WO2024127278A1

WO2024127278A1 - Cell agglomerate living material, methods of production and uses thereof

Info

Publication number: WO2024127278A1
Application number: PCT/IB2023/062636
Authority: WO
Inventors: João Filipe COLARDELLE DA LUZ MANO; Vítor Manuel ABREU GASPAR; Matilde RODRIGUES LAGARTO; Pedro OLIVEIRA LAVRADOR; Beatriz SILVA MOURA
Original assignee: Universidade de Aveiro
Current assignee: Universidade de Aveiro
Priority date: 2022-12-13
Filing date: 2023-12-13
Publication date: 2024-06-20
Anticipated expiration: 2025-06-13
Also published as: EP4634366A1

Abstract

The present disclosure relates to a cell agglomerate material comprising a modified hematopoietic cell; and a chemically functionalized biomaterial tethered to the surface of the modified hematopoietic cell; wherein the modified hematopoietic cell displays at its surface a chemical moiety that is covalently bound the chemically functionalized biomaterial. The disclosure also relates to a cell agglomerate material for use in medicine, in particular for use in the treatment of cancer or immune system diseases, to the use of said cell agglomerate material as a drug screen platform or disease modelling platform, and method of production thereof.

Description

D E S C R I P T I O N

CELL AGGLOMERATE LIVING MATERIAL, METHODS OF PRODUCTION AND USES THEREOF

TECHN ICAL FI ELD

[0001] The present disclosure relates to the in vitro generation of artificial living agglomerates, preferably of hematopoietic tissue cells, comprising metabolic glycoengineered cells, preferably lymphoid or myeloid lineage cells, interconnected by a cell tethering biomaterial, combination, methods of production and uses thereof.

[0002] Provided herein are metabolic glycoengineered cells, preferably hematopoietic tissue-derived cells, using non-natural monosaccharide sugars; a chemical functionalized cell tethering biomaterial, preferably a chemical functionalized glycosaminoglycan, and their covalent crosslinking compositions; a method to produce in vitro artificial living agglomerates displaying a high cell-biomaterial ratio and living biological functions; an injectable/mouldable agglomerate for cell therapy; an in vitro, ex vivo method for disease modelling and therapies screening; a kit for implantation for repair, treatment, or diagnostic of diseases.

[0003] The present disclosure also relates to the fabrication of an agglomerated of a cell-derived responsive platform capable of releasing the artificially agglomerated living cells as unitary elements along time, preferably an agglomerated lymphoid and/or myeloid cell-derived responsive platform capable of releasing the artificially agglomerated living cells as unitary elements along time.

BACKGROUND

[0004] Representing the fundamental units of life, cells play a pivotal role in the preservation of living organism's integrity. These might behave as single units or may develop specialized functions and cooperate with distinct types of cells and surrounding extracellular matrix (ECM), forming larger and more complex assemblies. Such structures are hierarchically organized, giving rise to tissues, and ultimately, to multicellular organisms. Across multicellular organisms, hematopoietic tissue-derived cells such as lymphoid and myeloid lineage cells are highly plastic and bioactive cellular lineages, that naturally live in suspension, as individualized units and respond dynamically to their surrounding environment when required. Lymphoid lineage and myeloid lineage cells are also responsible for mounting protective responses to exogenous pathogens, are involved in tissue repair and regeneration processes and on the removal of malignantly transformed cells, among other activities [1], [0005] Lymphoid and myeloid lineage cells modulatory and environmental-responsive behaviour, evolving nature, plasticity, as well as their natural engagement in pathologies treatment has sparked increasing interest for their widespread use in tissue repair, new therapies development and in vitro/ex vivo disease modelling applications, biosensing, as well as many other areas.

[0006] Engineered living materials (ELMs) can be defined as multi-dimensional tri-dimensional (3D) platforms produced of living cellular entities (living/biotic component), of different origins, including eukaryotic or prokaryotic; combined with non-living materials (abiotic component) and that aim to leverage, emulate and/or explore the intrinsic biological activity naturally encoded in its fundamental building blocks. In ELMs, cells are generally combined with supporting abiotic materials using bottom- up or top-down approaches. In general, the cellular (living/biotic) component is always present at a higher amount than its abiotic counterpart. Particularly, eukaryotic living materials comprising living cells benefit from the reciprocity and interplay between the extracellular matrix (ECM)-mimetic bioactive biomaterials and unit cells, resulting in materials with well-defined physicochemical properties and similar innate properties as an organic living entity, such as organized self-assembly, biological self-healing, microenvironment memory, longevity/aging patterns and biomolecules synthesis, at a spectrum of different length scales. ELMs have wide applicability as biomedical sensors, drug delivery systems, tissue-engineered platform, therapeutics, disease models, among several others [2],

[0007] Within ELMs, cellular functions are mostly governed by cell-cell and cell-biomaterials interactions or by surrounding environmental cues, behaving as a multi stimuli-responsive systems in which the fundamental living building blocks work cooperatively within the 3D architecture and interact with its surrounding environment.

[0008] Living cell units can be used to form living materials comprising cell agglomerates where cells are mostly combined and cooperatively exerting their biological activity. The production of living materials comprising agglomerated lymphoid and myeloid lineage cells is currently not possible since their biologically encoded activity does not naturally allow their agglomeration into 3D structures as they mostly exist as suspension units in biological fluids. This limits the use of the cells for fabricating ELMs that can be processed by bottom-up/top-down engineering approaches into 3D/four- dimensional (4D - i.e., time evolving constructs), injectable/implantable platforms, disease models and other uses thereof.

[0009] The cell glycocalyx is a multifunctional organelle located on the extracellular space of the cell membrane that embodies a set of molecules, in which monosaccharides emerge as the fundamental building blocks. In turn, the interconnected sugar units composing the well-known glycans can remain free or form glycoconjugates - glycosylated structures derived from the attachment of glycans with proteins and lipids. [4,5] Besides acting as a physical barrier, mammalian cells' glycocalyx is the first component to contact with the external surroundings, allowing an interplay between the extracellular environment and the intracellular machinery. [4] This bilateral communication renders a dynamic remodelling of the glycocalyx composition, which is indisputable considering certain cell-cell recognition processes such as tumour progression or pathogen invasion. [5] Cell membrane sialoglycans, comprise sialic acids that usually settle on the outlying edges of the glycans structures. [6,7]

[0010] These facts are disclosed in order to illustrate the technical problem addressed by the present disclosure.

G EN ERAL DESCRI PTI ON

[0011] The present disclosure relates to a method to produce engineered living materials comprising cells, in particular hematopoietic tissue derived cells (i.e., lymphoid and/or myeloid lineage cells) linked via covalent crosslinking to a chemically functionalized biomaterial that act as a cell tethering material, yielding living artificial 3D/4D cell agglomerates of lymphoid and/or myeloid cells. For the scope of the present application, the term "cell agglomerate material", or "artificial tissue", refers to the disclosed engineered living materials comprising cells linked via covalent crosslinking to a chemically functionalized biomaterial. The term "immunoids" refers to a cell agglomerate material obtained using lymphoid and/or myeloid cells.

[0012] Surprisingly, the disclosed method allowed the production of functional living materials comprising agglomerated lymphoid and myeloid lineage cells (immunoids), which naturally exist as suspension units, supporting the applications of such ELMs for numerous applications. The obtained cell agglomerate materials are assembled by a methodology that involves covalent bonds formation between transiently metabolic glycoengineered cells displaying chemical groups on their surface glycoproteins and a biomaterial, functionalized with a complementary chemical group which react with each other orthogonally. The cell agglomerate materials are quasi-al l-cel lular agglomerates owing to their high cell-to-biomaterial ratio, and display living features of cell agglomerates and the transient glycoengineering leads to the release of living unit cells from the cell agglomerate materials along time, evidencing the stimuli-responsive features of these living artificial cell agglomerates.

[0001] The present disclosure relates to the combination of metabolic glycoengineering, preferably on lymphoid or myeloid tissue cells, with a chemically reactive cell tethering biomaterial. Figure 1 depicts the concept and methodology for the assembly of such living architectures.

[0013] In an embodiment, the cell agglomerate material comprises stem cells, such as adipose tissue- derived mesenchymal stromal cells (hASCs), endothelial cells, such as umbilical vein endothelial cells (hUVECs), fibroblasts, such as dermal fibroblasts (hDFs), cancer cells, such as pancreatic ductal epithelioid carcinoma cells (hPANC-1), or hematopoietic tissue derived lineage cells.

[0014] In an embodiment, hematopoietic tissue derived lineage cells are of myeloid or lymphoid lineages including natural killer cells, mast cells, myeloblasts, basophils, neutrophils, eosinophils, monocytes, megakaryocytes, thrombocytes, lymphoid progenitor cells, myeloid progenitor cells, multipotent hematopoietic stem cells B, T lymphocytes, macrophages, including all their subpopulation types and/or phenotypically differentiated derivatives, and combinations thereof.

[0015] An aspect of the present disclosure relates to metabolic glycoengineered hematopoietic tissue-derived cells, namely, the glycoengineering of lymphoid and myeloid cells with non-natural monosaccharide sugar molecules comprising orthogonal chemistry chemical entities that will be installed in cells surface proteins, wherein the said proteins are cell surface glycoproteins.

[0016] Cell surface glycoproteins (i.e., N-linked and O-linked glycoproteins, glycosylphosphatidylinositol (GPI) anchored) constitute a plethora of biomacromolecules that govern several key biological processes, including cell-cell communication, cell-extracellular microenvironment communication, tissue development and disease onset/progression. Particularly, cell surface sialoglycans are involved in several biological and pathological processes: (i) for instance, through mediating intercellular communication and the events that arise from it; (ii) may also act as ligands for intrinsic receptors since sialic acids interact with lectins, antibodies, or enzymes, eliciting immune responses related with cell adhesion, lymphocyte homing and leukocyte migration to inflammation areas, and (iii) may act as ligands for extrinsic receptors in pathological events, allowing pathogens to mask their antigens from the detection of immune cells [3],

[0017] In an embodiment, naturally occurring surface sialoglycoproteins, preferably on hematopoietic tissue-derived cell lineages, are functionalized with non-natural monosaccharide sugar molecules displaying specific chemical moieties in their chemical structure and that enable orthogonal chemoselective chemistry, preferentially a moiety that enables biorthogonal click-chemistry. The clickchemistry moiety in non-natural monosaccharide sugar molecules can be an azide, diazirine, alkyne, alkene, norbornene, tetrazine, trans-cyclooctene, or others thereof with orthogonal or other reactivity thereof.

