactotriose, lactotetraose, neolactotetraose, gangliotriose, gangliotetraose, galabiose, and 9-O-acetyl- GD3. Numerous antigens of clinical significance bear carbohydrate determinants. One group of such antigens comprises the tumour-associated mucins (Roussel, et al., Biochimie 70, 1471 , 1988).

[0195] Generally, mucins are glycoproteins found in saliva, gastric juices, etc., that form viscous solutions and act as lubricants or protectants on external and internal surfaces of the body. Mucins are typically of high molecular weight (often >1 ,000,000 Dalton) and extensively glycosylated. The glycan chains of mucins are O-linked (to serine or threonine residues) and may amount to more than 80% of the molecular mass of the glycoprotein. Mucins are produced by ductal epithelial cells and by tumours of the same origin, and may be secreted, or cell-bound as integral membrane proteins (Burchell, et al., Cancer Res., 47, 5476, 1987; Jerome, et al., Cancer Res., 51 , 2908, 1991).

[0196] Autoantigens

[0197] In some examples, the epitope is derived from or part of an autoantigen. In other words, the antigen may comprise an auto or self-epitope.

[0198] The term “autoantigen” refers to an endogenous antigen that stimulates the production of autoantibodies, as in an autoimmune reaction. Examples of autoantigens include insulin, GAD, Hsp, nuclear antigens, acetylcholine receptor, myelin basic protein, myelin oligodendrocyte glycoprotein, proteolipid protein, myelin associated glycoprotein, glomular basement membrane protein, thyrotropin receptor, 3-beta-hydroxysteroid dehydrogenase, thyroglobulin, thyroid peroxidase, thyroid stimulating hormone receptor; thyroglobulin, thyroid peroxidase, thyroid peroxidase; transglutaminase, pyruvate dehydrogenase, cytochrome P4502D6, 21 -alpha-hydroxylase, tyrosinase, type IV collagen, and Scl-70.

[0199] In some examples, autoantigens may be presented by leukocytes as part of the immune response. Accordingly, in the context of leukocytes, autoantigens may be endogenous.

[0200] In some examples, autoantigen epitopes may be presented by leukocytes as part of the immune response. Accordingly, in the context of leukocytes, autoantigen epitopes may be endogenous.

[0201] Surface Presented Binding Proteins And Surface Presented Binding Protein-Ligand Complexes In some examples, the surface presented entities described herein may be surface presented binding proteins. The term surface presented binding protein refers to any protein that at least a portion of which is located on the outer surface of a cell and is capable of being bound by or binding to a cognate partner. A “leukocyte surface presented binding protein” refers to an endogenous surface presented binding protein on a leukocyte. For example, surface presented binding proteins may be cell surface receptor proteins presented on the outer surface of a cell. In another example, surface presented binding proteins may be cell adhesion molecules. In some examples, the surface antigens and epitopes thereof described above may be comprised within surface presented binding proteins (e.g. surface presented receptors).

[0202] “Cell adhesion molecules” (CAMs) are a subset of cell surface proteins that are involved in the binding of cells with other cells or with the extracellular matrix (ECM). CAMs help cells stick to each other and to their surroundings. CAMs are crucial components in maintaining tissue structure and function. There are four major superfamilies or groups of CAMs: the immunoglobulin super family of cell adhesion molecules (IgCAMs), Cadherins, Integrins, and the Superfamily of C-type of lectin-like domains proteins (CTLDs). Proteoglycans are also considered to be a class of CAMs.

[0203] “Cell surface receptor" refers to molecules that occur on the surface of cells, interact with the extracellular environment, and typically transmit or transduce (through signals) information regarding the environment intracellularly in a manner that can modulate cellular activity directly or indirectly, e.g., via intracellular second messenger activities or transcription from specific promoters, resulting in transcription of specific genes. One class of cell surface receptors includes membrane bound proteins, or complexes of proteins. Such receptors can have a variety of forms, but in general they comprise at least three domains; (a) a ligand-binding domain, which can be oriented either extracellularly or intracellularly; (b) a membrane-binding domain (usually a transmembrane domain); and (c) a signaling domain, which is responsible for propagating the downstream effects of the receptor.

[0204] Receptors employ a wide variety of means to transduce extracellular signals into the cell. A general mechanism involves a receptor binding a ligand, which activates the receptor to propagate a signal that eventually results in altered protein activity within the cell. Receptors may bind to various cognate partners. In some examples, the cognate partner is a ligand. In some examples, the cognate partner is another surface presented entity. For example, a surface presented binding protein may bind to an extracellular binding partner and / or to a protein that is also comprised within the cell membrane or presented on the surface of the cell. Examples of such cell surface receptors include hormone receptors, steroid receptors, cytokine receptors (such as I L1-a, I L- , IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10. IL-12, IL-15, IL-18, IL-21 , CCR5, CCR7, CCR-1-10, CCL20), chemokine receptors (such as CXCR4), adhesion receptors, growth factor receptors, (including, but not limited to, PDGF-R (platelet derived growth factor receptor), EGF-R (epidermal growth factor receptor), VEGF-R (vascular endothelial growth factor), uPAR (urokinase plasminogen activator receptor), ACHR (acetylcholine receptor), IgE-R (immunoglobulin E receptor), estrogen receptor, thyroid hormone receptor), integrin receptors (pi , 2, 3, 4, 5, 6, a1 , a2, a3, a4, a5, a6), MAC-1 ( 2 and cd11 b), aV 33, opioid receptors (mu and kappa), FC receptors, serotonin receptors (5-HT, 5-HT6, 5-HT7), p-adrenergic receptors, insulin receptors, leptin receptors, TNF receptors, statin receptors, FAS receptor, BAFF receptor, FLT3 ligand receptors, GMCSF receptors, pattern recognition receptors, calcitonin receptor like receptors, G-protein-coupled receptors, complement receptors, leukotriene receptors, prostaglandin receptors, natural cytotoxicity receptors, protein tyrosine phosphatase receptors, C-type lectin receptors, killercell immunoglobulin-like receptors, platelet activating factor receptors, selectins, matrix metalloproteinases, lipid receptors, T-cell receptors B-cell receptors and fibronectin receptors.

[0205] In some examples, surface presented binding proteins include one or more of G-protein- coupled chemokine receptors, Fc-receptors, cytokine receptors, and / or innate immune receptors. For example, when the cell is a granulocyte.

[0206] In some examples, surface presented binding proteins include one or more pattern recognition receptors (PRRs), including scavenger receptors, toll-like receptors (TLRs). For example, CSF1 R, CCR2, CX3CRI , F4 / 80, MerTK, Tim4, scavenger receptor Al, MARCO, CD36, CD163, CD206, Dectin 1 , CD169, TREM2 / DAP12, CD127, CD64, huCRIg(L), CR3, CD14, CD369 and TLR-4. and calcitonin receptor like receptors (CLRs). For example, when the cell is a macrophage or a dendritic cell.

[0207] In some examples, surface presented binding proteins include one or more of pattern recognition receptors (PRRs), (such as Toll-like receptors, C-type lectins, Nod-like receptors, and RIG-like receptors); G-protein-coupled receptors (such as Formyl-peptide receptors, classical chemoattractant receptors, and / or chemokine receptors); Fc-receptors (such as Fcy- receptors, Fca-receptors and / or Fce-receptors); adhesion receptors (such as selectins and selectin ligands, and / or integrins); and / or Cytokine receptors (such as type I cytokine receptors, type II cytokine receptors, IL-1 R family receptors, and / or TNFR family receptors). For example, RAGE, Mac-1 / aM 2, CD40, CD127, SLC44A2, PSGL-1 , or CXCR2. For example, when the cell is a neutrophil. In some examples, surface presented binding proteins include one or more of immunoglobulin receptors (Fc receptors); complement receptors (such as CR1 / CD35, CR3, and / or CD88); cytokine receptors (such as lL-3R, IL-5R, IL-1a, IL-2, IL-4, IFN-a, and TNF-a and / or GM-CSF), leukotriene receptors (such as CysLTI R and CysLT2R; and / or LTB4 receptor); prostaglandin receptors (such as PGD2 type 2 receptor); platelet activating factor receptor (PAF); and tolllike receptors (such as TLR7 and / or TLR8). For example, Fea, FCyRI-lll, FCERI-I I, CD80, CD86, CCR1 , CCR3, CCR5, CCR6, CCR8, CCR9, CXCR2, CXCR4, CD4, CD11 b, CD11c, CD47, CD48, CD50, CD58, CD66, CD127, CD69, CD101 , and F4 / 80. For example, when the cell is a eosinophil.

[0208] In some examples, surface presented binding proteins include one or more of cytokine receptors (such as IL-3R, IL-5R, and / or GM-CSFR); chemokine receptors (such as CCR2 and / or CCR3); complement receptors (such as CD11 b, CD11c, CD35, and / or CD88); prostaglandin receptors (such as CRTH2); Fc receptors (such as FCERI and / or FcyRllb); and / or TLRs. For example, IL-18R, IL-3R, CD127, ST2, FCERI , TLR4, TLR2 / 1 , TLR2 / 6, TLR5 and FcyRllb. For example, when the cell is a basophil.

[0209] In some examples, surface presented binding proteins include one or more of C-type lectin family receptors (such as Ly49 and / or CD94 / NKG2); natural cytotoxicity receptors; Toll-like receptors (such as TLR-1 , TLR-2, TLR-3, TLR-5, TLR-6, TLR-4, TLR-8, TLR-9 and / or TLR-7); Fc receptors (such as FcylllA / CD16); killer-cell immunoglobulin-like receptors; and / or immunoglobulin-like receptors (such as ILT or LIR). For example, NKRP1A, KIR, CD94, NKG2A, TIGIT, CD96, DNAM1 , CD127, CRTAM, NKG2D, NKp46, NKp44, NKp30, CD16, LFA-1 and CD27. For example, when the cell is a natural killer cell.

[0210] In some examples, surface presented binding proteins include one or more of T-cell receptors (TCRs); immunoglobulin receptors (Fc receptors); complement receptors; TNF-receptors (such as CD27 and / or CDw137 / 4-1 BB); protein tyrosine phosphatase receptors (such as CD45); CD4, CD5, CD6, CD8, CD28, CD43, and / or CD152 (CTLA-4). For example CD28, CD2, CD4, CD127, TCR proteins and complexes, LFA-1 , Integrin b7, and CD45. For example, when the cell is a T cell.

[0211] In some examples, surface presented binding proteins include one or more of immunoglobulin receptors (such as CD14, CD16, FCER1A); N-formyl peptide receptor 2; chemokine receptors (such as GPR159, CXCR4, IL17RA, C3AR1 , C5AR1 , CCR1 CCR2, CCR4, CCR5, CCR8, CX3CR1 , CXCR1 , CXCR2, CXCR3, CXCR4, and / or CXCR6); poliovirus receptor; cytokine receptors (such as IL10RA, IL10RB, IL13RA1 , IL15RA, IL17RA, IL2RB, IL2RG, IL3RA, IL6R, IL6ST, and / or IL9R); protein tyrosine phosphatase receptor Type C; Scavenger receptor class B member 1 ; Angiopoietin-1 receptor; Macrophage colony-stimulating factor 1 receptor; colony-stimulating Factor 2 Receptor Beta; Granulocyte-macrophage colonystimulating factor receptors; granulocyte colony-stimulating factor receptor; and / or toll-like receptors (such as TLR-6, TLR-1 , TLR-2, and / or TLR-3). For example, CD40, CD37, CD81 , CD87, CD9, CD127, CD37, CD82, CD80, CD86, CCR7, CCR1 , CCR2, CD369, TLR7, TLR9, NKG2D and c-Kit. For example, when the cell is a monocyte.

[0212] In some examples, surface presented binding proteins include one or more of integrins (such as pi , (32, and / or P3); toll-like receptors (such as TLR 1 , TLR-2, TLR-4 and / or TLR-6); matrix metalloproteinases (MMPs); selectins (such as P-selectin, C-type lectin-like receptor-2 (CLEC-2) and / or CD 72); transmembrane receptors (such as Adenosine Diphosphate (ADP) receptors and / or thrombin receptors); prostaglandin receptors (such as thromboxane receptors, prostacyclin (PGI2), PGD2 and / or PGE2 receptors); lipid receptors (such as platelet activating factor (PAF) receptors ad / or Lysophosphatidic acid receptors); immunoglobulin receptors (such as GPVI, FcyRIIA (CD32), FCERI receptors (CD23), Junctional adhesion molecules (JAM), Platelet-endothelial cell adhesion molecule-1 (PECAM-1 , CD31), and / or TREM-Like transcript-1 (TLT-1); protein tyrosine kinase receptors (such as thrombopoietin receptors, leptin receptors, tyrosine kinase with immunoglobulin and epidermal growth factor homology-1 receptors; insulin receptors, and / or platelet-derived growth factor receptors). For example, a2pi , CAR, CLEC-2, GPIIbllla, TLR 1 , TLR-2, TLR-4, TLR-6, GPIba, a2b 3, GPVI, DC-SIGN, CR2, CCR1 , CCR3, CCR4, CXCR4, CLEC2, CD36, SR-B1 , gC1qR, C3aR, C5aR, Siglec7, Siglec-9, SHT2A, CD127, PAR4, PAR1 , avp3 and P-selectin. For example, when the cell is a thrombocyte.

[0213] In some examples, surface presented binding proteins include one or more of chemokine receptors (such as GPR159, CCR4, CXCR4, CXCR5 and / or CCR6); B cell receptors (BCR); TNF receptors (such as CD40); immunoglobulin receptors (such as FCGR2A, FCGR2B, and / or FCRL5); tyrosine-protein kinase receptors (such as FLT3); Toll-like receptors; cytokine receptors (such as IL10RA, IL10RB, IL13RA1 , IL15RA, IL17RA, IL18R1 , IL1 R1 , IL1 RAP, IL2RB, IL2RG, IL4R, IL5RA, IL6R, IL6ST, and / or IL7R). For example, TACI, BCMA, CD20, CD80, CD86, BCRs, CD40, CD19, CD22, TLR-1 , TLR-2, TLR-4, TLR-5, TLR-6, TLR-8, TLR- 10, CR1 , CR2, Dec-1. CR3, CR4, CD127 and BR3. For example, when the cell is a B- lymphocyte.

[0214] In some examples, the surface presented binding proteins are preserved or maintained bound to their cognate binding partner (i.e. bound to a ligand). Ligands for binding proteins such as those described above are known in the art. In addition, ligands of binding proteins (such as the receptors described herein) may be determined by any suitable methods known in the art, such as fluorescence polarization (FP), fluorescence resonance energy transfer (FRET), surface plasmon resonance (SPR), radioligand assays, immunoprecipitation, and solid phase binding assays.

[0215] Surface Presented Ligands

[0216] In some examples the methods provided herein preserve ligands presented on the surface of a cell. The term “surface ligand” or “cell surface ligand” refers to a chemical or structure which will bind to a cognate receptor (such as a receptor protein). A “leukocyte surface ligand” refers to an endogenous surface ligand on a leukocyte. In some examples, the epitopes and antigens described above may be comprised within a surface presented ligand.

[0217] Cell surface presented ligands, such as leukocyte surface presented ligands, include protein molecules (such as lipoproteins, glycoproteins, receptor proteins, glycopeptides), lipids, and / or carbohydrates.

[0218] For example, ligands may include immune checkpoint receptor ligands, lectins, sialic acid molecules, and / or p-glucan carbohydrates.

[0219] For example, ligands may include one or more of LLT1 , MHC proteins, HLA proteins, CD155, CD112, CD113, CD111 , CD112, Necl-2, MIC / B, ULBP1-6, Rae-1 , MULTI , H60, pathogen proteins (such as viral or bacterial surface presented proteins), B7-H6, BAT3, ICAMs, CD70, HVEM, CEACAM-1 , phosphatidylserine, PD-L1 , PD-L2, CD47, CD24, Jagged 1 , Jagged 2, Delta-like ligands, CD40L, GPVI, allb 3, GPIba, selectins, CD47, PSGL-1 , MadCAM-1 , CD34, GlyCAM-1 , CD80, CD48, CD63, LYVE-1 , podoplanin, VCAMs, FTL3 ligand, CD28, NKG2DL, JAM-A, JAM-B, JAM-C, CD62L, CD137L, ephrin-A, and ephrin-B.

[0220] It will be understood that in some examples the surface presented proteins described above and / or post-translational modifications thereof may also be ligands.

[0221] Preservation of Surface-Presented Entities

[0222] The methods described herein preserve surface presented epitopes, binding proteins, binding protein-ligand complexes and / or ligands of cells (i.e. surface presented entities). Preservation or preserving of epitopes refers to maintaining one or more of the epitopes presented at a cell’s surface upon, during and / or after (i.e. after release from a hydrogel) entrapment in a hydrogel as described herein. Maintenance of the surface presented epitopes is used to refer to epitopes that remain intact and functional on the surface of the cell or cells. Functionality of an epitope may be defined by the ability of the epitope to be bound by a cognate partner, such as an antibody. Functionality may also be defined by the ability of the epitope to elicit an antigenic response in vitro or in vivo. Methods to detect binding and ergo functionality of an epitope (or an antigen that includes the epitope) are well known in the art. Examples of such methods include enzyme-linked immunosorbent assay (ELISA), Enzyme-linked immunospot (ELISPOT), Western blot methods, immunocytochemistry methods, flow cytometry, fluorescence-activated cell sorting (FACS), immunoprecipitation methods and radioimmunoassay methods.

[0223] Preservation or preserving of ligands refers to maintaining one or more of the ligands presented at a cell’s surface upon, during and / or after (i.e. after release from a hydrogel) entrapment in a hydrogel as described herein. Maintenance of the surface presented ligands is used to refer to ligands that remain intact and functional on the surface of the cell or cells. Functionality of a ligands may be defined by the ability of the ligands to be bound by a cognate partner, such as a cognate receptor protein.

[0224] Preservation or preserving of surface presented binding proteins refers to maintaining one or more of the binding proteins presented at a cell’s surface upon, during and / or after (i.e. after release from a hydrogel) entrapment in a hydrogel as described herein. Maintenance of the surface presented binding proteins is used to refer to binding proteins that remain intact and functional on the surface of the cell or cells. Functionality of a binding protein may be defined by the ability of the binding protein to bind to a cognate partner, such as a cognate ligand.

[0225] Preservation or preserving of surface presented binding protein-ligand complexes refers to maintaining one or more of the binding protein-ligand complexes presented at a cell’s surface upon, during and / or after (i.e. after release from a hydrogel) entrapment in a hydrogel as described herein. Maintenance of the surface presented binding protein-ligand complexes is used to refer to binding protein-ligand complexes that remain intact and functional on the surface of the cell or cells. Functionality of a binding protein-ligand complex may be defined by the ability of the binding protein-ligand complex to remain in a state wherein the binding protein is bound to its cognate ligand.

[0226] In some examples, the methods provided herein preserve at least 50% of each epitope present on a cell’s surface in a functional state. For example, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 99% of each epitope at the cell’s surface in a functional state relative to a control.

[0227] In some examples, additionally or alternatively, the methods provided herein preserve at least 50% of all epitopes present on a cell’s surface in a functional state. For example, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 99% of all epitopes at the cell’s surface in a functional state relative to a control.

[0228] In some examples, the methods provided herein preserve at least 50% of each ligand present on a cell’s surface in a functional state. For example, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 99% of each ligand at the cell’s surface in a functional state relative to a control.

[0229] In some examples, additionally or alternatively, the methods provided herein preserve at least 50% of all ligands present on a cell’s surface in a functional state. For example, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 99% of all ligands at the cell’s surface in a functional state relative to a control.

[0230] In some examples, the methods provided herein preserve at least 50% of each binding protein present on a cell’s surface in a functional state. For example, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 99% of each binding protein at the cell’s surface in a functional state relative to a control.

[0231] In some examples, additionally or alternatively, the methods provided herein preserve at least 50% of all binding proteins present on a cell’s surface in a functional state. For example, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 99% of all binding proteins at the cell’s surface in a functional state relative to a control.

[0232] In some examples, the methods provided herein preserve at least 50% of each binding protein-ligand complex present on a cell’s surface in a functional state. For example, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 99% of each binding protein-ligand complex at the cell’s surface in a functional state relative to a control.

[0233] In some examples, additionally or alternatively, the methods provided herein preserve at least 50% of all binding protein-ligand complexes present on a cell’s surface in a functional state. For example, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 99% of all binding protein-ligand complexes at the cell’s surface in a functional state relative to a control.

[0234] The number of functional epitopes, binding proteins, binding protein-ligand complexes and / or ligands at a cell’s surface after release from a hydrogel may be provided in comparison to a control. For example, the control may be a cell or cells that have not undergone entrapment in a hydrogel as described herein (i.e. have not been subjected to a method of the invention). The level of functional epitopes, binding proteins, binding protein-ligand complexes and / or ligands may be expressed as a percentage of each or all epitopes, binding proteins, binding protein-ligand complexes and / or ligands present at a cell’s surface after entrapment and release in comparison to the total number of each or all epitopes, binding proteins, binding protein-ligand complexes and / or ligands present on the cell’s surface prior to entrapment. In some examples, the number of functional epitopes, binding proteins, binding protein-ligand complexes and / or ligands may be in comparison to a cell that has been preserved using a method of the prior art. For example the control may be a cell or cells that have been preserved by a method of the prior art. For example, by comparison of the total number of each or all epitopes, binding proteins, binding protein-ligand complexes and / or ligands present on the cell’s surface after entrapment and release from a hydrogel as described herein to the total number of each or all epitopes, binding proteins, binding protein-ligand complexes and / or ligands present on the cell’s surface after preservation using a method of the prior art.

[0235] In some examples, preserving epitopes, binding proteins, and / or ligands alternatively or additionally includes maintaining a level expression of epitopes, binding proteins, and / or ligands by the cell or cells. For example, the level of expression of each or all epitopes binding proteins, and / or ligands prior to carrying out methods of the invention may be substantially the same or similar to level of expression after entrapment in a hydrogel as described herein. In some examples, the level of expression of epitopes, binding proteins, and / or ligands may be in comparison to a cell that has been preserved using a method of the prior art. For example, the control may be a cell or cells that have been preserved by a method of the prior art. For example, by comparison of the level of expression of each or all epitopes, binding proteins, and / or ligands of the cell or cells after entrapment and release from a hydrogel as described herein to the level of expression of each or all epitopes, binding proteins and / or ligands expressed after preservation using a method of the prior art.

[0236] In some examples, the methods provided herein preserve at least 50% of expression of each epitope. For example, a level of expression of each epitope at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 99% relative to a control.

[0237] In some examples, alternatively or additionally the methods provided herein preserve at least 50% of expression of all epitopes. For example, a level of expression of all epitopes at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 99% relative to a control.

[0238] In some examples, the methods provided herein preserve at least 50% of expression of each ligand. For example, a level of expression of each ligand at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 99% relative to a control.

[0239] In some examples, alternatively or additionally the methods provided herein preserve at least 50% of expression of all ligands. For example, a level of expression of all ligands at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 99% relative to a control.

[0240] In some examples, the methods provided herein preserve at least 50% of expression of each binding protein. For example, a level of expression of each binding protein at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 99% relative to a control.