[0018] In an embodiment the non-natural monosaccharide sugar molecule is selected from a list of N-substituted or O-substituted mannosamine, galactosamine, glucosamine, acetyl-glucosamine, trehalose, neuraminic acid, including their acylated sugars or tetraacylated sugars derivatives, or other derivative sugars thereof.

[0019] In an embodiment the non-natural sugar molecules dose for glycoengineering hematopoietic tissue-derived cells is at least 0.1 pM, preferably in the range of 1 pM - 500 pM. [0020] In an embodiment glycoengineering of hematopoietic tissue-derived cells is performed with at least 5 minutes of sugar incubation in cells.

[0021] In an embodiment the cell tethering biomaterial, is selected from a list comprising basement membrane extracts of animal or human origin, gelatin methacrylate; any chemical modification of animal or human platelet lysates, including platelet lysates methacrylate; poly(ethylene glycol) (PEG), poly(lactic acid) (PLA), Poly (s- caprolactone) (PCL), fucoidan, chitosan, cellulose, glycogen, pectin, alginate, hyaluronic acid (HA), including their oxidized versions, poly— lactic acid-co-glycolic acid (PLGA), decellularized extracellular matrix from heathy or tumour tissues, fibronectin, collagen; or mixtures thereof.

[0022] In an embodiment the cell tethering biomaterial is chemically functionalized with a chemically reactive group that is an orthogonally reactive group to that of metabolic glycoengineered cell surface glycoproteins.

[0023] In an embodiment the biomaterial orthogonally reactive group functionalization is selected from a list comprising azide, diazirine, alkyne, alkene, norbornene, furan, maleimide, tetrazine, including dipyridyl-tetrazine trans-cyclooctene, dibenzocyclooctynol, or similar organic molecules, thereof.

[0024] In an embodiment the biomaterial orthogonally reactive group degree of functionalization is of at least 10%.

[0025] Another aspect of the present disclosure relates to a method for obtaining hematopoietic tissue derived lineage cells living artificial immunoid described in the present disclosure comprising the following steps: preparing a cellular suspension of metabolic glycoengineered cells; preferably incubated with the sugar for 24 hours; preparing a chemically functional tethering biomaterial; promoting cells and biomaterials mixture for covalent bonds formation preferably for 4 hours of incubation for handleable living immunoids production; wherein the immunoids preferentially comprise a density of at least 1 million cells per cubic centimetre;

[0026] In an embodiment living immunoids precursor units - glycoengineered hematopoietic tissue- derived cells and cell tethering biomaterial - are combined into a homogeneous mixture for chemically driven self-assembly, for at least 10 seconds.

[0027] In an embodiment, the disclosed cell agglomerate materials, in particularthe living immunoids, are freestanding living materials comprising agglomerated cells, in particular hematopoietic tissue derived cell lineages, and cell tethering biomaterials combination that can be moulded as a 3D platform for injection in moulds or implantation.

[0028] The present disclosure also relates to the fabrication of an immunoid capable of releasing living cells as free, non-aggregated units along time.

[0029] Another aspect of the present disclosure relates to an injectable, mouldable cell agglomerate formulation for cell therapy.

[0030] Another aspect of the present disclosure relates to an in vitro/ex vivo method for disease modelling and therapies screening.

[0031] Another aspect of the present disclosure relates to a kit for injection/implantation for repair, treatment, or diagnostic of diseases.

[0032] For the scope of the present disclosure, therapies include marketed drugs or drugs under development, nanoparticles, immunotherapy, radiotherapy, combinatory therapies, and others thereof.

[0033] In an embodiment, the present disclosure relates to a living artificial agglomerate - immunoid- comprising sugar-based metabolic glycoengineered hematopoietic tissue-derived lineage cells transiently tethered to a biomaterial via bioorthogonal click chemistry covalent linkages wherein the metabolic glycoengineered cells are obtained by using non-natural sugar derivatives; wherein said hematopoietic tissue-derived lineage cells are of human origin; wherein said cell tethering biomaterial is modified with orthogonal click chemistry moieties; wherein the said living agglomerates are mostly comprised by viable cells; wherein said transiently referrers to a covalent linkage that degrades over time.

[0034] In an embodiment the non-natural sugar derivative is a tetraacylated mannosamine derivative functionalized with azide moieties.

[0035] In an embodiment, the sugar dose administered to the cells is in the range of 1 pM - 500 pM.

[0036] In an embodiment, the sugar is administered to the cells for at least 10 seconds.

[0037] In an embodiment the orthogonally reactive moieties are dibenzocyclooctyne.

[0038] In an embodiment the orthogonally reactive moieties degree of functionalization is of at least 10%.

[0039] In an embodiment, the decellularized extracellular matrix is from human origin, preferably an autologous matrix.

[0040] In an embodiment the degree of functionalization with orthogonally reactive moieties is at least 10%. [0041] In an embodiment the metabolic glycoengineered cells glycocalyx is functionalized with an azide in the sialic acid residues.

[0042] In an embodiment the artificial agglomerates tissue is comprised of either lymphoid or myeloid lineage cells.

[0043] In an embodiment the agglomerate has cell-to-weight/volume of biomaterial of at least 1:0.01.

[0044] In an embodiment the agglomerate comprises a volumetric cell density of at least, 2 million cells per cubic centimetre, preferably 1 million cells per cubic centimetre.

[0045] In an embodiment the agglomerate releases less than 4% of total hematopoietic derived lineage cell units at day 0. In a further embodiment, the agglomerate releases at least 50% of total hematopoietic derived lineage cell units at day 7, preferably 60% of total hematopoietic derived lineage cell units at day 7, more preferably at least 70% of total hematopoietic derived lineage cell unit, at day 7.

[0046] In an embodiment the agglomerate is injectable and mouldable to any shape.

[0047] In an embodiment, the present disclosure also relates to a kit for therapeutics screening for use in the therapy, diagnostic, or treatment of human diseases, or for evaluating the efficiency of a compound for immune diseases, comprising the artificial agglomerates and released immune cell units.

[0048] An aspect of the present disclosure relates to an in vitro or ex vivo method for therapeutics screening for use in the therapy, diagnostic, or treatment of immune system diseases, or for evaluating the efficiency of a compound for immune diseases, cancer, tissue repair, comprising the use of the artificial tissues.

[0049] Another aspect of the present disclosure relates to an injectable platform for locally and systemically treating cancer comprising the use of the disclosed artificial tissues.

[0050] An aspect of the present disclosures comprises an implantable cell-releasing platform for localized and immunomodulation of tissues comprising the use of the disclosed artificial tissues.

[0051] The present disclosure relates to a cell agglomerate material comprising 60 to 95 % wtdry of modified cells; and 5 to 40 % wtdry of a chemically functionalized biomaterial tethered to the surface of the modified cells; wherein the modified cells display at their surface a chemical moiety that is covalently bound the chemically functionalized biomaterial.

[0052] In an embodiment, the chemical moiety is selected from azide, diazirine, alkyne, alkene, norbornene, tetrazine, trans-cyclooctene, or combinations thereof. [0053] In an embodiment, the chemical moiety is comprised in a surface glycoprotein of the modified cells, preferably a modified sialoglycoprotein.

[0054] In an embodiment, the modified surface glycoprotein comprises a non-natural sugar, functionalized with the chemical moiety.

[0055] In an embodiment, the non-natural sugar is functionalized with a group selected from azide, diazirine, alkyne, alkene, norbornene, tetrazine, trans-cyclooctene, or combinations thereof.

[0056] In a further embodiment the non-natural sugar is selected from a list comprising N-substituted mannosamine, O-substituted mannosamine, galactosamine, glucosamine, acetyl-glucosamine, trehalose, neuraminic acid, acylated sugars derivatives of neuraminic acid, tetraacylated sugars derivatives of neuraminic acid, tetraacylated mannosamine derivatives, or combinations thereof.

[0057] In an embodiment the non-natural sugar is tetraacylated mannosamine derivative functionalized with azide chemical moiety, in particular N-azidoacetylmannosamine-tetraacylated.

[0058] In an embodiment, the modified cells are hematopoietic cells, stem cells, such as adipose stem cells, endothelial cells, fibroblasts, cancer cells, or mixtures thereof.

[0059] In an embodiment, the modified cells are hematopoietic cells, preferably of lymphoid or myeloid lineage. In a further embodiment, the modified cells are selected from a list comprising: natural killer cells, mast cells, myeloblasts, basophils, neutrophils, eosinophils, monocytes, megakaryocytes, thrombocytes, lymphoid progenitor cells, myeloid progenitor cells, multipotent hematopoietic stem cells B, T lymphocytes, macrophages, including all their subpopulation types and/or phenotypically differentiated derivatives, and combinations thereof.

[0060] In an embodiment, the chemically functionalized biomaterial is selected from a list comprising basement membrane extracts of any animal or human origin, gelatin, poly(lactic acid), Poly (s- caprolactone), fucoidan, chitosan, laminarin, cellulose, glycogen, pectin, hyaluronic acid, chemically modified hyaluronic acid, poly— lactic acid-co-glycolic acid, decellularized extracellular matrix, fibronectin, collagen; or mixtures thereof.

[0061] In an embodiment, the chemically functionalized biomaterial is a decellularized extracellular matrix from human origin, preferably an autologous matrix.

[0062] In an embodiment the chemically functionalized biomaterial comprises a chemically reactive group that is orthogonally reactive to the chemical moiety comprised in the modified cell.

[0063] In an embodiment the chemically functionalized biomaterial is functionalized with a reactive group selected from a list comprising azide, diazirine, alkyne, alkene, norbornene, furan, thiols, maleimide, tetrazine, dibenzocyclooctynol, dibenzocyclooctyne, strained alkyne, or combinations thereof. [0064] In an embodiment, the chemically functionalized biomaterial is functionalized with dibenzocyclooctyne. In a further embodiment, the chemically functionalized biomaterial is hyaluronic acid functionalized with dibenzocyclooctyne.