[0241] In some examples, alternatively or additionally the methods provided herein preserve at least 50% of expression of all binding proteins. For example, a level of expression of all binding proteins at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 99% relative to a control.

[0242] Methods of determining the level of expression of epitopes (or antigens including the epitopes), binding proteins and / or ligands are well known in the art. For examples, methods include flow cytometry, Northern blotting, quantitative polymerase chain reaction (qPCR), DNA microarray, and RNA-Seq.

[0243] In some examples, preserving epitopes, binding proteins, binding protein-ligand complexes and / or ligands alternatively or additionally includes maintaining presentation of each or all of the surface epitopes, binding proteins and / or ligands at the cell’s surface. That is to say that the amount of expressed epitope (or antigen including the epitope), binding protein and / or ligand by a cell subjected to the methods of the invention that is processed by the cell and presented on the cell’s surface is maintained at a level that is the same as or similar to a cell that has not undergone a method of the invention. In some examples, the level of preservation may be compared to a cell or cells that have been preserved using methods of the prior art.

[0244] The level of presented epitopes, binding proteins, binding protein-ligand complexes and / or ligands may be determined by comparing the level of expression of an epitope, binding protein and / or ligand to the amount of epitope, binding protein and / or ligand present on a cell’s surface. The level of expression and amount of epitope, binding protein and / or ligand presented on cell’s surface may be determined using the methods described above.

[0245] Hydrogels

[0246] The invention has been exemplified using alginate hydrogels. However, the invention also applies to other reversibly cross-linked hydrogels with the equivalent mechanical properties. Alternative hydrogels that may be equally used within the context of the invention are described in more detail below.

[0247] The hydrogels provided herein are reversibly cross-linked hydrogels. As used herein a “reversibly cross-linked hydrogel” refers to a hydrogel that is formed by reversible cross-linking (i.e. the cross-linking can be reversed such that the hydrogel reverts back to a solution). Reversal of the cross-linking enables the cells to be released from the hydrogel (e.g. at the point of use / after transportation or storage is complete). Examples of reversibly cross-linked hydrogels are well known in the art. Accordingly, suitable hydrogels may readily be identified by a person of skill in the art.

[0248] The hydrogel referred to herein comprises a hydrogel-forming polymer (i.e. at least one hydrogel forming polymer) having a cross-linked or network structure or matrix; with an interstitial liquid. The hydrogel is capable maintaining the structural integrity and / or functionality of the cells and surface epitopes, binding proteins, binding protein-ligand complexes and / or ligands thereof contained within. Preferably, the hydrogel is semi- permeable.

[0249] The term "hydrogel-forming polymer" refers to a polymer which is capable of forming a crosslinked or network structure or matrix under appropriate conditions, wherein an interstitial liquid and cells may be retained within such a structure or matrix. The hydrogel may comprise internal pores.

[0250] Initiation of the formation of the cross-linked or network structure or matrix may be by any suitable means, depending on the nature of the polymer.

[0251] The polymer will in general be a hydrophilic polymer. It will be capable of swelling in an aqueous liquid. In one example, the hydrogel-forming polymer is collagen. In this example, the collagen hydrogel comprises a matrix of collagen fibrils which form a continuous scaffold around an interstitial liquid and the cells. Dissolved collagen may be induced to polymerise / aggregate by the addition of dilute alkali to form a gelled network of cross-linked collagen fibrils. The gelled network of fibrils supports the original volume of the dissolved collagen fibres, retaining the interstitial liquid. General methods for the production of such collagen gels are well known in the art (e.g. W02006 / 003442, W02007 / 060459 and W02009 / 004351). The collagen which is used in the collagen gel may be any fibril-forming collagen.

[0252] Examples of fibril-forming collagens are Types I, II, III, V, VI, IX and XI. The gel may comprise all one type of collagen or a mixture of different types of collagen. Preferably, the gel comprises or consists of Type I collagen. In some examples of the invention, the gel is formed exclusively or substantially from collagen fibrils, i.e. collagen fibrils are the only or substantially the only polymers in the gel. In other examples of the invention, the collagen gel may additionally comprise other naturally-occurring polymers, e.g. silk, fibronectin, elastin, chitin and / or cellulose. Generally, the amounts of the non- collagen naturally-occurring polymers will be less than 5%, preferably less than 4%, 3%, 2% or 1 % of the gel (wt / wt). Similar amounts of non-natural polymers may also be present in the gel, e.g. peptide amphiphiles, polylactone, polylactide, polyglycone, polycaprolactone and / or phosphate glass.

[0253] In some examples, the hydrogel-forming polymer is alginic acid or an alginate salt of a metal ion. Preferably, the metal is a Group 1 metal (e.g. lithium, sodium, or potassium alginate) or a Group 2 metal (e.g. calcium, magnesium, barium or strontium alginate). In some examples, the polymer is calcium alginate or sodium alginate or strontium alginate. In some examples, the polymer is calcium alginate.

[0254] One factor which determines alginate gel permeability is the mannuronic (M) and guluronic (G) acid contents of the gel. Gels with a high M:G ratio have a small intrinsic pore size. The M:G ratio may be manipulated to increase the permeability of gels as necessary to improve the preservation of the cells within the hydrogel. In some examples, the G content of the alginate gel is 0- 30%. In some examples, the M content is preferably 30- 70%. In some examples, the gel is an alginate gel with a M content of 50-70% or 60-70% and the gel additionally comprises or a pore enhancer (also referred to herein as a porogen). In some examples, the pore size increasing agent is hydroxyethyl cellulose (HEC). In this example, HEC may be used in the preparation of the hydrogel; it is then completely, substantially completely or partially removed from the hydrogel prior to use. Concentrations of HEC in the hydrogel (during preparation) include 0.5 - 3.0% HEC, for example, 1.0 - 2.5%, and 1 .2 - 2.4% HEC. In some examples, the concentration of HEC in the hydrogel (during preparation) is 1.2% or 2.4%. (Concentrations are given as weight %). The HEC may be suspended in the gels as micelles. Removal of the HEC may be attained by washing the hydrogel in a suitable aqueous solvent or buffer, e.g. tissue culture medium. In some examples, the hydrogelforming polymer is an alginate. In some examples, the cells can be coated first with a different hydrogel-forming polymer as described herein followed by a further coating of an alginate. In other examples, the hydrogel forming polymer is a mixture of alginate and another hydrogelforming polymer. In some examples, the alginate is modified (e.g. with peptides).

[0255] In some examples, the hydrogel-forming polymer is a cross-linked acrylic acid-based (e.g. polyacrylamide) polymer.

[0256] In some examples, the hydrogel-forming polymer is a cross-linkable cellulose derivative, a hydroxyl ether polymer (e.g. a poloxamer), pectin or a natural gum.

[0257] In some examples, the hydrogel is not thermo-reversible at physiological temperatures, i.e. the sol-gel transition of the hydrogel cannot be obtained at a temperature of 0 - 40 °C.

[0258] The structure of the hydrogel may be changed by varying the concentration of the hydrogel forming polymer in the hydrogel. The structure affects the robustness of the gel and its handling properties. Preferred concentrations of the hydrogel-forming polymer in the hydrogel are 0.1- 5% (weight of polymer to volume of interstitial liquid), and include for example 0.1- 0.4%, 0.2-0.4%, 0.4-0.5%, 0.5-0.7%, 0.7-1.1 %, 1.1 -1.3%, 1.3-2.2%, 2.2-2.6%, 2.6-3.0%, 3.0- 3.5%, 3.5-4.0%, 4.0-4.5% and 4.5-5.0% (or any combination thereof e.g. 0.1 -0.5%, 0.2 to 0.7% etc).

[0259] In one example, the viscosity of the non-gelled hydrogel solution is up to 500 mPa.s, Optionally, the viscosity of the non-gelled hydrogel solution is between 5 and 200 mPa.s (preferably between 5 and 100 mPa.s).

[0260] In other examples, the concentration of the hydrogel-forming polymer in the hydrogel is above 0.25%, 0.3%, 0.4%, 0.5% or 0.6%. In other examples, the concentration of the hydrogel forming polymer in the hydrogel is below 5%, 4.5%, 4.0%, 3.5%, 3.0%, 2.6%, 2.4%, 1.5%, 1.4%, 1.3% or 1.2%. In some preferred examples, the concentration of the hydrogel-forming polymer in the hydrogel is about 0.3%, about 0.6% or about 1.2%. In some particularly preferred examples, the concentration of the hydrogel-forming polymer in the hydrogel is about 1 %. In some particularly preferred examples, the hydrogel is formed from about 1% sodium alginate or from about 1% calcium alginate. In some examples, the gelling of the hydrogel is facilitated using a compound comprising a multivalent metal cation, e.g. using calcium chloride. In particular, calcium chloride (e.g. 50- 200 mM calcium chloride, preferably 75-120 mM calcium chloride) may be used to gel alginate hydrogels.

[0261] In other examples, an alternative metal chloride is used, e.g. magnesium or barium or strontium chloride. Alternatively, other multivalent cations may be used, e.g. La3+ or Fe3+.

[0262] In some examples, the multivalent cation is selected from one or more of calcium, strontium, magnesium, barium, iron, or lanthanum or salts or solvates thereof. In some examples, the multivalent cation is a combination of two or more multivalent cations. For example, the multivalent cation may be a combination of calcium and strontium.

[0263] In some examples, the multivalent cation may be included in the hydrogel. Therefore, in some examples, the hydrogel comprises one or more multivalent cations. For example, hydrogel may include calcium and strontium.

[0264] The invention further provides a process for preparing a hydrogel, comprising the step of gelling the hydrogel-forming polymer in the presence of a Group 2 metal salt selected from the group consisting of magnesium and calcium salts.

[0265] In some examples, the hydrogel comprises cross-linked alginate. For example, the hydrogel may comprise cross-linked calcium-alginate, strontium-alginate, barium-alginate, magnesium alginate and / or sodium-alginate. In one example, the hydrogel may comprise cross-linked calcium-alginate, optionally wherein the hydrogel comprises cross-linked calcium-alginate and sodium-alginate. Accordingly, in one example, a reversibly cross-linked calcium-alginate hydrogel (optionally with sodiumalginate) is provided, wherein the hydrogel comprises a sample, wherein the sample comprises cells as described herein.

[0266] In any of the examples described herein, the cross-linked alginate may be from about 0.1 % (w / v) to about 5.0% (w / v) calcium alginate. For example, the cross-linked alginate may be from about 0.5% (w / v) to about 3.0% (w / v), 1.0 % (w / v) to about 2.5% (w / v), about 1.5% (w / v) to about 2.0% (w / v) calcium alginate, or any range therebetween.

[0267] The interstitial liquid may be any liquid in which polymer may be dissolved and in which the polymer may gel. Generally, it will be an aqueous liquid, for example an aqueous buffer. Suitable aqueous buffers are described elsewhere herein. In some examples, the interstitial liquid may include blood plasma or a portion thereof. Blood plasma refers to the liquid component of blood comprising water, sugars, fats, proteins and salts. In some examples, the interstitial liquid is blood plasma. In some examples, the interstitial liquid may include one or more of anticoagulant agents selected from anticoagulant citrate dextrose solution (ACD), sodium citrate, sodium heparin, lithium heparin and / or ethylenediaminetetraacetic acid (EDTA). In some examples, the interstitial liquid is blood plasma and includes one or more anticoagulant agents.

[0268] The liquid may contain an antibiotic. In some examples, the hydrogel is sterile, i.e. aseptic. In some examples, the liquid does not contain animal-derived products, e.g. foetal calf serum or bovine serum albumin.

[0269] The hydrogels may be produced in any suitable size. For ease of transportation, however, the hydrogels may be less than 10 cm in length, less than 8, 5, 3, or 1 cm in length. The thickness of the hydrogel may be 0.1 - 3 cm, for example, 0.1 - 2 cm, 0.5 - 1 .5 cm, or about 1.0 cm.

[0270] The volume of the hydrogels may be 50 pl- 500 ml, for example, 0.1 - 300 ml, 0.2 - 200 ml or 0.2 - 100 ml.

[0271] The volume of the hydrogels may be 0.2 - 100 ml, for example, 0.2 - 50ml, 0.2 - 25 ml or 0.2 - 10 ml. In some examples, the volume of the hydrogel is 0.4 - 5ml, for example, 0.4 - 4ml, or 0.4 - 3ml. In some examples, the volume may be about 0.5ml or about 3ml.

[0272] In some examples, the hydrogel is in the form of a thin layer, disc or sheet. Hydrogels in such forms are shown herein to enhance cell viability during hypothermic storage. In some examples, the gel is in the form of a disc or thin layer. The disc may for example, have a diameter of 5-50 mm or 10-50 mm, for example, 10-30 mm, 15-25 mm, or about 19 mm. The thickness of the thin layer, disc or sheet may be 0.1 - 5mm, for example, about 0.5-2.0 mm, about 1.0 or 1.5 mm, or about 1 , 2, 3, 4 or 5 mm. In some examples, the final volume of hydrogel in the disc is about 200 pl to 1 ml, for example, 200-600 pl, 300-500 pl or 400-450 pl. With regard to the discs, the hydrogel polymer concentration may be about 1.2% due to the increased structural stability provided by this concentration. In some examples, the hydrogel (e.g. a disc) is an uncompressed hydrogel, i.e. it has not been subjected to an axial compressing force.

[0273] In some examples, the hydrogel is in the form of a block. A block refers to a piece of gel that is three dimensional portion of gel which in comparison to a sheet has a relatively large thickness. The block may be any shape such as cuboid, cylindrical, spherical or pyramidal. In some examples, the block has a size configured to be received within a receptacle. For example, the receptacle may be a tube, a flask, a dish, a vessel or a plate. For example, the receptacle may be a blood collection tube.

[0274] The hydrogels described herein may be formed using any suitable means. One non-limiting means for forming the hydrogels described herein in is the use of reagents available in the field, for example SwabReady™. In this example, gel beads comprising calcium-alginate are used and additional alginate is added with the sample to induce further crosslinking. These hydrogels comprise two hydrogel forming polymers (sodium alginate and calcium alginate). Such hydrogels are clearly encompassed by the claims, as they comprise a reversibly crosslinked hydrogel.

[0275] The method include preparing a sample comprising cells for storage or transportation from a first location to a second location or specified location. The method comprises the steps of: (a) contacting the cells or sample including the cells with a hydrogel-forming polymer; and (b) polymerising the polymer to form a reversibly cross-linked cell-containing hydrogel.

[0276] In one example, the method includes preserving leukocyte surface presented entities in a sample comprising one or more leukocytes. The method comprises the steps of: (a) contacting the sample comprising one or more leukocytes with a hydrogel-forming polymer; and (b) polymerising the polymer to form a reversibly cross-linked leukocyte-containing hydrogel wherein at least one or more leukocytes is entrapped or encapsulated in the hydrogel.

[0277] Suitable samples are described elsewhere herein.

[0278] The cells or sample containing the cells (e.g. a whole blood sample) may be contacted with a hydrogel-forming polymer using any appropriate means. For example, cells or the sample may be mixed with a solution that contains the hydrogel forming polymer (prior to polymerization / aggregation or prior to crosslinking of a hydrogel-forming polymer). As would be clear to a person of skill in the art, contacting with “a hydrogel-forming polymer” encompasses contacting the cells or sample one hydrogel-forming polymer, or more than one (e.g. two) hydrogel-forming polymers. For example, the cells or sample may be contacted with strontium alginate and calcium alginate during formation of the hydrogel.

[0279] The cells or samples may be contacted with the hydrogel-forming polymer whilst within a sealable receptacle (such that e.g. once the hydrogel is formed, the receptacle can be sealed ready for storage and / or transportation), or it may be contacted with the hydrogel forming polymer before the cells or sample are placed in a sealable receptacle. Suitable receptacles are described elsewhere herein.

[0280] The method then comprises polymerising the cell-polymer to form a reversibly cross-linked cell-containing hydrogel wherein the cells are within the hydrogel. Methods for polymerising the cell-polymer to form a reversibly cross-linked cell-containing hydrogel are well known in the art, and differ depending on the polymer used. For example, polymerisation of an alginate solution (to form an alginate hydrogel) may be induced by a chemical agent such as calcium chloride.

[0281] As used herein, the terms “polymerising” and “gelling” the hydrogel are used interchangeably to refer to the change in state of the hydrogel-forming polymer from a liquid to a hydrogel.

[0282] The hydrogel is gelled under appropriate cell-compatible conditions, i.e. conditions which are not detrimental or not significantly detrimental to the structural integrity and / or functionality of the cells and the surface epitopes, binding proteins, binding protein-ligand complexes and / or ligands thereof. In some examples, the hydrogels are prepared under cGMP (current Good Manufacturing Practice) conditions.

[0283] Within the context of the invention, the cells that are entrapped or encapsulated by a hydrogel of the invention may be packaged in a sealed receptacle. As used herein, a “sealed receptacle” refers to a container that can maintain a seal against the continuous flow of gases or liquids. For example, the sealed receptacle may be a watertight and / or air-tight container e.g. a plastic container. Non-limiting examples of appropriate sealed receptacles include a sealed vial or cryovial or tissue culture flask, optionally together with an appropriate buffer (e.g. a salt based buffer). In other examples, the hydrogel may be contained within a sealed bag or blood collection tube.

[0284] In one example, the sealed receptacle is selected from a tube, a flask, a dish, a vessel or a plate (e.g. a plate comprising a plurality of wells). For example, the plate may be selected from a 4-, 6-, 8-, 12-, 24-, 48-, 96-, 384-, 1536- well plate. Appropriate receptacles are well known in the art. The receptacle may be sealed using a lid (e.g. a screw fit lid) or another means (e.g. adhesive film, or tape etc).

[0285] In one example, the method may comprise mixing the cells (e.g. cells in a whole blood sample) with an aqueous buffer prior to step (a).

[0286] The method may further comprise sealing the cell-containing hydrogel into a receptacle for storage or transportation from the first location to the second location. The receptacle within which the cell-containing hydrogel was formed may be a receptacle that is suitable for storage or transportation from the first location to the second location - in this example, the method may merely comprise sealing the hydrogel into the receptacle in which it was formed. In another example, the method may comprise placing the formed hydrogel into a suitable receptacle, before sealing the receptacle. In this context, the method may comprise packaging and sealing the cell -containing hydrogel into the receptacle for storage or transportation from the first location to the second location. Examples of suitable receptacles are discussed elsewhere and include a vial, tube, flask, dish, vessel or plate. Several means for sealing the cell-containing hydrogel into the receptacle are known in the art (e.g. the using a lid, adhesive film, or tape etc).

[0287] Accordingly, the cells may be placed within the receptacle prior to step (a) of the method e.g. the hydrogel-forming polymer may be contacted with the cells whilst the cells are located within the receptacle that is suitable for storage or transportation. Alternatively, the cells may be placed within the receptacle after step (a) of the method e.g. the hydrogel-forming polymer may be contacted with the cells i.e. in a sample (and optionally polymerised as per step (b)) before the cells are placed within the receptacle that is suitable for storage or transportation.

[0288] The hydrogel is gelled under appropriate cell-compatible conditions, i.e. conditions which are not detrimental or not significantly detrimental to the viability of the cells.

[0289] For storage, transportation or delivery of the cells in the hydrogel, the hydrogel must be appropriately packaged. The methods of the invention therefore comprises packaging the cellcontaining hydrogel in a receptacle. for storage or transportation from the first location to the second location and sealing the receptacle. Suitable receptacles have been described elsewhere herein.

[0290] The cell-containing hydrogel may be in contact with (e.g. fully or partially immersed in) an appropriate media in the sealed / sealable receptacle. Suitable media include cell or tissue culture media, e.g. supplemented DMEM media.

[0291] The method may be used to store and / or transport the cell-containing hydrogel for any suitable period of time. For example, the cell-containing hydrogel may be stored and / or transported for at least 6 hours, at least 12 hours, at least 24 hours. Typically, the cell -containing hydrogel may be stored and / or transported for at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days etc. For example, the cell - containing hydrogel may be stored and / or transported for at least 3 days, or at least 5 days. For example, the cell -containing hydrogel may be stored and / or transported for 3 days. The cell -containing hydrogel may be stored and / or transported for at least 7 days etc. The cellcontaining hydrogels may be stored and / or transported for up to 10 or 20 weeks. Preferably, the cells are stored in the hydrogel for up to 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 weeks before being released from the hydrogels. More preferably, the cells are stored in the hydrogel for up to 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 days before being released from the hydrogels.

[0292] In some examples, the cell-containing hydrogel or method may be used to store (and therefore preserve surface epitopes, binding proteins, binding protein-ligand complexes and / or ligands of cells) the cell-containing hydrogel for any suitable period of time. For example, the cellcontaining hydrogel or method may be used to store samples collected from patients until the sample can be analysed. For example, storing samples at a research clinic before scheduling their processing. For example, the cell-containing hydrogel may be stored for at least 10 minutes, at least 20 minutes, at least 30 minutes, at least 40 minutes, at least 50 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 9 hours, at least 10 hours, at least 11 hours, at least 12 hours, at least 24 hours, at least 36 hours, at least 48 hours, at least 72 hours or more. For example, least 24 hours, at least 36 hours, at least 48 hours, or at least 72 hours. In some examples, the cell-containing hydrogel may be stored for about 24 hours. In some examples, the cell-containing hydrogel may be stored for about 72 hours.

[0293] The cell described herein may be transported within the hydrogel (and sealed receptacle) by any suitable means, e.g. by post or courier, which might include transportation by automotive means, e.g. by car, van, lorry, motorcycle, aeroplane, etc. Preferably, the transportation is by post or courier.

[0294] The second location or designated location is preferably a location which is remote from the first location, e.g. at least 1 mile, preferably more than 5 miles, from the first location. In some examples, the second location is with the same building, facility or complex. For example, the second location be a different department. For example, the second location may be about 1 m, 5m, 10m, 100m, 200m, 300m, 400m, 500m, or more from the first location.

[0295] Transportation from a first location to a second location or specified location may take at least 10 minutes, at least 20 minutes, at least 30 minutes, at least 40 minutes, at least 50 minutes, at least 1 hour, at least 2 hours, at least 5 hours, at least 12 hours, at least 24 hours etc. For example, transportation from a first location to a second location or specified location may take about 3 days.

[0296] In some examples, the cells within the hydrogels (and sealed receptacle) are stored or transported under cell culture conditions (e.g. about 37 °C, about 5% CO2 and about 95% humidity). In some examples, they are stored or transported under chilled conditions, e.g. 4- 6 °C, preferably about 4 °C. In a particular example, they are refrigerated when stored or transported (which is defined as from 2-8 °C (Ell Pharmacopoeia)). In another example, they are stored or transported cool (defined as from 8-15°C)).

[0297] In other examples, they are stored or transported under ambient conditions, e.g. 10-25 °C, preferably 15-20 °C. In some examples, the ambient temperature may be up to 30 °C (i.e. 10 to 30°C), or even up to 40 °C. In yet other examples, they are stored or transported at about 37 °C.

[0298] In some examples, they are stored or transported at Controlled Room Temperature (CRT) (which is defined as from 15 to 25°C). They may be stored or transported cool or at CRT (i.e. from 8 to 25°C).