[0065] In an embodiment the chemically functionalized biomaterial is hyaluronic acid functionalized with dibenzocyclooctyne and the modified cells display at their surface an azide chemical moiety.

[0066] In an embodiment, the degree of functionalization of the chemically functionalized biomaterial is at least 10%, preferably 12 to 15%.

[0067] In an embodiment, the mass ratio between the modified cells and the chemically functionalized biomaterials is at least is at least 1:0.2; preferably, the mass ratio between the modified cells and the chemically functionalized biomaterials ranges from 1:0.2 to 1:0.02. In an embodiment, it is considered that each agglomerate comprises 100 to 1000 mg of cells per mL (average cell weight around 1 ng) and a biomaterial solution of 20mg/mL.

[0068] In an embodiment, the cell agglomerate material comprises at least 1 million cells per cubic centimetre, preferably at least 2 million cells per cubic centimetre of material.

[0069] In an embodiment, the disclosed cell agglomerate material is implantable, injectable and/or mouldable, preferably in situ.

[0070] In an embodiment, the covalent bound between the modified cells and the chemically functionalized biomaterial is degradable.

[0071] In an aspect, the present disclosure relates to a kit comprising the disclosed cell agglomerate material. In an embodiment, the kit is for use in tissue engineering, tissue repair, biomedical sensors, drug delivery systems, immunotherapy or diagnostics.

[0072] The present disclosure also relates to a cell agglomerate material as disclosed for use in medicine.

[0073] In an embodiment, the cell agglomerate material is for use in the treatment of cancer, treatment of immune system diseases, immunotherapy, tissue engineering, cell delivery, wound healing, or tissue repair.

[0074] In a further embodiment, the cell agglomerate material is for use as biomedical sensors, drug delivery systems, or diagnostics.

[0075] An aspect of the present disclosure relates to the use of the disclosed cell agglomerate material as a drug screening platform, cell culture platform, or disease modelling platform.

[0076] The present disclosure also describes a method for obtaining the disclosed cell agglomerate material, the method comprising the following steps: incubating a pool of cells with a functionalized non-natural sugar to obtain a suspension of modified cells; mixing the suspension of modified cells with a chemically functionalized biomaterial for covalent bond formation, wherein the mass ratio between the cells and the biomaterial ranges from 1:0.2 to 1:0.02; incubating the suspension of cells with the chemically functionalized biomaterial for at least 4 hours at 37 °C to obtain the cell agglomerate material.

[0077] In an embodiment, the cell agglomerate material comprises 100 mg of cells to 20 mg of biomaterial per mL

[0078] In an embodiment, the functionalized non-natural sugar is incubated in the pool of cells for at least 24 h.

[0079] In an embodiment, the suspension of hematopoietic cells is incubated with at least 0.1 pM of the non-natural sugar, preferably with 1 pM - 500 pM of the non-natural sugar, more preferably 25 pM - 75 pM of the non-natural sugar.

BRI EF DESCRI PTI ON OF TH E DRAWI N GS

[0080] The following figures provide preferred embodiments for illustrating the disclosure and should not be seen as limiting the scope of invention.

[0081] Figure 1: Bioorthogonal click chemistry of naturally-in-suspension cells for the obtention of living materials and brief description of the workflow.

[0082] Figure 2: Embodiment of fluorescence and confocal microscopy analysis of metabolic glycoengineered THP-1, Jurkat and human dendritic cells, with increasing concentrations of the azido sugar derivative N-azidoacetylmannosamine-tetraacylated (Ac4ManNAz), i.e., 0 pM, 10 pM, 50 pM and 100 pM, after 24 h incubation. Blue channel - Cells' nuclei (Hoechst 33342). Green channel - Cell surface azides labelled with DBCO-PEG4-RhodllO. Scale bars represent 100 pm.

[0083] Figure 3: Embodiment of flow cytometry analysis of metabolic glycoengineered THP-1 cells. (A) Representative dot plots of THP-1 incubated with different Ac4ManNAz doses following a 24 h incubation period. (B) Fluorescence intensity analysis of increasing Ac4ManNAz administered doses in THP-1 cells. Data are presented as mean ± s.d., n = 3. (C) Representative histograms of THP-1 incubated with different Ac4ManNAz doses, for 24 h. Control — pristine THP-1 cells representing a negative control. 10 pM Ac4ManNAz - cells functionalized with 10 pM Ac4ManNAz. 50 pM Ac4ManNAz — cells functionalized with 50 pM Ac4ManNAz. 100 pM Ac4ManNAz — cells functionalized with 100 pM Ac4ManNAz.

[0084] Figure 4: Embodiment of A) Metabolic activity analysis of glycoengineered THP-1 cells with different concentrations of Ac4ManNAz at day 1, day 3 and day 7 after functionalization. Data are presented as mean ± s.d., n = 4, and statistical differences were determined by one-way ANOVA with Tukey's multiple comparisons test, where: n.s = non-significant, ** p < 0.01, *** p < 0.001 and **** p < 0.0001. (B) Metabolic activity analysis of glycoengineered Jurkat cells with different concentrations of Ac4ManNAz at day 1, day 3 and day 7 after functionalization. Data are presented as mean ± s.d., n = 12, and statistical differences were determined by one-way ANOVA with Tukey's multiple comparisons test, where: n.s = non-significant, *** p < 0.001 and **** p < 0.0001.

[0085] Figure 5: Embodiment of (A) ¹H-NMR spectroscopy of HA, HA-TBA and HA-DBCO. (B) Methodology for the assembly of cell agglomerates. (C) Micrographs of cell agglomerates (cell density of 100 x 10⁶ cells per mL

[0086] Figure 6: Embodiment of assembly of the cell agglomerate material (cell density of 100 x 10⁶ cells per mL) with relatively reduced Ac4ManNAz concentrations (10 pM, 25 pM and 50 pM). Culture medium transferring of (A) 10 pM cell agglomerate material (B) 25 pM and 50 pM cell agglomerate material. (C) 25 pM and 50 pM cell agglomerate material appearance after 3 days in culture medium.

[0087] Figure 7: Embodiment of assembly of cell agglomerate material (cell density of 100 x 10⁶ cells per mL) with relatively high Ac4ManNAz concentrations (75 pM and 100 pM). (A) 75 pM and 100 pM cell agglomerate material removal from the PDMS mold depicting their handling stability. (B) 75 pM and 100 pM cell agglomerate material appearance after 3 days in culture medium.

[0088] Figure 8: Embodiment of confocal microscopy analysis of 75 pM sugar-incubated THP-1 immunoids viability on day 3, day 7 and day 14 of maturation in standard culture medium. Each image is a result of slice stacking from the cell agglomerate material (immunoids) with 2 mm height and cell density of 100 x 10⁶ cells per mL. PI — propidium iodide, nucleic acids labelling. Calcein-AM — live cells labelling. Scale bars represent 200 pm.

[0089] Figure 9: Embodiment of confocal microscopy analysis of 75 pM Ac4ManNAz-incubated Jurkat immunoids viability on day 1, day 3, day 7, day 21 and day 28 of maturation in standard culture medium. Each image is a result of slice stacking from the cell agglomerate (immunoids) with 2 mm height and cell density of 100 x 10⁶ cells per mL. Red channel - propidium iodide, nucleic acids labelling. Green channel - Calcein-AM, live cells labelling. Scale bars represent 300 pm (day 1) and 100 pm.

[0090] Figure 10: Embodiment of cell-releasing profile of 75 pM cell agglomerate material, namely THP-1 immunoids. (A) Cumulative percentage of released cells (shed cells) in day 0 — immediate release -, day 3 and day 7 of culture). Data are presented as mean ± s.d., n = 3, and statistical differences were determined by one-way ANOVA with Tukey's multiple comparisons test, where: *** p < 0.001 and **** p < 0.0001. (B) Table describing the fraction of released cells on day 0, day 3 and day 7 from three independent cell agglomerates (n = 3). (C)/(D) Fluorescence microscopy analysis of released cells from cell agglomerates after 7 days of maturation in standard culture medium. Scale bars represent 200 pm and 100 pm, respectively. Red channel (PI)— propidium iodide, nucleic acids labelling. Green channel (Calcein-AM) — live cells labelling.

[0091] Figure 11: Embodiment of A) Fluorescence microscopy analysis of metabolic glycoengineered ASCs, with increasing Ac4ManNAz concentrations, after 24 h incubation. Blue channel - Cells' nuclei (DAPI). Green channel - Cell surface azides labelled with DBCO-PEG4-RhodllO. Scale bars represent 150 pm; B) Flow cytometry analysis of metabolic glycoengineered ASCs, as a function of the concentration of Ac4ManNAz (10 pM, 50 pM or 100 pM Ac4ManNAz); C) Cell density tunability of ASCs living materials; D) Self-healing of ASCs living materials. Scale bars represent 5 mm. (E) Confocal imaging of fluorescently-labelled cell agglomerates' halves self-healed into cuboids after 7 days. Normalized line fluorescence intensity profile across the constructs. Green channel - DiO-hASCs. Red channel - DiD-hASCs. Scale bars, 1 mm.

[0092] Figure 12: Embodiment of confocal imaging of fluorescently-labelled heterotypic cell agglomerates (hASCs and Jurkat). Green channel - CMFDA-THP-1. Red channel - DiD-hASCs. Scale bars represent 200 pm.

[0093] Figure 13: Embodiment of results of A) ELISA human IL-2 quantification of freestanding Jurkat GFP cells, freestanding 100 pM Ac4ManNAz-incubated Jurkat GFP cells and Jurkat GFP immunoids. (B)/(C) Confrontation assay: Jurkat immunoids and GelMA hydrogels containing A549 cells were placed together, (i) Control: A549 GelMA 5% hydrogel (100 pL, 10⁸ cells per mL). (ii) Migration of Jurkat towards A549 hydrogel: Jurkat immunoids with 1 and 3 days of maturation placed directly in contact with A549 GelMA hydrogels, (iii) A549 GelMA 5% hydrogel with free Jurkat living material (no contact). Red channel - DiD, Jurkat cells. Green channel - Phalloidin green, actin filaments of Jurkat and A549 cells. Blue channel - DAPI, Jurkat and A549 nuclei. Scale bars represent 200 pm.