[0299] In yet other examples, they are stored or transported at hypothermic temperatures (i.e. below about 35 °C, typically in the range of O to 32 °C). In one example, they are stored or transported between CRT and 32°C (i.e. 15 to 32°C). In another example, they are stored or transported cool, at CRT or up to 32°C (i.e. from 8 to 32°C).

[0300] In some examples, the methods include fulfilling an order or request. The order or request may be received by any suitable means, e.g. via the internet, email, text message, telephone or post.

[0301] The methods described herein may further comprise dispatching the sealed receptacle for transportation from the first location to the second location or to a specified location. As used herein, “dispatching” refers to releasing the receptacle for transport (e.g. releasing the receptacle to the courier for transport / delivery to the intended destination). Dispatch therefore does not include transport of the sealed receptacle to the second location per se.

[0302] Aspects of the invention described elsewhere (e.g. suitable receptacles, hydrogels, nucleic acids, buffers) apply equally here.

[0303] The hydrogel referred to herein is one from which the cells can be released. In other words, after the preservation or storage and / or transport of the cells contained therein, the hydrogel is capable of being dissociated thus allowing the release or removal of all or substantially all of cells which were previously retained therein. The hydrogel is dissociated under appropriate cell-compatible conditions, i.e. conditions which are not detrimental or not significantly detrimental to the cells.

[0304] Preferably, the hydrogel is dissociated by being chemically disintegrated or dissolved. For example, alginate gels may be disintegrated in an appropriate alginate dissolving buffer (e.g. 0.055 M sodium citrate, 0.15 M NaCI, pH 6.8). Another suitable alginate dissolving buffers are well known to a person skilled in the art.

[0305] Uses

[0306] Also provided herein is the use of the hydrogels described herein for preserving surface presented epitopes of one or more cells. The cells may be cells as described herein. In one example, the one or more cells are leukocytes.

[0307] In some examples, the hydrogels described herein are for use in methods of preserving surface presented epitopes of one or more cells, for example, for use in preserving surface presented cell epitopes.

[0308] Also provided herein is the use of the hydrogels described herein for preserving surface presented ligands of one or more cells. The cells may be cells as described herein. In one example, the one or more cells are leukocytes.

[0309] In some examples, the hydrogels described herein are for use in methods of preserving surface presented ligands of one or more cells, for example, for use in preserving surface presented cell ligands.

[0310] Also provided herein is the use of the hydrogels described herein for preserving surface presented binding proteins of one or more cells. The cells may be cells as described herein. In one example, the one or more cells are leukocytes.

[0311] In some examples, the hydrogels described herein are for use in methods of preserving surface presented binding proteins of one or more cells, for example, for use in preserving surface presented cell ligands.

[0312] Also provided herein is the use of the hydrogels described herein for preserving surface presented binding protein-ligand complexes of one or more cells. The cells may be cells as described herein. In one example, the one or more cells are leukocytes.

[0313] In some examples, the hydrogels described herein are for use in methods of preserving surface presented binding protein-ligand complexes of one or more cells, for example, for use in preserving surface presented cell ligands.

[0314] In some examples, the hydrogels described herein may be for use in the methods described herein. The ability of the hydrogels described herein to preserve surface epitopes is a previously unknown property. By carrying out investigations into the preservation of surface epitopepresenting cells, such as the immune cells (such as leukocytes) described herein, the inventors unexpectedly found that the hydrogels preserve surface cell epitopes and the presentation thereof. In addition, the methods described herein preserve surface presented binding proteins, binding protein-ligand complexes and / or ligands.

[0315] The preservation of epitopes has a number of advantages and uses. For example, cells collected from a subject may be preserved and sent to a location for further analysis. For example, the cells may be used for diagnostic purposes. For example, the preserved epitopes may be used to identify surface markers indicative of a disease state. Preserving the epitopes by the methods and uses described herein may allow for more accurate and precise detection of epitopes (i.e. markers) indicative of a disease or condition in the subject. In addition, the levels of surface expression of epitope containing antigens after carrying out methods described herein may more accurately reflect in vivo levels of expression.

[0316] In some examples, the preserved epitopes may be better preserved for use in analysis methods such as flow cytometry. “Flow cytometry” as used herein refers to a method and a process whereby cells within a sample can be detected and identified when transversing past a detector within an apparatus containing a detecting source and a flowing apparatus. The surface presented epitopes may allow for detection of specific cells and cell types. Such methods may also allow determination of cell numbers, cell function, cell characteristics, and / or detection of diseases such as cancers (e.g. leukemia or lymphoma), immune diseases (e.g. autoimmune diseases and immunodeficiencies or infections). By providing cells with better preserved surface markers, more accurate results may be obtained. Therefore, in some examples, there is provided the use of cells having surface epitopes preserved by the methods described herein for improving characterisation of the cells using flow cytometry.

[0317] In some examples, the cells with surface epitopes preserved by the methods described herein may be subjected to fluorescence-activated cell sorting (FACS). FACS is a technique used to separate cells according to their content of particular molecules of interest. The molecule of interest can be specific for a type of cell or for particular cell state. The molecule of interest can be fluorescently labelled directly by binding to a fluorescent dye, or by binding to a second molecule, which has been fluorescently labelled, e.g., an antibody or lectin that has been fluorescently labelled and that specifically binds to the molecule of interest. By improving the preservation of surface epitopes presented on cells, more accurate FACS results may be obtained. In some examples, cells including surface epitopes preserved by the methods described herein may be used in methods of immunohistochemistry. As used herein, the term “immunohistochemistry” refers to a method of determining the presence or distribution of an antigen in a sample by detecting interaction of the antigen with a specific binding agent, such as an antibody. A sample is contacted with an antibody under conditions permitting antibodyantigen binding. Antibody-antigen binding can be detected by means of a detectable label conjugated to the antibody (direct detection) or by means of a detectable label conjugated to a secondary antibody, which binds specifically to the primary antibody (indirect detection).

[0318] In some examples, cells including surface epitopes preserved by the methods described herein may be used in methods of quality control testing. For example, the surface epitopes may be used to help determine the health of cells that have been preserved. In some examples, the cells may be tested to determine quality of the cells prior to use in cell therapy or immunotherapy. “Cell therapy” means any therapy wherein autologous or allogenic cellular material (e.g. cells per se) is administered to a patient and includes adoptive immunotherapy or autologous or allogenic transplantation. Adoptive immunotherapy (also referred to as “adoptive cellular therapy”) is a treatment used to help the immune system fight diseases, such as cancer and infections with certain viruses. As per the NCI (National Cancer Institute) definition of adoptive immunotherapy, T cells are collected from a patient and isolated and expanded ex vivo in the laboratory, which increases the number of T cells that are able to kill cancer cells or fight infections. These T cells are given back to the patient to help the immune system fight disease.

[0319] Enriching

[0320] Also provided herein is the use of the hydrogels described herein for obtaining a mixed cell population that is enriched for a target cell. Data which particularly supports this aspect of the invention is found in example 2.

[0321] The method for obtaining a mixed cell population that is enriched for a target cell comprises:

[0322] (a) contacting a mixed population of cells comprising the target cell with a hydrogel forming polymer;

[0323] (b) polymerising the polymer to form a reversibly cross-linked cell-containing hydrogel wherein the mixed population of cells comprising the target cell is entrapped or encapsulated in the hydrogel; and

[0324] (c) storing the reversibly cross-linked cell-containing hydrogel of (b) to obtain a cell population that is enriched for the target cell.

[0325] The step of contacting cells with a hydrogel forming polymer is described elsewhere herein and applies to this method. In one example, the mixed cell population may be a mixed population of leukocytes.

[0326] In another example, the method for obtaining a mixed leukocyte population that is enriched for a target leukocyte comprises:

[0327] (a) contacting a mixed population of leukocytes comprising the target leukocyte with a hydrogel forming polymer;

[0328] (b) polymerising the polymer to form a reversibly cross-linked leukocyte-containing hydrogel wherein the mixed population of cells comprising the target leukocyte is entrapped or encapsulated in the hydrogel; and

[0329] (c) storing the reversibly cross-linked leukocyte-containing hydrogel of (b) to obtain a leukocyte population that is enriched for the target leukocyte.

[0330] The step of polymerising the polymer to form a reversibly cross-linked cell-containing hydrogel is described elsewhere herein and applies to this method.

[0331] The step of storing the reversibly cross-linked cell-containing hydrogel is described elsewhere herein and applies to this method.

[0332] As used herein, the term “enriched” refers to the increase of the proportion of a sample component of a sample relative to the other sample components. Enriching can be the result of increasing the proportion of a sample component or reducing the proportion of other sample components. For example, as used herein, a mixed cell population that has been enriched for a target cell means that the proportion of the target cell has increased relative to the proportion of a non-target cell in the mixed cell population. In one example, the target cell may be a leukocyte.

[0333] As used herein, the term “mixed cell population” refers to a population of at least two or more cells comprising at least one target cell and at least one non-target cell. As used herein, “nontarget cell” refers to any other cell in the mixed population of cells that is not the target cell.

[0334] The enrichment may otherwise be considered as sorting the target cell from the non-target cell of a mixed population of cells. In enrichment, the target cell may be isolated from the non- target cell or vice versa.

[0335] Suitably, the mixed cell population may be enriched for a target cell such that there is at least about 1% more, at least about 2% more, at least about 3% more, at least about 4% more, at least about 5% more, at least about 10% more, at least about 20% more, at least about 30% more, at least about 40% more, or at least about 50% more target cell in the cell population after storing according to step (c) relative to the amount of target cell pre-enrichment (i.e. at the start of step (a)). The proportion of non-target cell in the cell population would have decreased accordingly. For example, if a mixed cell population has been enriched for a target cell such that there is about 20% more target cell relative to pre-enrichment, then there is about 20% less non-target cell than there was pre-enrichment.

[0336] Suitably, the mixed cell population may be enriched for a target cell such that there is at least about 1% less, at least about 2% less, at least about 3% less, at least about 4% less, at least about 5% less, at least about 10% less, at least about 20% less, at least about 30% less, at least about 40% less, or at least about 50% less non-target cell in the cell population after storing according to step (c) relative to the amount of non-target cell pre-enrichment (i.e. at the start of step (a)). The proportion of target cell in the cell population would have increased accordingly.

[0337] In another example, a mixed cell population may comprise about 40% non-target cell and about 60% target cell pre-enrichment. If there is about 20% more target cell relative to non- target cell after the method has been applied, then the enriched cell population will have about 80% target cell and about 20% non-target cell.

[0338] The mixed population of cells may comprise any of the cells described elsewhere herein. The mixed population of cells may comprise immune cells as described elsewhere herein. The mixed population of cells may comprise leukocytes as described elsewhere herein.

[0339] Suitably, the immune cells may be human cells as described herein. In one example, the immune cells may be leukocytes. The leukocytes may be selected from one or more of: lymphocytes, monocytes and / or granulocytes. Lymphocytes, monocytes, and granulocytes are described elsewhere herein.

[0340] The mixed population of cells may be comprised in or obtained from a whole blood sample. Whole blood samples are described elsewhere herein.

[0341] The target cell of the mixed cell population may be selected from lymphocytes and / or monocytes. However, the skilled person would appreciate that the target cell of the mixed cell population is not limited to lymphocytes and / or monocytes.

[0342] The non-target cell of the mixed cell population may be granulocytes. However, the skilled person would appreciate that the non-target cell of the mixed cell population is not limited to granulocytes.

[0343] Suitably, the cross-linked cell-containing hydrogel wherein the mixed population of cells comprising the target cell is entrapped or encapsulated in the hydrogel may be stored for a period of time. Suitably, the period of time may be at least about 12 hours, at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 1 week, or at least about 2 weeks. Suitably, the period of time may be at least about 3 days. Suitably, the cross-linked cell-containing hydrogel wherein the mixed population of cells comprising the target cell is entrapped or encapsulated in the hydrogel may be stored at from about 2 °C to about 3 °C, 2 °C to about 4 °C, 2 °C to about 5 °C, 2 °C to about 6 °C, 2 °C to about 7 °C, or 2 °C to about 8 °C. Suitably, the cross-linked cell-containing hydrogel wherein the mixed population of cells comprising the target cell is entrapped or encapsulated in the hydrogel may be stored at from about 2 °C to about 8 °C. Suitably, the cross-linked cellcontaining hydrogel wherein the mixed population of cells comprising the target cell is entrapped or encapsulated in the hydrogel may be stored at about 4 °C.

[0344] Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. For example, Singleton and Sainsbury, Dictionary of Microbiology and Molecular Biology, 2d Ed., John Wiley and Sons, NY (1994); and Hale and Marham, The Harper Collins Dictionary of Biology, Harper Perennial, NY (1991) provide those of skill in the art with a general dictionary of many of the terms used in the invention. Although any methods and materials similar or equivalent to those described herein find use in the practice of the present invention, the preferred methods and materials are described herein. Accordingly, the terms defined immediately below are more fully described by reference to the Specification as a whole. Also, as used herein, the singular terms "a", "an," and "the" include the plural reference unless the context clearly indicates otherwise. Unless otherwise indicated, nucleic acids are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively. It is to be understood that this invention is not limited to the particular methodology, protocols, and reagents described, as these may vary, depending upon the context they are used by those of skill in the art.

[0345] Aspects of the invention are demonstrated by the following non-limiting examples.

[0346] EXAMPLES

[0347] EXAMPLE 1

[0348] MATERIALS AND METHODS

[0349] Protocol for Epitope Preservation Testing on BloodReady stored whole blood.

[0350] Day 0 Sample Acquisition

[0351] On Day 0, whole blood samples were obtained from healthy volunteers. Three different conditions were tested: fresh, BloodReady, and CytoChex. A volume of 3 mL whole blood was used per condition. For CytoChex, the blood was drawn directly into the preservation tube.

[0352] Dav 0 BloodReady Encapsulation For storage in BloodReady, BloodReady beads had been prepared in 10 mL collection tubes. BloodReady beads were collected from the fridge and any excess fluid was removed from the beads. For each encapsulation, 3 mL blood was mixed with 0.75 mL of Gel A. The blood and Gel A mixture was then added to the BloodReady beads slowly and inverted once to allow adequate mixing of the beads and blood. The sample was then left for 15 minutes at RT to allow gelation to occur. Following encapsulation, the BloodReady samples were returned to the fridge for storage at 2-8°C for 3 days.

[0353] Day 0 Fresh Blood Epitope Staining

[0354] For staining of fresh blood leukocyte surface epitopes, 100 pL of fresh whole blood was added to 50 wells of 2 x 96-deep well plates. A CD45 antibody was then added to each well. The corresponding PE antibody was then added to the corresponding well (see Table 1 for list of antibodies). The 96wp was then left for blood surface epitope staining for 30 minutes at RT. Following staining, 2 mL of warmed PharmLyse solution was then added and the plates were incubated for 20 minutes at RT to allow RBC-lysis to occur. Following lysis, the plates were spun down at 500 xg for 5 minutes at RT. The residual volume was aspirated and discarded, avoiding the cell pellet. 2 mL of wash buffer was added to each well and the plates were spun down at 500 xg for 5 minutes at RT. The residual volume was aspirated and discarded, avoiding the cell pellet. 2 mL of wash buffer was added to each well and the plates were spun down at 500 xg for 5 minutes at RT. The residual volume was removed, avoiding the cell pellet, and cells were resuspended in 300 pL of wash buffer. Samples were then run on Northern Lights to analyse epitope expression.

[0355] Dav 3 BloodReady Release

[0356] The BloodReady preserved blood and Cyto-Chex preserved blood were released following manufacturer’s guidelines (Cyto-Chex preserved blood was released directly from preservation tube).

[0357] After 3 days’ storage, BloodReady samples were collected from the fridge for release. To release the encapsulated whole blood, 6 mL of dissolution buffer (0.2M Citrate) was added to the bottom of the sample tube to dislodge the beads. The beads were left to dissolve for 15 minutes at RT on the rocker at 40rpm. Released blood samples were then filtered to remove dissolved beads by pipetting the blood sample through a 100 pm filter into a 50 mL falcon tube. The released sample was then used for subsequent downstream applications. For PBMC isolation, SepMate tubes were used according to manufacturer’s instructions (STEMCELL Technologies).

[0358] Dav 3 Stored Blood Epitope Staining For staining of stored blood leukocyte surface epitopes, 150 pL of BloodReady preserved blood was added to 50 wells of 2 x 96-deep well plates, and 100 pL of Cyto-Chex preserved blood was added to 50 wells of 2 x 96-deep well plates. CD45 antibody was then added to all wells. The corresponding PE antibody was then added to the corresponding well in both plates (see Table 1 for list of antibodies). The BloodReady and CytoChex plates were then left for blood surface epitope staining for 30 minutes at RT. Following staining, 2 mL of warmed PharmLyse solution was then added and the plates were incubated for 20 minutes at RT to allow RBC-lysis to occur. Following lysis, the plates were spun down at 500 xg for 5 minutes at RT. The residual volume was aspirated and discarded, avoiding the cell pellet. 2 mL of wash buffer was added to each well and the plates were spun down at 500 xg for 5 minutes at RT. The residual volume was aspirated and discarded, avoiding the cell pellet. 2 mL of wash buffer was added to each well and the plates were spun down at 500 xg for 5 minutes at RT. The residual volume was removed, avoiding the cell pellet, and cells were resuspended in 300 pL of wash buffer. Samples were then run on Northern Lights to analyse epitope expression. Data was compared between Day 0 fresh blood, 3 Day BloodReady preserved blood and 3 Day CytoChex preserved blood. Data was analysed using FCS Express Software.

[0359] Table 1 : Epitope Antibodies Tested (all PE-conjugated)

[0360] Results & Discussion

[0361] It can be seen from Figures 1A to D that BloodReady preserves the expression of >90% epitopes tested on lymphocytes, >80% on monocytes and >70% on granulocytes. In comparison with prior art systems Cyto-Chex the methods of the invention showed better preservation of several epitopes to Cyto-Chex.

[0362] In Figures 1A and 3 it can be seen that methods of the invention showed better preservation of, for example, CD62L, CD317, CD82, CD367 and Integrin b7 in lymphocytes in comparison to prior art methods. CD62L (L-Selectin CD62L) plays a pivotal role in controlling the traffic of lymphocytes and neutrophils from peripheral lymph nodes to sites of inflammation (1).

[0363] Cryopreservation induces a profound decrease of CD62L expression (2). Integrin b7 functions in the homing of T cells to intestinal sites (3).

[0364] In Figures 1 B and 2 it can be seen that methods of the invention showed better preservation of, for example, CD62L, CD20, CD10, CD127, CD87, and Integrin b7 in monocytes in comparison to prior art methods. CD62L is a cell adhesion molecule playing a role in regulating the recruitment of monocytes to tissue from the blood during inflammation (4). CD87 facilitates CD11 b / CD18-mediated adhesion of human monocytes.

[0365] In Figures 1C and 4 it can be seen that methods of the invention showed better preservation of, for example, CD62L, CD317, CD87, CD369, CD367 and CD282 in granulocytes in comparison to prior art methods.

[0366] This data has important implications for clinical diagnostics and monitoring that relies on reproducible and accurate immunophenotyping of whole blood specimens by flow cytometry. Alongside it’s clinical use, immunophenotyping allows epidemiological prediction of disease risk, and has aided in the development and implementation of multiple immunotherapies. Flow cytometric immunophenotyping is conducted at analysis centres, which are often distant from the site of whole blood collection, and therefore transportation of samples is required (5). The processing of diagnostic samples can however be delayed by transportation, as well as by personnel and equipment availability. Since whole blood stability is limited after 24h following collection (6, 7), this can have several consequences for the outcome of flow cytometric analysis. Strategies have been implemented to extend the time to analysis, including various fixative reagents such as CytoChex described herein. The assessment of leukocyte epitope preservation documented here shows that BloodReady technology outperforms CytoChex in surface epitope stabilisation. A loss of marker expression can lead to impaired discrimination of leukocyte subsets over time, rendering samples unsuitable for detailed immunophenotyping and in turn clinical utility. Fixation is also reported to compromise the relative distribution of leukocytes, leading to under and over representation of certain populations (8, 9).

[0367] BloodReady, offers a simple and effective solution for preserving whole blood leukocyte populations and their respective immunophenotype profile for more than 24 hours (e.g.3 days). This solution is advantageous over Cyto-Chex, providing improved preservation of markers for more than 24 hours. This offers a solution to overcome the logistical challenges of whole blood processing and transportation, without limiting the quality and informativeness of data obtained. The downstream applications of whole blood preserved by BloodReady are unlimited, unlike fixative solutions which restrict subsequent functional assays.

[0368] EXAMPLE 2MATERIALS AND METHODS

[0369] Protocol for Multiplex Antibody Staining on BloodReady stored whole blood.

[0370] Day 0 Sample Acquisition

[0371] On Day 0, whole blood samples were obtained from healthy volunteers. Two different conditions were tested: fresh and BloodReady. A volume of 3 mL whole blood was used per condition. Dav 0 BloodReady Encapsulation

[0372] For storage in BloodReady, BloodReady beads had been prepared in 10 mL collection tubes. BloodReady beads were collected from the fridge and, for each encapsulation, 3 mL blood was mixed with 0.75 mL of Gel A. The blood and Gel A mixture was then added to the BloodReady beads slowly and inverted once to allow adequate mixing of the beads and blood. The sample was then left for 15 minutes at RT to allow gelation to occur. Following encapsulation, the BloodReady samples were returned to the fridge for storage at 2-8°C for 3 days.

[0373] Day 0 Fresh Blood Multiplex Staining

[0374] For multiplex antibody staining of fresh blood immune cells, 100 pL of fresh whole blood was added to a 5 mL Falcon™ Round-Bottom Polypropylene Test Tube. A 33-colour antibody panel was then added to the blood (see Table 2 for list of antibodies). The falcon tube was then left for blood immune cell surface epitope staining for 30 minutes at RT. Following staining, 2 mL of warmed PharmLyse solution was then added and the falcon tube was incubated for 20 minutes at RT to allow RBC-lysis to occur. Following lysis, the falcon tube was spun down at 500xg for 5 minutes at RT. The residual volume as aspirated and discarded, avoiding the cell pellet. 2 mL of wash buffer was added to the blood sample and the falcon was spun down at 500xg for 5 minutes at RT. The residual volume was removed, avoiding the cell pellet, and cells were resuspended in 300 pL of wash buffer. Samples were then run on Northern Lights to analyse immune cell populations.

[0375] Dav 3 BloodReady Release

[0376] After 3 days’ storage, BloodReady samples were collected from the fridge for release. To release the encapsulated whole blood, 6 mL of dissolution buffer (0.2M Citrate) was added to the bottom of the sample tube to dislodge the beads. The beads were left to dissolve for 15 minutes at RT on the rocker at 40rpm. Released blood samples were then filtered to remove dissolved beads by pipetting the blood sample through a 100 pm filter into a 50 mL falcon tube. The released sample was then used for subsequent downstream applications. For PBMC isolation, SepMate tubes were used according to manufacturer’s instructions (STEMCELL Technologies).