[0094] Figure 14: Embodiment of Jurkat cell agglomerate following removal from the assembly mould. Cell agglomerate prepared with 100 pMAc4ManNAz and cell density of lx 10⁹ cells per mL.

[0095] Figure 15: Embodiment of optical images of 100 pM Ac4ManNAz-incubated Jurkat cell agglomerates in standard culture medium (RPMI) at day 0, day 1 and day 7 of maturation. Cellreleasing profile develops during the maturation of the cell agglomerates. Scale bars represent 500 pm for day 0 and day 1 of maturation, and 100 pm for day 7.

[0096] Figure 16: Embodiment of results of A) Live/Dead fluorescence microscopy analysis of hASC- based cell agglomerates fabricated with different geometries, namely a, cylinder, b, hexagon and c, prism, matured for 24 h.; B) micrographs and Live/Dead fluorescence microscopy analysis of discshaped cell agglomerates matured for 24 h comprising different human cell building blocks, namely a, hUVEC, b, hDF and, c, hPANC-1; Data demonstrates the cytocompatible cell-to-cell orthogonal crosslinking regardless of construct shape. Blue channel - cells nuclei. Green channel - calcein-AM, live cells. Red channel - propidium iodide, dead cells. Scale bars, 200 pm; C) confocal imaging of heterotypic cell agglomerates comprising two different cell types (i.e., hASCs and hDFs) that were combined at varying ratios (i.e., 0, 25, 50, 75, 100%). The distinct cellular building blocks (i.e., hASCs and hDFs) were fluorescently-labelled with DiD and DiO fluorophores, respectively. Green channel - DiO-hDFs. Red channel - DiD-hASCs. Scale bars, 100 pm.

[0097] Figure 17: Embodiment of in vivo analysis of application of cell agglomerate materials for wound healing. A, Treatment timeline and schematic representation of the wound model used. B, Representative micrographs of excisional wounds over time. Scale bars, 1 cm. c, Representative traces showing the progression of wound bed closure during treatment. D, Quantification of wound areas across different groups over time, relative to the initial wound area. Data represented as mean ± s.d., n = 5. ****p < 0.0001 (Day 7 cell agglomerate material with hASCs vs Sham, and Day 12 cell agglomerate material with hASCs vs Sham), one-way ANOVA with Tukey's multiple comparisons test. E, Representative hematoxylin & eosin (H&E) staining in tissue cross-sections showcasing wound re- epithelial ization profile over time. Dotted lines are placed on the right of wound boundaries. Scale bars, 250 pm. F, Quantification of wound re-epithelialization (%). Data represented as mean ± s.d., n = 5. *p = 0.0333, **p = 0.0033, two-tailed unpaired Welch's t-test. G, Quantification of granulation tissue thickness. Data represented as mean ± s.d., n = 5. *p = 0.0263, ***p = 0.0005, two-tailed unpaired Welch's t-test. H, Quantification of neovascularization in wound sites, expressed as blood vessel number. Data represented as mean ± s.d., n = 5. *p = 0.0167, two-tailed unpaired Welch's t-test.

DETAI LED DESCRI PTION

[0098] The present disclosure relates to a cell agglomerate material comprising a modified hematopoietic cell; and a chemically functionalized biomaterial tethered to the surface of the modified hematopoietic cell; wherein the modified hematopoietic cell displays at its surface a chemical moiety that is covalently bound the chemically functionalized biomaterial. The disclosure also relates to a cell agglomerate material for use in medicine, in particular for use in the treatment of cancer or immune system diseases, to the use of said cell agglomerate material as a drug screen platform or disease modelling platform, and method of production thereof.

[0099] Surprisingly, the obtained cell agglomerate material allows the release of living cells as free, non-aggregated units along time. Thus, the disclosed cell agglomerate material can be used as a cell releasing platform, for localized delivery of cells in vivo, promoting immunomodulation of tissues in situ and in vivo. In an embodiment, the agglomerate releases at least 70% of total hematopoietic derived lineage cell unit, after 7 days of incubation. [00100] In an embodiment, the disclosed cell agglomerate material was prepared using a human monocytic cell line derived from an acute monocytic leukaemia patient (THP- 1 cell line). THP-1 cells were cultured in RPMI-1640 medium, supplemented with sodium bicarbonate (1.2 g L ¹), 4-(2- hydroxyethyl)-l-piperazineethanesulfonic acid (HEPES, 2.4 g L ¹), 10 %(v/v) fetal bovine serum (FBS) and 1 % antibiotic (ATB). Cell manipulation was performed under fully aseptic conditions in a Class II biological safety cabinet, and cells were cultured in an incubator, at 37 °C, within a humidified 5 % CO2 atmosphere. Routinely, THP-1 cells were cultured in suspension in non-adherent T-flasks. Cell growth was monitored every other day by using an optical contrast microscope. When appropriate, cell counting was performed in a Neubauer chamber to determine maximum cell density before passaging (8 x 10⁵ — 1 x 10⁶ cells per mL). For cell expansion, cells were harvested through centrifugation (250 g, 5 min, room temperature), counted and distributed in non-adherent T-175 cm² cell culture flasks, at a density of 5 x 10⁵ cells per mL, in a total volume of 40 mL to promote cell expansion. Cell culture media was replenished every 3 to 4 days depending on cell density.

[00101] For the scope and interpretation of the present disclosure, the term "room temperature" relates to a temperature ranging from 20°C to 25°C, preferably 21°C.

[00102] In another embodiment, the cell agglomerate material was prepared using an immortalized T lymphocyte cell line derived from an acute T cell leukaemia patient (Jurkat cell line), cultured using the same protocol as described for THP-1 cells. The green-fluorescent Jurkat cell line, stably transfected with the turboGFP protein, was cultured similar to both THP-1 cells and Jurkat cells, with the addition of G418 as selection antibiotic at 3 pL/mL every 2 weeks of culture.

[00103] In yet another embodiment, hDCs were cultured in tissue culture plates at a density of 1-2 x 10⁶ cells/mL in RPMI-1640 medium supplemented with 10% FBS, 1% ATB, 1% L-Glutamine, and a cocktail of granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-4 to induce dendritic cell differentiation. Cell manipulation was performed as previously described under fully aseptic conditions in a Class II biological safety cabinet, and cells were cultured in an incubator, at 37 °C, within a humidified 5 % CO2 atmosphere. As a standard procedure, hDCs were maintained in suspension culture using non-adherent T-flasks and monitored using an optical contrast microscope. Culture medium was replenished every 2-3 days by carefully replace half of the medium with fresh medium containing GM-CSF and IL-4. After 5-7 days in culture, immature dendritic cells were counted using a haemocytometer and overall viability was assessed using trypan blue.

[00104] In a further embodiment, human adipose tissue-derived mesenchymal stromal cells (hASCs) were isolated from subcutaneous adipose tissue obtained from liposuction procedures, processed and phenotypically validated as known in the state of art. Human umbilical vein endothelial cells (hUVECs) were isolated from umbilical cord vein, processed and phenotypically validated as known in the state of art. The aforementioned human tissues were obtained in Aveiro from Hospital do Baixo Vouga and Hospital da Luz, after approval of the corresponding competent Ethics Committee. Informed consent was obtained from all subjects. Human primary dermal fibroblasts (hDFs, ATCC® PCS-201-012™) and human pancreatic ductal epithelioid carcinoma cell line (hPANC-1, ATCC® CRL-1469™) were purchased from LGC Standards S.L.U. hASCs were routinely cultured in a-MEM (Thermo Fisher Scientific), hUVECs were maintained in Medium 199 (Thermo Fisher Scientific), hDFs were cultured in Medium 106 (Thermo Fisher Scientific) and hPANC-1 were maintained in RPMI 1640 medium (Thermo Fisher Scientific). Unless otherwise specified, a-MEM and RPMI 1640 were supplemented with sodium bicarbonate (2.2 g L-l, Sigma-Aldrich Merck KGaA), 10 % (v/v) heat-inactivated fetal bovine serum (FBS, South America origin, Thermo Fisher Scientific) and 1 % of antibiotic-antimycotic mixture (Streptomycin, Amphotericin B and Penicillin, Thermo Fisher Scientific). Medium 199 was supplemented with sodium bicarbonate (2.2 g L ¹), 20 % FBS, 1 % antibiotic-antimycotic mixture, 1 % GlutaMAX™ (Gibco). In addition, the complete medium 199 was freshly supplemented with heparin sodium salt (100 pg mL-1, from porcine intestinal mucosa, PanReac AppliChem) and endothelial cell growth supplement (40 pg mL ¹, from bovine neural tissue, Sigma-Aldrich Merck KGaA). Culture medium was exchanged every 3 to 4 days. Cells were subcultured before reaching confluence by using trypsin-EDTA solution (0.25 %, Sigma-Aldrich Merck KGaA). In brief, hASCs (ranging from passage 4 to 9), hDFs (ranging from passage 3 to 12), hUVECs (ranging from passage 4 to 6) and hPANC-ls (ranging from passage 4 to 9) were used.

[00105] In an embodiment, the chemical functionalization of cell membrane sialoglycoproteins with bioorthogonal azides was prompted through a metabolic engineering technique consisting of the allocation of non-natural mannosamine monosaccharides functionalized with azide (-N3) groups. For this purpose, THP-1 cells were seeded in p-Slide 8 well chambers at a density of 50 x 10³ cells per well and incubated for 24 h with complete culture medium and different concentrations of the azido sugar derivative (Ac4ManNAz), particularly, 0 (untreated control), 10, 50 and 100 pM. Subsequently, the cells were washed twice with Dulbecco's phosphate-buffered saline (dPBS, pH 7.4) and then incubated for 1 h, at room temperature (RT), with a nuclear labelling probe (Hoechst 33342, 20 pg mL ¹) and DBCO- PEG4-RhodllO (20 pM in serum free RPMI 1640). Cells were then washed two more times with dPBS and then carefully immersed in dPBS in the p-Slide 8 well chambers. To validate the successful azide- labelling, modified cells were imaged in a fluorescence microscope (Figure 2). Azide groups detection was performed by using an azide reactive fluorescent probe, DBCO-PEG4-RhodllO (20 pM, 1 h incubation, washed twice).