[0377] Day 3 Stored Blood Multiplex Staining

[0378] For multiplex antibody staining of stored fresh blood immune cells, 150 pL of fresh whole blood was added to a 5 mL Falcon™ Round-Bottom Polypropylene Test Tube. A 33-colour antibody panel was then added to the blood (see Table 2 for list of antibodies). The falcon tube was then left for blood immune cell surface epitope staining for 30 minutes at RT. Following staining, 2 mL of warmed PharmLyse solution was then added and the falcon tube was incubated for 20 minutes at RT to allow RBC-lysis to occur. Following lysis, the falcon tube was spun down at 500xg for 5 minutes at RT. The residual volume as aspirated and discarded, avoiding the cell pellet. 2 mL of wash buffer was added to the blood sample and the falcon was spun down at 500xg for 5 minutes at RT. The residual volume was removed, avoiding the cell pellet, and cells were resuspended in 300 pL of wash buffer. Data was compared between Day 0 fresh blood and 3 Day BloodReady preserved blood. Data was analysed using FCS Express Software.

[0379] Table 2: Antibodies Tested

[0380] Results & Discussion

[0381] It can be seen from Figure 5 that BloodReady preserves the relative proportions of major leukocyte populations of T cells, NK cells, B cells, and monocytes in the blood.

[0382] T cells (CD3+) show a non-significant increase of 4.3%. Delineation of the T cell population shows that Helper T cells (CD4+) increase by 3.3%, Cytotoxic T cells (CD8+) increase by 1.1 %, Regulatory T cells (CD25+ CD127- / dim) decrease by 0.2%, while Gamma delta T cells (TCRyb+) and Natural Killer T cells (CD56+) remain unchanged after 3-day storage in BloodReady.

[0383] B cells (CD19%) show an increase of 4.5%. Delineation of the B cell population shows that Transitional B cells (CD27- lgD+) increase by 3.8%, Non-Switched B Cells (CD27+ lgD+) increase by 0.3%, and Switched B cells (CD27+ IgD-) increase by 0.2%.

[0384] NK Cells (CD56+ CD16+) increase by 1.6%. Subdivision of NK cells showed that CD57+ NK cells decrease by 0.2%, Early NK cells (CD56+ CD16-) remain unchanged, and Mature NK cells (CD56+ CD16+) increase by 1.2%.

[0385] Lymphoid Cells (ILC) (CD127+) show an increase of 0.14%, which when subdivided into ILCs CD2+ CD4+, and ILCs CD2+ CD4- show increases of 0.1% and 0.06%, respectively.

[0386] Monocytes (CD4+ HLA-DR-) increase by 4.1%. Subdivision of monocytes show that Classical Monocytes (CD14+ CD16-) increase by 3.6%, Intermediate Monocytes (CD14+ CD16+) decrease by 0.02%, and Non-Classical Monocytes decrease (CD14- CD16+) by 0.06%.

[0387] Granulocytes, which encompass Neutrophils, Eosinophils, and Basophils are the only population that show a significant change in their proportion after storage in BloodReady. Granulocytes decrease by 18.6% compared to fresh blood, suggesting a sensitivity of this cell type to hypothermic storage.

[0388] BloodReady maintains live populations of T Cells, B Cells, NK Cells, ILCs, and monocytes after 3 days storage at 4°C. The accurate delineation of major leukocytes into immune cell subpopulations shows that important cell markers that are used in the characterisation of these cells are retained after storage in BloodReady. This in turn allows successful flow cytometry applications on whole blood after storage in BloodReady, for use in immunophenotyping, cell monitoring, and diagnosis. The reader's attention is directed to all papers and documents which are filed concurrently with or previous to this specification in connection with this application and which are open to public inspection with this specification, and the contents of all such papers and documents are incorporated herein by reference.

[0389] All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and / or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and / or steps are mutually exclusive.

[0390] Each feature disclosed in this specification (including any accompanying claims, abstract and drawings), may be replaced by alternative features serving the same, equivalent, or similar purpose, unless expressly stated otherwise. Thus, unless expressly stated otherwise, each feature disclosed is one example only of a generic series of equivalent or similar features.

[0391] The invention is not restricted to the details of any foregoing embodiments. The invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.

[0392] REFERENCES

[0393] 1. Klaus Ley, in Microcirculation, 2008

[0394] 2. Costantini A, Mancini S, Giuliodoro S, Butini L, Regnery CM, Silvestri G, Montroni M. Effects of cryopreservation on lymphocyte immunophenotype and function. J Immunol Methods. 2003 Jul;278(1-2):145-55.

[0395] 3. DeNucci CC, Pagan AJ, Mitchell JS, Shimizu Y. Control of alpha4beta7 integrin expression and CD4 T cell homing by the betal integrin subunit. J Immunol. 2010 Mar 1 ;184(5):2458-67

[0396] 4. Rutkowska E, Kwiecieh I, Ktos K, Rzepecki P, Chciatowski A. Intermediate Monocytes with PD-L1 and CD62L Expression as a Possible Player in Active SARS-CoV-2 Infection. Viruses. 2022 Apr 15;14(4):819. doi: 10.3390 / v14040819. PMID: 35458548; PMCID: PMC9031659.

[0397] 5. Diks A, Bonroy C, Teodosio C, Greenland R, de Mooij B, de Maertelaere E. Impact of blood storage and sample handling on quality of high dimensional flow cytometric data in multicenter clinical research. J Immunol Methods. 2019;475.

[0398] 6. Wong K, Sandlin R, Carey T. The Role of Physical Stabilization in Whole Blood Preservation. Sci Rep. 2016;6(21023).

[0399] 7. Johnson R, Overlee B, Sagen J. Peripheral blood mononuclear cell phenotype and function are maintained after overnight shipping of whole blood. Sci Rep. 12(1):19920. Pinto L, Trivett M, Wallace D. Fixation and cryopreservation of whole blood and isolated mononuclear cells: Influence of different procedures on lymphocyte subset analysis by flow cytometry. Cytom Part B Clin Cytom. 2004;63:47-55. Sakkestad S, Skavland J, Hanevik K. Whole blood preservation methods alter chemokine receptor detection in mass cytometry experiments. J Immunol Methods.

[0400] 2019;476(112673).

[0401] Embodiments

[0402] Embodiment 1. A method of preserving surface presented entities of one or more cells, the method comprising

[0403] (a) contacting the one or more cells with a hydrogel-forming polymer;

[0404] (b) polymerising the polymer to form a reversibly cross-linked cell-containing hydrogel wherein at least one or more cells is entrapped or encapsulated in the hydrogel.

[0405] Embodiment 2. The method of embodiment 1 , wherein the method further comprises: packaging and sealing the cell-containing hydrogel in a water tight or air tight receptacle.

[0406] Embodiment 3. The method of embodiment 2, wherein the method further comprises: storing the packaged cell-containing hydrogel for a period of time; optionally wherein the period of time is at least 24 hours; and / or transporting the packaged cell-containing hydrogel from a first location to a second location.

[0407] Embodiment 4. A method of transporting one or more cells comprising surface presented entities, wherein surface presented entities of the one or more cells are preserved, comprising:

[0408] (a) preparing the one or more cells for transportation and preserving the surface presented entities by

[0409] (i) contacting the one or more cells with a hydrogel-forming polymer;

[0410] (ii) polymerising the polymer to form a reversibly cross-linked cellcontaining hydrogel wherein at least one or more cells is entrapped or encapsulated in the hydrogel;

[0411] (iii) packaging and sealing the cell-containing hydrogel in a water tight or air tight receptacle; and

[0412] (b) transporting the packaged cell-containing hydrogel of step (a) from a first location to a second location.

[0413] Embodiment 5. A method for fulfilling an order or request for one or more cells comprising surface presented entities, the method comprising:

[0414] (a) preparing the one or more cells for transportation and preserving the surface presented entities by

[0415] (i) contacting the one or more cells with a hydrogel-forming polymer; (ii) polymerising the polymer to form a reversibly cross-linked cellcontaining hydrogel wherein at least one or more cells is entrapped or encapsulated in the hydrogel;

[0416] (iii) packaging and sealing the cell-containing hydrogel in a water tight or air tight receptacle; and

[0417] (b) dispatching the packaged cell-containing hydrogel of step (a) for transportation; or transporting the cell-containing hydrogel of step (a) to a location specified in the order or request.

[0418] Embodiment 6. The method any preceding embodiment, wherein the method further comprises: releasing the one or more cells from the hydrogel optionally at a or the second location or a or the specified location.

[0419] Embodiment 7. The method of any preceding embodiment, wherein the one or more cells comprise a population of cells individually dispersed in the hydrogel.

[0420] Embodiment 8. The method of any preceding embodiment, wherein the one or more cells are comprised in or obtained from a whole blood sample.

[0421] Embodiment 9. The method of any preceding embodiment, wherein the one or more cells comprise immune cells; optionally wherein the one or more cells are human cells.

[0422] Embodiment 10. The method of embodiment 9, wherein the immune cells are selected from one or more of: lymphocytes, monocytes and / or granulocytes.

[0423] Embodiment 11. The method of any preceding embodiment, wherein the surface presented entities comprise one or more of : cell surface epitopes; cell surface binding proteins, optionally wherein the cell surface binding proteins comprise cell surface receptors; cell surface ligands; and / or cell surface binding protein-ligand complexes.

[0424] Embodiment 12. The method of embodiment 11 , wherein the cell surface epitopes are comprised within one or more surface antigens and wherein the surface antigens comprise one or more cell associated antigens; optionally wherein the cell associated antigen is one or more of: CD278, CD46, CD2, B2 microglobulin, CD10, CD100, CD156c, CD16, CD162, CD170, CD1172a b, CD11a, CD11 b, CD126, CD13, CD179a, CD18, CD182, CD191 , CD194, CD14, CD141 , CD218a, CD244, CD25, CD27, CD127, CD29, CD41 , CD42b, CD5, CD55, CD58, CD298, CD3, CD31 , CD317, CD33, CD61 , CD62L, CD62P, CD63, CD81 , CD82, CD87, CD35, CD354, CD367, CD28, CD4, CD9, CD93, CD95, CD97, CD99, HLA-E, Siglec-7, CD102, CD184, CD197, CD217, CD282, CD36, CD369, CD371 , CD45RO, CD7, CD84, CD88, HLA-A, HLA-B, HLA-C, HLA- DR, CD20, CD22, CD45RA, CD45RB, CD49f, Integrin b7, CX3CR1 , CCR10, CD15, CD114, CD183, CD19, CD137L, CD56, CD314, CD34, CD8, GRP56, MERTK, Siglec-8, CD24, and / or CD243.

[0425] Embodiment 13. The method of embodiment 11 or 12, wherein the cell surface epitopes are comprised within one or more surface antigens and wherein: the one or more cells comprise lymphocytes and the surface antigens comprise one or more of CD62L, CD317, CD82, CD367 and / or Integrin b7; the one or more cells comprise monocytes and the surface antigens comprise one or more of CD62L, CD20, CD10, CD127, CD87 and / or Integrin b7; the one or more cells comprise granulocytes and surface antigens comprise one or more of CD62L, CD317, CD87, CD369, CD367 and / or CD282.

[0426] Embodiment 14. The method of any one of embodiments 11 to 13, wherein the surface epitopes are comprised within one or more surface antigens and wherein the surface antigens comprise one or more of: a tumour antigen; a pathogen associated antigen; an allergenic antigen; a disease associated antigen; and / or an autoantigen.

[0427] Embodiment 15. The method of any preceding embodiment, wherein preserving comprises: maintaining the surface presented entities of the one or more cells in a functional state at the surface of the one or more cells; and / or maintaining a level of expression of each or all surface presented entities by the one or more cells; and / or maintaining presentation of each or all of the surface presented entities by the one or more cells. Embodiment 16. The method of any preceding embodiment, wherein the hydrogel comprises cross-linked alginate, optionally wherein the hydrogel comprises cross-linked calciumalginate, strontium alginate, barium-alginate, magnesium-alginate and / or sodium-alginate or combinations thereof.