[00106] In an embodiment, metabolic glycoengineering of THP-1 was validated via flow cytometry analysis in which cells were incubated with different concentrations of Ac4ManNAz, 0 (untreated control), 10, 50 and 100 pM, having 2.5 x 10⁶ cells per condition. After a 24 h incubation period, the azide-modified cells followed the same DBCO-PEG4-RhodllO labelling protocol as previously described. The resulting cell pellet was resuspended with dPBS and sieved with a cell strainer (40 pm) before the analysis. Flow cytometry analysis was performed through the collection of 5 x 10⁴ events in the region of interest regarding DBCO-PEG4-RhodllO-labelled cells (Figure 3).

[00107] In another embodiment, the introduction of non-natural mannosamine monosaccharides carrying azide (-N3) groups into Jurkat cells and human DCs, which prompted the chemical functionalization of cell membrane sialoglycoproteins, through a metabolic engineering technique was performed as previously described for THP-1 cells. To further validate azide labelling of the cell membrane, Jurkat and hDCs were imaged through fluorescence and confocal microscopy, respectively (Figure 2). In yet another embodiment, the same protocol was applied to hASCs (Figure 11), hUVECs, hDFs, and hPANC-1.

[00108] In an embodiment, the cytocompatibility of azide functionalization was evaluated by measuring the metabolic activity of azide-functionalized THP-1 cells. The metabolic activity was assessed in three separate time-points, 1, 3 and 7 days, following cells' metabolic glycoengineering with different sugar concentrations (Figure 4A). Metabolic activity was evaluated by using the non- reactive CellTiter 96® Aqueous One Solution cell proliferation assay. In brief, THP-1 cells were seeded in a 96-well plate (non-adherent) at a density of 50 x 10³ cells per well (n = 10) and incubated in complete culture medium with different Ac4ManNAz concentrations, 0 (untreated control), 10, 25, 50, 75 and 100 pM, having as well a 0.1 % DMSO control group. Then, each cell-containing well was incubated with the assay reagent and left for 2 h in the incubator, covered from light, according to the manufacturer's instructions. The absorbance of the formazan salt was then analysed in a multi-mode microplate reader equipped with a tungsten halogen lamp, X = 490 nm. The absorbance retrieved for the blank (i.e., culture medium incubated with the assay reagent) acted as a correction value. The other conditions were then normalized to the untreated control group. Similarly, the metabolic activity of azide-functionalized Jurkat cells was assessed after 1, 3 and 7 days of culture, following cells' metabolic glycoengineering with a range of sugar concentrations — 0 (untreated control), 10, 25, 50, 75 and 100 pM of Ac4ManNAz. The metabolic activity was evaluated by using the non-reactive CellTiter 96® Aqueous One Solution cell proliferation assay.

[00109] In an embodiment, high molecular weight hyaluronic acid (HA, MW: 1.5-1.8 Mda), a naturally occurring glycosaminoglycan, was selected to act as the backbone for azide-cell attachment, which functionalization was attained through the conjugation of dibenzocyclooctyne (DBCO) moieties in a two-step chemical reaction. Firstly, hyaluronic acid sodium salt was converted to the tetrabutylammonium salt via acidic ion exchange to become soluble in organic solvents, including dimethyl sulfoxide (DMSO). The hyaluronic acid sodium salt (1 g, 0.5 % w/v in ultrapure water) was added to a proton exchange resin (3 g, DowexTM 50WX8 100-200,) and stirred at 800 rpm, for 24 h, at RT. The polymer solution was then centrifuged (3500 g, 10 min), titrated to pH 7.03 with tetrabutylammonium (TBA, 40 wt.% in water), frozen at -80 °C and ultimately freeze-dried, for 5 days. Hyaluronic acid-TBA formation was evaluated by ¹H-NMR spectroscopy to identify the characteristic signals of the salt in HA backbone (Figure 5A). The second part of this strategy consisted of the chemical functionalization of hyaluronic acid with DBCO units via covalent bond. In short, hyaluronic acid-TBA salt (0.41 g, 0.5 % w/v in DMSO) was combined with DBCO-PEG4-NH2 (0.050 g, 0.15 molar equivalents to monomer) and placed in a round bottom flask. Then, benzotriazole-1- yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP, 0.047 g, dissolved in DMSO — 2 mL) was added to the previous solution and further stirred at 900 rpm during 48 h in an inert atmosphere (N2) at room temperature and covered from light. The reaction was quenched by adding double distilled deionized and filtered (0.2 pm filter) water and transferred to a dialysis tubing (molecular weight cut-off 6000- 8000 Da), which was kept dialyzing against NaCI (0.1 M) for 10 days, and then, with ultrapure water for 3 days. The obtained mixture was further neutralized (pH = 7.4) with sodium bicarbonate (1 M in ultrapure water), frozen at -80 °C and freeze-dried for 7 days.

[00110] In an embodiment, the validation of the DBCO-hyaluronic acid synthesis was investigated through proton nuclear magnetic resonance (¹H NMR) spectra recorded on a 400 MHz spectrometer. Prior to spectra acquisition, samples were dissolved in deuterated water, D2O (containing 0.05 wt.% 3- (trimethylsilyl)propionic-2,2,3,3-d4 acid, sodium salt), and transferred into 5 mm NMR tubes and analysed with 1 second relaxation delay, 512 scans and 2 dummy scans. The degree of DBCO substitution was established through the analysis of the corresponding DBCO peaks (6 = 7.75 — 7.25 ppm, 8 protons), having as internal reference the hyaluronic acid acetamide peak (6 = 2.0 ppm, 3 protons). In an embodiment, the degree of substitution (D.S.) was measured asl2% (Figure 5A).

[00111] In an embodiment, the assembly of the 3D living materials, in the present disclosure termed as cell agglomerate material (or immunoids if obtained using lymphoid and/or myeloid cells), required prior azide functionalization on cells' surface sialoside glycoproteins, such as on THP-1 cells, before combining the cell tethering biopolymer (as depicted in Figure 5B). Briefly, for immunoids assembly, a culture of THP-1 supplemented with complete culture medium aimed for a density of 500 x 10³ cells per mL, then having a supplementation of 75 pM Ac4ManNAz, with a 24 h incubation period. Functionalized cells were then harvested, washed with dPBS and the obtained pellet was further resuspended in HA-DBCO (2 % w/v, in serum-free culture medium) reaching a homogenous mixture with the pre-designed cell density of 100 x 10⁶ cells per mL. Sterile polydimethylsiloxane (PDMS, Dow Corning) homemade moulds with disc-shaped reservoirs served as containers for immunoids precursor solution, which was left to self-assemble in the incubator, at 37 °C, 5 % CO2, for 4 h. Afterwards, the immunoids constructs were transferred to 12-well plates and cultured in complete culture medium (3 mL), with the last being exchanged every two days (Figure 5C). The negative control for the immunoids formation followed two different strategies, whereas the first consisted of the resuspension of azide- functionalized THP-1 cells with unmodified hyaluronic acid (2 % w/v, in serum-free culture medium with ATB) resulting in a final cell density of 100 x 10⁶ cells per mL, followed by a 4 h incubation period. As the second approach for the negative control, pristine THP-1 cells were resuspended in DBCO- hyaluronic acid (2 % w/v, in serum-free culture medium) at a final cell density of 100 x 10⁶ cells per mL, incubating for 4 h.

[00112] In another embodiment, the assembly of the Jurkat and Jurkat GFP immunoids required prior azide functionalization of cells' surface sialoside glycoproteins before combining with the cell tethering HA-DBCO biopolymer by following the experimental design of THP-1 3D living materials, having the constructs in this case been assembled with 75 pM and 100 pM of Ac4ManNAz. In yet another embodiment, cell agglomerates were also obtained using hASCs, hUVECs, hDFs and hPANC-1, or combinations thereof (Figure 11, Figure 12 and Figure 16). These cells were surface functionalized with azide groups following the aforementioned metabolic glycoengineering process. Briefly, cells were cultured in their respective suitable standard culture medium until reaching 70% confluence. Culture medium was supplemented with 100 pM Ac4ManNAz and then added to each cell type. After glycoengineering, azide-bearing cells were washed with warm dPBS, detached using trypsin-EDTA, neutralized in their respective culture medium (equal volume) and passed through a cell strainer (100 pm) to remove remaining cell aggregates. After centrifugation (300 g, 5 min), azide bearing cell pellets were gently resuspended in DBCO-hyaluronic acid (2 % w/v, in serum-free culture medium) to yield solutions with pre-determined cell densities (10, 20, 40, 80 and 100 x 10⁶ cells/cm³). The obtained precursor solutions were then distributed into sterile polydimethylsiloxane elastomer (PDMS, Sylgard® 184, Dow Corning) molds containing multiple geometrical configurations (i.e., cylinder, hexagon and triangular prisms). After self-assembling (3 h, cell culture incubator), the resulting cell agglomerates were transferred to 12-well plates (non-adherent, VWR) and cultured under standard conditions with frequent media exchange every 2 days.

[00113] In an embodiment, the total mass content distribution of cells and DBCO-hyaluronic acid in dry cell agglomerate materials (i.e., using 2 % w/v DBCO-hyaluronic acid) with densities ranging 10 to 100 x 10⁶ cells/cm³ was calculated assuming 3.5 ng weight per mammalian cell. In an embodiment of results, cell agglomerate materials comprise 60 to 95 % wtdry of cells and 5 to 40 % wtdry of the functionalized biomaterial, in particular HA-DBCO. Thus, these materials comprise a high cell density, with more than half of its dry mass being cell material.

[00114] In an embodiment to assure the cytocompatibility of the intercellular bioorthogonal crosslinking methodology, immunoid's viability was additionally evaluated by performing a Live/Dead assay. For this purpose, the THP-1 disc-shaped immunoids were assembled as described above and further analysed in three time-points, specifically, day 3 day 7 and day 14 after maturation. Immunoids were washed with dPBS and incubated (30 min, at 37 °C, 5 % CO2) in dPBS containing Calcein-AM (5 pM in dPBS) and propidium iodide (7.5 pM in dPBS, PI). Subsequently, immunoids were gently washed twice with dPBS and imaged in a high-resolution laser-scanning confocal microscope, equipped with gallium arsenide phosphide (GaAsP) and photomultiplier (PMT) detectors. In a further embodiment, the cytocompatibility of cell agglomerates obtained with other cell types, such as Jurkat immunoids, was evaluated by performing a Live/Dead assay, following the already established protocol for THP-1 cells, using a high-resolution laser-scanning confocal microscope, equipped with GaAsP and PMT detectors (Figure 8).

[00115] In an embodiment, the morphology of the THP-1 immunoids was evaluated through F-actin and nuclei staining. THP-1 disc-shaped immunoids were assembled as previously described and analysed in day 7 and day 14 of maturation in complete culture medium. Prior to the staining, the constructs were washed with dPBS and fixed at day 7 and day 14 of maturation in formaldehyde (4 % v/v in dPBS) during 1 h, washed with dPBS, and then permeabilized with 0.1 % Triton X-100 for 10 minutes at room temperature, followed by dPBS washing. The immunoids were labelled with Flash Phal loidin Red 594 (1.65 pM in dPBS) and left incubating for 30 min, at room temperature. Afterwards, the immunoids were washed with dPBS, and the cell nuclei were stained with DAPI (5.71 pM in dPBS) for 10 min, and then washed three times with dPBS for further imaging in a high-resolution laserscanning confocal microscope, equipped with GaAsP and PMT detectors.

[00116] In an embodiment, the cell-releasing profile from immunoids under physiological conditions was analysed. The transient nature of the Ac4ManNAz modification in THP-1 immunoids translates into cell shedding throughout the maturation time in culture medium. Cell shedding was evaluated through the ratio between released cells into the medium and the total cell amount of the living materials in three time-points, day 0, day 3 and day 7, also resorting to a Live/Dead assay in the last time-point to assess the viability of the released cells. In brief, for days 0, 3, and 7, immunoids complete culture medium was retrieved and centrifuged at 300 g for 5 min, and the obtained cell pellet was resuspended and stained with Trypan Blue (0.4 % v/v) for further cell counting in a Neubauer chamber. On day 7 of maturation, culture medium was retrieved and centrifuged, and the supernatant discarded, while the cells were incubated (30 min, at 37 °C, 5% CO2) with a dPBS solution containing Calcein-AM and PI (5 pM and 7.5 pM, respectively). Afterwards, the cell suspension was centrifuged, the supernatant discarded, and THP-1 cells were washed with dPBS and centrifuged one more time to remove Calcein and PI remains. The obtained pellet was resuspended with 200 pL of dPBS and seeded in 8 well chambers, and further imaged in a fluorescence microscope.

[00117] In an embodiment, selective cell surface glycoengineering was used as a pivotal strategy towards the final objective of assembling hematopoietic cells, in particular cell of lymphoid or myeloid lineage (such as THP-1 immune cells) in living constructs. [00118] In an embodiment selected azido-containing monosaccharide for glycosylation, N- azidoacetylmannosamine-tetraacylated (Ac4ManNAz), has convenient cytocompatible nature, endows cells with bioorthogonality as these selectively bind to the cell membrane sialoglycans, which fundamental units — sialic acids — usually settle on the outlying edges of the glycans structures.

[00119] In an embodiment of cell surface functionalization of THP-1 cells, initially various concentrations of Ac4ManNAz were administered for a 24 h period, under standard culture conditions (i.e., complete culture medium, 37 °C and 5 % CO2 atmosphere). The visualization of azide-labelled cell membranes was prompted by the administration of a fluorescent azide-reactive probe (DBCO- PEG4- RhodllO) into the functionalized THP-1 cells and then further analysed by fluorescence imaging. This approach takes advantage of the bioorthogonality of click-chemistry moieties (i.e., via strain-promoted azide-alkyne cycloaddition (SPAAC)), providing a highly selective labelling of cells and low background fluorescence, rendering it a valuable methodology to investigate cell surface modification via metabolic glycoengineering with azide sugars. Microscopy analysis (Figure 1) uncovered that fluorescence signal was directly proportional to Ac4ManNAz concentration. A dose of 100 pM, led to an almost full coverage of labelled cells. In contrast, the control group containing pristine (nonmodified) THP-1 cells has not shown any DBCO-related fluorescence as anticipated, considering that the sialoglycans of these cells were azide-free. THP-1 cells' morphology was also not affected throughout the administration of increasing Ac4ManNAz concentrations, keeping their characteristic spheroidal shape.

[00120] In an embodiment, a complementary flow cytometry analysis (Figure 3) supported the previous data concerning the successfully glycoengineered cells. The gating approach was based in two parameter density plots, using forward and side scattering, identifying the monocyte population and excluding debris (Figure 3). Ac4ManNAz-administered groups displayed a clear shift of their dot plots compared to the control group, indicating cells glycocalyx functionalization. These data also supported the previous results regarding the influence of azide-functionalization on THP-1 cells morphology, which was not affected by metabolic glycoengineering. The fluorescence intensity of DBCO-PEG4- RhodllO was investigated, having as expected higher fluorescence values — a consequence of increasing Ac4ManNAz concentrations (Figure 3). Both analyses (microscopy and flow cytometry) support that proper azide-functionalization was achieved, with immune cells presenting the selected mannosamine derivatives at their surface for subsequent intercellular HA-DBCO SPAAC coupling.

[00121] In another embodiment, the mannosamine derivative was administered to both Jurkat and hDCs, following the rationale used for THP-1 cell surface functionalization. Azide-labelled cell membranes were visualized using the DBCO- PEG4-RhodllO probe through fluorescence and confocal microscopy, respectively. Microscopy analysis (Figure 2) disclosed a similar behaviour to the one of azide-labelled THP-1 cells, regarding the coverage of labelled cells as the incubated azide shifts concentrations.

[00122] In an embodiment a characterization of azide-functionalized cells metabolic activity was performed. The engagement of the azido-functionalized mannose towards the biosynthetic machinery of THP-1 cells encouraged a metabolic activity analysis at 3 time-points: 1, 3 and 7 days of culture after Ac4ManNAz incubation. The data revealed that in the three time-points all the groups displayed higher percentages of metabolic activity compared to the control group (pristine THP-1 cells), indicating that both the DMSO group and the Ac4ManNAz-treated groups overstimulated cells' metabolism, confirming the unnatural sugar's hijacking of the sialic acid biosynthetic pathway as well. Regarding the metabolic activity results, the significant increase observed in the DMSO group on day 3 (Figure 4A) of culture might be correlated with cell membrane permeation. The abnormal influx of molecules within THP-1 cells caused by DMSO triggers intracellular mechanisms to compensate for the sensed metabolic imbalance. [8] From day 3, there is a noticeable difference between the 75 pM and 100 pM Ac4ManNAz- treated groups, with the latter exhibiting a significant decrease on its metabolic activity, also verified on day 7 of culture. This indicates that installing a significant number of non-native moieties can negatively affect cells biological functions and ultimately result in cell death, demonstrating a clear dose-response effect in this methodology. The corresponding cells metabolic activity when exposed to 25 pM, 50 pM and 75 pM of Ac4ManNAz, remained stable along the analysed time-points, simultaneously demonstrating efficient cell surface functionalization and noncytotoxicity. The highest Ac4ManNAz concentration (100 pM) translated into a decreased metabolic activity. Following this rationale, the selected Ac4ManNAz concentration for further THP-1 immunoids assembly was 75 pM given its compromise between the efficacy of azide functionalization and cell cytocompatibility.

[00123] In another embodiment, the effects of azide functionalization in Jurkat cells metabolic activity were assessed at 1, 3 and 7 days of culture after Ac4ManNAz incubation. The obtained data (Figure 4B) revealed no significant effects from the 3^rd day of culture regarding the highest concentrations of the mannosamine derivative towards the cells' metabolic activity. For this reason, subsequent analysis related with the assembly of living materials comprised 75 pM and 100 pM of Ac4ManNAz.

[00124] In an embodiment of optimization of quasi-all cellular, preferably myeloid, cell-based immunoids (assembly and characterization), the second element for cell agglomerate assembly, HA- DBCO, was then synthesized and used to provide a structural matrix for azide-functionalized cells to anchor and organize into a 3D macrotissue-dense construct in a totally artificial mode since these cells naturally exist in suspension as free units. The biopolymer HA was selected owing to its ubiquitous presence in the ECM of native human tissues, while the DBCO functionalization units were leveraged to establish the connection between cell surface azides and the HA framework. Subsequently, the crosslinking by the Strain-promoted alkyne-azide cycloaddition (SPAAC) bioorthogonal covalent coupling was tested through the combination of HA-DBCO with azide-functionalized THP-1 immune cells. The incubation of various Ac4ManNAz concentrations, namely 10 pM, 25 pM, 50 pM, 75 pM and 100 pM was also investigated. After the 4 h incubation period, glycoengineered cells were able to selfassemble into bulky tridimensional structures quasi-all formed by cells (100 x 10⁶ cells per mL).

[00125] Notably, the concentration of administered Ac4ManNAz, ranging from 10 pM to 100 pM, proved to have a substantial impact on integrity of cell agglomerates, at first on the PDMS mould removal and throughout its maturation period (Figures 6). Immunoid prepared with THP-1 incubated with 10 pM of Ac4ManNAz immediately lost their disc-shaped morphology during extraction from the mould, exhibiting highly soft mechanical properties and water texture, which was compromised while subjected to mechanical stress, e.g., dissolving itself during transfer to the culture medium (Figure 6A). Regarding the assemblies with intermediate concentrations 25pM and 50 pM, these held most of their structure after mould removal (Figure 6B), wherein the 50 pM immunoids being more handleable than those incubated with 25 pM of the modifying sugar. However, after 3 days in culture medium, the 3D constructs spontaneously shed into fragments (Figure 4C) owing to the low doses of administered azido-sugar and poor intercellular bridging, thus yielding short-term assemblies. For higher Ac4ManNAz concentrations, i.e. 75 pM and 100 pM, immunoids were more robust than the previous assemblies, contributing to easier handling (Figure 7). In an embodiment, the immunoids were left in culture medium, and after 3 days the differences amongst them were readily perceived, denoting the disassembly of the 100 pM immunoid given the already proven cytotoxic effect of the administered 100 pM Ac4ManNAz dosage for THP-1 cells (Figure 5B). In an embodiment for better results, the concentration of 75 pM is selected to assemble the subsequent immunoids since it presents the most adequate compromise between structural stability and cell activity for the analysed THP-1 cells (Figure 7B).