[0428] Embodiment 17. The method of embodiment 15, wherein the cross-linked alginate comprises from about 0.1 % (w / v) to about 5.0% (w / v) calcium alginate.

[0429] Embodiment 18. The method of any preceding embodiment, wherein polymerisation is induced by a chemical agent; optionally wherein the chemical polymerisation agent comprises calcium chloride and strontium chloride.

[0430] Embodiment 19. The method of one of embodiments 2 to 18, wherein the sealed receptacle is a sealed storage vial or transport tube, or wherein the receptacle is a cell culture vessel; optionally wherein the cell culture vessel is selected from a cell culture tube, blood collection tube, a cell culture flask, a cell culture dish or a cell culture plate comprising a plurality of wells; further optionally wherein the cell culture plate comprising a plurality of wells is selected from a 4-, 6-, 8-, 12-, 24-, 48-, 96-, 384-, 1536- well cell culture plate.

[0431] Embodiment 20. The method of any preceding embodiment, wherein the surface epitopes are preserved for at least 24 hours.

[0432] Embodiment 21. The method of any one of embodiments 6 to 20, wherein the one or more cells are viable after release.

[0433] Embodiment 22. Use of a reversibly cross-linked hydrogel to preserve surface presented entities of one or more cells.

[0434] Embodiment 23. The use of embodiment 22, wherein the reversibly cross-linked hydrogel comprises a hydrogel as defined in any one of embodiments 15 to 18.

[0435] Embodiment 24. The use of embodiment 22 or 23, wherein: the one or more cells comprise one or more cells as defined in any one of embodiments 7 to 10, 12 and 21 ; and / or the surface epitopes comprises one or more epitopes as defined in any one of embodiments 11 to 13.

[0436] Embodiment 25. A reversibly cross-linked hydrogel for use in a method of preserving surface presented entities of one or more cells, wherein the method is according to any one of embodiment 1 to 21.

[0437] Embodiment 26. A method of obtaining a cell population that is enriched for a target cell, the method comprising:

[0438] (a) contacting a mixed population of cells comprising the target cell with a hydrogel forming polymer;

[0439] (b) polymerising the polymer to form a reversibly cross-linked cell-containing hydrogel wherein the mixed population of cells comprising the target cell is entrapped or encapsulated in the hydrogel; and

[0440] (c) storing the reversibly cross-linked cell-containing hydrogel of (b) to obtain a cell population that is enriched for the target cell.

[0441] Embodiment 27. The method of embodiment 26, wherein the mixed population of cells are comprised in or obtained from a whole blood sample.

[0442] Embodiment 28. The method of any one of embodiments 26 or 27, wherein the mixed population of cells comprise immune cells; optionally wherein the immune cells are human cells.

[0443] Embodiment 29. The method of any one of embodiments 26 to 28, wherein the immune cells are selected from one or more of: lymphocytes, monocytes and / or granulocytes.

[0444] Embodiment 30. The method of any one of embodiments 26 to 29, wherein the target cells are selected from lymphocytes and / or monocytes.

[0445] Embodiment 31. The method of any one of embodiments 26 to 30, wherein the hydrogel is stored in step (c) for a period of time; optionally wherein the period of time is at least 3 days; further optionally at from about 2 °C to about 8 °C.

Claims

Claims1. A method of preserving leukocyte surface presented entities in a sample comprising one or more leukocytes, the method comprising(a) contacting the sample comprising one or more leukocytes with a hydrogel-forming polymer;(b) polymerising the polymer to form a reversibly cross-linked leukocyte-containing hydrogel wherein at least one or more leukocytes is entrapped or encapsulated in the hydrogel.

2. The method of claim 1 , wherein the method further comprises: packaging and sealing the leukocyte-containing hydrogel in a water tight or air tight receptacle.

3. The method of claim 2, wherein the method further comprises: storing the packaged leukocyte-containing hydrogel for a period of time; optionally wherein the period of time is at least 24 hours; and / or transporting the packaged leukocyte-containing hydrogel from a first location to a second location.

4. A method of transporting a sample comprising one or more leukocytes, wherein the leukocytes comprise leukocyte surface presented entities, and wherein the leukocyte surface presented entities of the one or more leukocytes are preserved, comprising:(a) preparing the sample comprising one or more leukocytes for transportation and preserving the surface presented entities by(i) contacting the sample comprising one or more leukocytes with a hydrogel-forming polymer;(ii) polymerising the polymer to form a reversibly cross-linked leukocytecontaining hydrogel wherein at least one or more leukocytes is entrapped or encapsulated in the hydrogel;(iii) packaging and sealing the leukocyte-containing hydrogel in a water tight or air tight receptacle; and(b) transporting the packaged leukocyte-containing hydrogel of step (a) from a first location to a second location.

5. A method for fulfilling an order or request for a sample comprising one or more leukocytes, wherein the leukocytes comprise leukocyte surface presented entities, the method comprising:(a) preparing the sample comprising one or more leukocytes for transportation and preserving the leukocyte surface presented entities by(i) contacting the sample comprising one or more leukocytes with a hydrogel-forming polymer;(ii) polymerising the polymer to form a reversibly cross-linked leukocytecontaining hydrogel wherein at least one or more leukocytes is entrapped or encapsulated in the hydrogel;(iii) packaging and sealing the leukocyte-containing hydrogel in a water tight or air tight receptacle; and(b) dispatching the packaged leukocyte-containing hydrogel of step (a) for transportation; or transporting the leukocyte-containing hydrogel of step (a) to a location specified in the order or request.

6. The method any preceding claim, wherein the method further comprises: releasing the one or more leukocytes from the hydrogel optionally at a or the second location or a or the specified location.

7. The method of any preceding claim, wherein the one or more leukocytes comprise a population of leukocytes individually dispersed in the hydrogel.

8. The method of any preceding claim, wherein the one or more leukocytes are comprised in or obtained from a whole blood sample.

9. The method of any preceding claim, wherein the one or more leukocytes are human cells.

10. The method of any preceding claim, wherein the one or more leukocytes are selected from one or more of: lymphocytes, monocytes and / or granulocytes.

11. The method of any preceding claim, wherein the leukocyte surface presented entities comprise one or more of : cell surface epitopes; cell surface binding proteins, optionally wherein the cell surface binding proteins comprise cell surface receptors; cell surface ligands; and / or cell surface binding protein-ligand complexes.

12. The method of claim 11 , wherein the cell surface epitopes are comprised within one or more surface antigens and wherein the surface antigens comprise one or more cell associated antigens; optionally wherein the cell associated antigen is one or more of: CD278, CD46, CD2, B2 microglobulin, CD10, CD100, CD156c, CD16, CD162, CD170, CD1172a b, CD11a, CD11 b, CD126, CD13, CD179a, CD18, CD182, CD191 , CD194, CD14, CD141 , CD218a, CD244, CD25, CD27, CD127, CD29, CD41 , CD42b, CD5, CD55, CD58, CD298, CD3, CD31 , CD317, CD33, CD61 , CD62L, CD62P, CD63, CD81 , CD82, CD87, CD35, CD354, CD367, CD28, CD4, CD9, CD93, CD95, CD97, CD99, HLA-E, Siglec-7, CD102, CD184, CD197, CD217, CD282, CD36, CD369, CD371 , CD45RO, CD7, CD84, CD88, HLA-A, HLA-B, HLA-C, HLA- DR, CD20, CD22, CD45RA, CD45RB, CD49f, Integrin b7, CX3CR1 , CCR10, CD15, CD114, CD183, CD19, CD137L, CD56, CD314, CD34, CD8, GRP56, MERTK, Siglec-8, CD24, and / or CD243.

13. The method of claim 11 or 12, wherein the cell surface epitopes are comprised within one or more surface antigens and wherein: the one or more leukocytes comprise lymphocytes and the surface antigens comprise one or more of CD62L, CD317, CD82, CD367 and / or Integrin b7; the one or more leukocytes comprise monocytes and the surface antigens comprise one or more of CD62L, CD20, CD10, CD127, CD87 and / or Integrin b7; the one or more leukocytes comprise granulocytes and the surface antigens comprise one or more of CD62L, CD317, CD87, CD369, CD367 and / or CD282.

14. The method of any of claims 11 to 13, wherein the surface epitopes are comprised within one or more surface antigens and wherein the surface antigens comprise one or more of: a tumour antigen; a pathogen associated antigen; an allergenic antigen; a disease associated antigen; and / or an autoantigen.

15. The method of any preceding claim, wherein preserving comprises: maintaining the leukocyte surface presented entities of the one or more leukocytes in a functional state at the surface of the one or more leukocytes; and / or maintaining a level of expression of each or all leukocyte surface presented entities by the one or more leukocytes; and / ormaintaining presentation of each or all of the leukocyte surface presented entities by the one or more leukocytes.

16. The method of any preceding claim, wherein the hydrogel comprises cross-linked alginate, optionally wherein the hydrogel comprises cross-linked calcium-alginate, strontium alginate, barium-alginate, magnesium-alginate and / or sodium-alginate or combinations thereof.

17. The method of claim 15, wherein the cross-linked alginate comprises from about 0.1 % (w / v) to about 5.0% (w / v) calcium alginate.

18. The method of any preceding claim, wherein polymerisation is induced by a chemical agent; optionally wherein the chemical polymerisation agent comprises calcium chloride and strontium chloride.

19. The method of one of claims 2 to 18, wherein the sealed receptacle is a sealed storage vial or transport tube, or wherein the receptacle is a cell culture vessel; optionally wherein the cell culture vessel is selected from a cell culture tube, blood collection tube, a cell culture flask, a cell culture dish or a cell culture plate comprising a plurality of wells; further optionally wherein the cell culture plate comprising a plurality of wells is selected from a 4-, 6-, 8-, 12-, 24-, 48-, 96-, 384-, 1536- well cell culture plate.

20. The method of any preceding claim, wherein the surface epitopes are preserved for at least 24 hours.

21. The method of any one of claims 6 to 20, wherein the one or more leukocytes are viable after release.

22. Use of a reversibly cross-linked hydrogel to preserve leukocytes surface presented entities of one or more leukocytes comprised in a sample.

23. The use of claim 22, wherein the reversibly cross-linked hydrogel comprises a hydrogel as defined in any one of claims 15 to 18.

24. The use of claim 22 or 23, wherein: the one or more leukocytes comprise one or more leukocytes as defined in any one of claims 7 to 10, 12 and 21 ; and / orthe surface epitopes comprises one or more epitopes as defined in any one of claims11 to 13.

25. A reversibly cross-linked hydrogel for use in a method of preserving leukocyte surface presented entities of one or more leukocytes, wherein the method is according to any one of claims 1 to 21.

26. A method of obtaining a leukocyte population that is enriched for a target leukocyte, the method comprising:(a) contacting a mixed population of leukocytes comprising the target leukocyte with a hydrogel forming polymer;(b) polymerising the polymer to form a reversibly cross-linked leukocyte-containing hydrogel wherein the mixed population of leukocytes comprising the target leukocyte is entrapped or encapsulated in the hydrogel; and(c) storing the reversibly cross-linked leukocyte-containing hydrogel of (b) to obtain a leukocyte population that is enriched for the target leukocyte.

27. The method of claim 26, wherein the mixed population of leukocytes are comprised in or obtained from a whole blood sample.

28. The method of any one of claims 26 or 27, wherein the mixed population of leukocytes are human cells.

29. The method of any one of claims 26 to 28, wherein the leukocytes are selected from one or more of: lymphocytes, monocytes and / or granulocytes.

30. The method of any one of claims 26 to 29, wherein the target leukocytes are selected from lymphocytes and / or monocytes.

31. The method of any one of claims 26 to 30, wherein the hydrogel is stored in step (c) for a period of time; optionally wherein the period of time is at least 3 days; further optionally at from about 2 °C to about 8 °C.

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