[00126] In a further embodiment, considering the identification of the optimal sugar dose and the production of stable cell agglomerates, in particular immunoids prepared with THP-1 and Jurkat cell, their cell viability was additionally characterized by Live/Dead assays at different maturation time points in vitro 3, 7 and 14 days to evaluate cellular activity across time. Overall, THP-1 immunoids exhibited a greater proportion of viable (green) cells to dead (red) cells throughout all the time-points. Also, THP-1 immunoids tridimensionality did not prompt necrotic core formation by having a uniform dispersion of viable and dead cells across the living material (Figure 8).

[00127] In an embodiment, to further corroborate the absence of significant cytotoxicity from the incubated concentrations of the mannosamine derivative towards the suspension living materials, construct viability was also evaluated through Live/Dead assays after 1,3, 5, 7, 21 and 28 days (Figure 9) of maturation, using Jurkat cells treated with 75 pM Ac4ManNAz for the assembly of Jurkat immunoids. The majority of the constructs' cells remained viable throughout their maturation in vitro, displaying an overall lesser number of dead cells.

[00128] In an embodiment, the releasing profile from the cell agglomerates, in particular the immunoids' cell releasing profile from the 3D platforms, was envisioned since metabolic glycoengineering of cells surface is biologically transient (i.e., not permanent as in other cell surface engineering techniques). In this sense, the release of living cell units from THP-1 immunoids was investigated at different timeframes. Immediately after their incubation in culture medium (day 0), and after 3 days and 7 days of maturation in culture. As the data corroborates, on day 0, the mean of released cells was roughly 2.5 % of total assembled cells, endowing the immunoids with a 97.5 % yield regarding successfully tethered cells (Figure 10A and 10B). The percentage of released cells slightly increased on day 3, an indication of continuous disassembly of the immunoids during their in vitro culture. Considerable cell-release was observed on day 7 of maturation, with approximately 70 % of the cells that comprised the original immunoid structure being present in culture medium in the form of free THP-1 cells (Figure 10A).

[00129] Interestingly, when performing a Live/Dead microscopy analysis on immunoids at day 7 the released cells were mostly viable indicating that the proposed methodology is suitable for promoting the release of fully functional THP-1 myeloid cells (Figure 10C and 10D). These findings also support the transient nature of metabolic glycoengineering as proposed in this methodology and with this tethering biomaterial. The fact that the 75 pM immunoid almost entirely tethers the available cells and achieves major cell-shedding at day 7 in culture medium, encourages this framework's biomedical applications that are discussed herein.

[00130] In another embodiment, adipose-derived stem cells (ASCs) were subjected to the disclosed bioorthogonal click reaction. Moreover, their uptake of Ac4ManNAz was assessed through fluorescence microscopy and cytometry (Figure 11A and 11B) and various cell densities were further tested to demonstrate the tunability of such cell-rich assemblies (Figure 11C). The use of hASCs relied in their known capacity of differentiation into various lineages and function specialization, particularly in wound healing by accelerating wound closure, re-epithelization, angiogenesis, and also acting as immune mediators. In this sense, the cell agglomerates prepared with modified hASCs were characterized regarding their self-healing capacity after disruption of the gel (Figure 11D) and by joining two frameworks assembled separately, as depicted in Figure HE with DiD and DiO stained living materials.

[00131] In another embodiment, heterotypic cellular constructs, herein exemplified by the incorporation of hASCs and THP-1 cells, confer distinct advantages in the domain of tissue engineering and regenerative medicine. hASCs, characterized by their multipotent differentiation capacity, specifically towards adipogenic lineages, are crucial orchestrators in tissue reparative processes. Concurrently, THP-1 cells, as a monocyte lineage, exhibit pronounced immunomodulatory competencies. The amalgamation of hASCs and THP-1 cells within a heterotypic living material engenders a cooperative interplay that amplifies therapeutic efficacy. Such cooperative interaction can contribute to the creation of a pro-regenerative milieu, characterized by amplified anti-inflammatory responses, immune homeostasis, and facilitation of tissue remodelling. The integration of hASCs and THP-1 cells within heterotypic constructs presents a paradigmatic approach in advancing regenerative medicine, propelling novel strategies for tissue restitution and functional rehabilitation. Following this rationale, a cell agglomerate as heterotypic living material was conceived, left in culture, and further imaged (Figure 12), having as cell units hASCs and THP-1 stained with DiD and green CMFDA, respectively, for better perception of the construct's heterogeneity. In yet another embodiment, heterotypic cellular constructs were obtained by preparing a cell agglomerate material using both hASCs and hDFs (Figure 16C).

[00132] In an embodiment, Jurkat GFP living materials were evaluated considering the effects that the artificial confinement and attachment had in the suspension cells (i.e., cells that are non-adhesive when cultured in vitro). Interleukin-2 exerts a ruling effect on T cells proliferation, as well as in the generation of effector and memory cells. [10] ELISA quantification of human IL-2 showcased the expression ranges of this cytokine in freestanding cells units compared to Jurkat immunoids (Figure 13A), having as control groups pristine Jurkat GFP cells and 100 pM Ac4ManNAz-treated Jurkat GFP cells, both in suspension. The overall results displayed a considerably higher detection of IL-2 in the Jurkat cell agglomerate material showing that the 3D assembly contributes to the activation of these cells, contrasting with the low absorbances of the freestanding Jurkat GFPgroups ultimately translating into the lack of detection of IL-2 (Table 2).

Table 2 — Interpolation of IL-2 concentration in analysed samples (pg/mL) from the mean corrected absorbances. Samples were recovered on day 1, day 3 and day 7 of culture; controls were performed using 5 million cells per construct (50 pL). Samples A, B and C - Jurkat immunoids assembled as previously described; K - free Jurkat cells in culture medium using the same cell density as for the immunoids (5xl0⁶ cells); KN3 - free Jurkat cells in culture medium using the same cell density as for the immunoids (5xl0⁶ cells), incubated with 100 pM Ac4ManNAz.

[00133] In an embodiment, the invasion dynamics of Jurkat T cells within a three-dimensional matrix of cancer cells was investigated. A549 cells (adenocarcinomic human alveolar basal epithelial cells) encapsulated in a 100 pL hydrogel of gelatin methacrylate (GelMA, 5% wt.%), were cultured in the presence of 10 pL DiD-stained Jurkat immunoids at a cell ratio of 1:1 (10⁸ cells per mL). Two distinct methods were employed to facilitate interaction: direct contact between the gelatin matrices and placing separate gels within the same well-plate. Prior to the confrontation between the cell agglomerates (immunoids) and A549 GelMA hydrogels, the maturation of Jurkat cell agglomerates was taken into account considering their expression levels of IL-2 - already presented in Figure 13A. Comparative analyses were conducted between two distinct maturation time points: one with a maturation period of 1 day and the other with a maturation period of 3 days. The results, depicted in Figure 13B and 13C revealed a significant difference in T cell migration between the two conditions. Specifically, the day 3 maturation model exhibited heightened migratory behaviour compared to the model with 1 day of maturation. This observation implies that an extended maturation period positively influences the migratory capability of T cells within the three-dimensional cancer cell matrix, underscoring the importance of temporal factors in modulating T cell functionality in this microenvironment.

[00134] In an embodiment of optimization of quasi-all cellular lymphoid cell-based immunoids (assembly and characterization), the assembly of lymphoid cell lineage-based immunoid living agglomerates formation via the proposed method was also investigated. Both a higher cell density (1 x 10 ⁹ cells per mL) and a higher dose of azide sugar were administered to Jurkat T-lymphocyte cells that were used as model cell lineages of lymphoid living cells (Figure 14). The results demonstrate that T cell immunoids are mechanically robust, handleable, stable in culture and that they also exhibit cell releasing capabilities along time in culture (Figure 15). Indeed, considering that T lymphocytes are significant players of adaptive immunity having the ability to recognize a broad spectrum of antigens from pathogens and tumours, their assembly as 3D agglomerates can be applicable for immunotherapy-based approaches (i.e., engineered CAR-T cells), and other medical applications that can benefit from a localized delivery of a significant density of T-cells including for repair, treatment diagnosis and modelling of human diseases in vitro/ex vivo.

[00135] In an embodiment, an in vivo murine excisional wound splinting model was used to assess the performance of the disclosed cell agglomerate materials in promoting wound repair. All experiments and animal procedures received ethical approval and were approved by the General Directorate for Food and Veterinary (DGAV, under internal reference 2021-42). BALB/c nude female mice, 7 - 8 weeks old and weighing ~15 - 18 g at the time of surgery, were purchased from Charles River Laboratories. All animals were acclimated for 1 week before the experiment. Animals were housed individually after surgery in type III cages with a transparent divisor to prevent wound and bandage abuse. Animals were kept at standard conditions (i.e., 20 - 22 °C, 45 - 65 % humidity), exposed to 12 h light/dark cycle and provided food (2014S, Envigo) and water (distilled type II) ad libitum. After surgery, soft food and recovery gel (ClearH2O) were also provided to animals. In an embodiment, the surgery was performed under aseptic conditions with aerosolized isoflurane (IsoVet®) anaesthesia. Buprenorphine (0.08 mg/kg, Bupaq®) was given pre-surgery and every 12 h for the following 3 days. Additional paracetamol (200 mg/kg/day ben-u-ron®) was administered through the water during 5 to 7 days. Eye lubricant (Siccafluid®) and temperature control were ensured during the whole procedure. Dorsal skin was disinfected with povidone-iodine (Betadine®) followed by a 70 v/v % ethanol rinse. Sterile biopsy punches (0 = 6 mm, Kai Medical) were then used to create full-thickness excisional wounds in the interscapular area, followed by placing of a silicone splint with suture sites (14 mm O.D., 7 mm I.D., Grace Bio-Labs) centred around the wound. Splints were secured with non-absorbable suture lines (Silk, 5-0 size, Thermo Fisher Scientific) and then covered with a transparent film dressing (Tegaderm™, 3M) to protect wounds from dryness and self-grooming damage. Typically, because mouse skin is mobile, contraction accounts for a large part of the wound closure process. In the tested model, silicone splints are inserted to immobilize the wound, preventing wound closure through contraction. As a result, wound healing occurs through re-epithelialization and granulation tissue formation, a process that is more relevant to that occurring in humans. Mice were randomly assigned to "cell agglomerate material" or "sham" experimental groups, cell agglomerate materials prepared with hASCs were implanted into mice from the "cell agglomerate material" experimental group wound sites, and wound closure was assessed over time (Figure 17a). At their pre-determined timepoints (i.e., 3, 7 and 12 days), animals were sacrificed by isoflurane anaesthesia followed by cervical dislocation. After euthanasia, optical micrographs were captured from each wound to assess the extent of wound closure. For wound area quantification, open wound areas from each excisional defect were determined in ImageJ. Moreover, wound trace masks were generated in ImageJ following polygon fitting to wounds perimeter. At different euthanasia time-points, skin lesion areas were retrieved, preserved in formaldehyde (4% v/v in dPBS), processed via paraffin embedding, followed by sectioning at 4 pm thickness and staining with hematoxylin (5 min, Epredia™ Richard-Allan Scientific™ Hematoxylin Gill 2, Thermo Fisher Scientific) and eosin (4 min, Epredia™ Richard-Allan Scientific™ Eosin-Y alcoholic, Thermo Fisher Scientific) for histopathological analysis. Full tissue sections were scanned using the NanoZoomer 2.0HT (Hamamatsu Photonics) and digitally converted into virtual slides for visualization. All measurements and quantifications were performed at a 40x magnification within the NDP.view software (v2.9.29, Hamamatsu Photonics). Histopathological analysis was performed on H&E-stained tissue sections by two independent histopathologists blinded to the identity of the experiments. To assess wound re-epithelialization, the lengths of the original wounds and newly formed epithelia (i.e., neoepidermis) were measured. The percentage of re-epithelialization was calculated by dividing the length of neoepidermis by the total wound length and multiplying it by 100. In addition, the thickness of the newly formed granulation tissue between the wound edges was measured. Granulation tissue thickness for each mouse is presented as the average of 5 different measurements along the wound bed. To quantify neovascularization in the vicinity of the wound, multiple random high power (40x) fields (HPFs, n = 3) were examined in each section, with subsequent counting of blood vessels containing red blood cells. The quantification is expressed as the number of blood vessels normalized to the number of HPFs. The scar elevation index was calculated by dividing the thickness of the tissue in the wound bed by the thickness of non-injured tissue. Thickness measurements were performed from the epidermis to subcutaneous white adipose tissue and presented as the average of different measurements for each mouse. Mice body weight was assessed at each pre-determined timepoint during the course of the experiment. Macroscopic observations and wound trace analysis revealed a significant acceleration of wound closure over time with the treatment with the cell agglomerate material (Figure 17b and c). Specifically, on day 7, group treated with the cell agglomerate material presented 27.0 ± 9.4 % of wound area compared to 79.0 ± 16.2 % in the sham group. By day 12, only 8.8 ± 6.4 % of the original wound remained in the treated mice in contrast with 61.4 ± 22.0 % in the sham group (Figure 17b). Histopathological analysis of tissue crosssections indicated that the cell agglomerate material led to significantly improved re-epithelialization of wound beds by day 7, ultimately resulting in full tissue re-epithelialization by day 12 (Figure 17e and f). To further characterize the wound healing process, epithelial thickness index in complete re- epithel ial ized wounds was measured. Epithelial index analysis showed that the treated mice displayed epidermal hypertrophy (> 105 % epithelial thickness index) with a decreasing trend over time, indicating that wound healing progressed successfully.

[00136] The disclosed agglomerated material shows tissue-like densities and features similar to key aspects of living systems. The cell agglomerate materials are rapidly assembled via glycosaminoglycan linkages established between living cells and are autonomously reinforced by de novo secreted extracellular matrix components during maturation. In essence, the interfacing of cells with glycosaminoglycan-derivatives to form living materials resembles the natural interactions established between cells glycocalyx and glycosaminoglycans in biological tissues. This approach offers volumetric and architectural control over the assembly of living constructs that can be customized with a wide variety of human cell types to harness the biofunctionality and heterogeneous composition of living tissues. In addition, state-of-the-art genetic engineering tools can also be used to further customize cellular functions and instructive behaviour at the intracellular level beyond their native phenotypes, yielding cellular building blocks arising from the same cell type but encoded with distinct functions. The programmability of the disclosed cell agglomerate materials is thus expanded at the single cell level and allow to install additional biofunctionalities (e.g., tailored secretome or overexpression of matrix components). On an upper level, the ability of these constructs to autonomously merge with tissue-mimetic hydrogels greatly expands the toolbox of hybrid materials and their resulting multifunctional properties. The vast library of existing hydrogels amenable to be interfaced with the cell agglomerate materials adds another layer of modularity and programmability in the form of integrating spatially-confined compartments with tailored biomaterial composition, distinct cellular contents and functionally-graded mechanical environments. The synergy of seamlessly combining cellrich constructs with different classes of biomaterials (i.e., hydrogels, fibres, capsules) gives rise to new classes of cell-governed materials with unique physicochemical properties and bioactivity that can be better suited for engineering living tissue analogues. This multileveled programmability was further demonstrated by the intrinsic tissue-integrative and self-merging features of the cell agglomerate materials that allow for the modular assembly of different macro-scale building blocks serving as specialized biofunctional units. In an embodiment, the disclosed cell agglomerate materials can be interfaced with emerging pre-vascularization technologies to generate increasingly larger, biofunctional and complex materials.

[00137] Additionally, the cell agglomerate material of the present disclosure, comprising agglomerated hematopoietic tissue lineage-derived cells, and tethering biomaterials constitute valuable living materials which not only condense naturally non-condensable cells, but are also capable of releasing these biofunctional units of interest in a suitable time-window that supports their viability and consequently their biological activity.

[00138] The term "comprising" whenever used in this document is intended to indicate the presence of stated features, integers, steps, components, but not to preclude the presence or addition of one or more other features, integers, steps, components, or groups thereof.

[00139] Where singular forms of elements or features are used in the specification of the claims, the plural form is also included, and vice versa, if not specifically excluded. For example, the term "a tissue" or "the tissue" also includes the plural forms "tissues" or "the tissues," and vice versa. In the claims articles such as "a," "an," and "the" may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include "or" between one or more members of a group are considered satisfied if one, more than one, or all the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention also includes embodiments in which more than one, or all the group members are present in, employed in, or otherwise relevant to a given product or process.

[00140] Where ranges are given, endpoints are included. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and/or the understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. It is also to be understood that unless otherwise indicated or otherwise evident from the context and/or the understanding of one of ordinary skill in the art, values expressed as ranges can assume any subrange within the given range, wherein the endpoints of the subrange are expressed to the same degree of accuracy as the tenth of the unit of the lower limit of the range.

[00141]The disclosure should not be seen in any way restricted to the embodiments described and a person with ordinary skill in the art will foresee many possibilities to modifications thereof. [00142]The above-described embodiments are combinable.

[00143]The following claims further set out particular embodiments of the disclosure.

[00144] The following documents are incorporated by reference:

1. Laiosa, C. V., Stadtfeld, M., & Graf, T. (2006). Determinants of lymphoid-myeloid lineage diversification. Annual review of immunology, 24, 705-738.

2. Lavrador, P., Gaspar, V. M., & Mano, J. F. (2021). Engineering mammalian living materials towards clinically relevant therapeutics. EbioMedicine, 74, 103717.

3. Ghosh, S. (2020). Sialic acid and biology of life: An introduction. Sialic acids and sialoglycoconjugates in the biology of life, health and disease, 1.

4. Mbeki, L (2020). The emerging role of the mammalian glycocalyx in functional membrane organization and immune system regulation. Frontiers in cell and developmental biology, 8, 253.

5. Chen, R., Li, L, Feng, L., Luo, Y., Xu, M., Leong, K. W., & Yao, R. (2020). Biomaterial-assisted scalable cell production for cell therapy. Biomaterials, 230, 119627.

6. Park, J., Andrade, B., Seo, Y., Kim, M. J., Zimmerman, S. C., & Kong, H. (2018). Engineering the surface of therapeutic "living" cells. Chemical reviews, 118(4), 1664-1690.

7. Cheng, B., Xie, R., Dong, L., & Chen, X. (2016). Metabolic remodeling of cell-surface sialic acids: Principles, applications, and recent advances. ChemBioChem, 17(1), 11-27.

8. Xiang, Y., Zhao, M. M., Sun, S., Guo, X. L., Wang, Q.., Li, S. A., ... & Zhang, Y. (2018). A high concentration of DMSO activates caspase-1 by increasing the cell membrane permeability of potassium. Cytotechnology, 70(1), 313-320.

9. Kim, M. et al. (2017) 'Multi-cellular natural killer (NK) cell clusters enhance NK cell activation through localizing IL-2 within the cluster', Scientific Reports, 7(1). doi:10.1038/srep40623.

10. Rollings, C.M. et al. (2018) 'lnterleukin-2 shapes the cytotoxic T cell proteome and immune environment-sensing programs', Science Signaling, 11(526). doi:10.1126/scisignal.aap8112.

Claims

C L A I M S

1. A cell agglomerate material comprising

60 to 95 % wtdryof modified cells; and

5 to 40 % wtdry of a chemically functionalized biomaterial tethered to the surface of the modified cells; wherein the modified cells display at their surface a chemical moiety that is covalently bound the chemically functionalized biomaterial.

2. The cell agglomerate material according to any of the previous claims wherein the chemical moiety is selected from azide, diazirine, alkyne, alkene, norbornene, tetrazine, trans-cyclooctene, or combinations thereof.

3. The cell agglomerate material according to any of the previous claims wherein the chemical moiety is comprised in a surface glycoprotein of the modified cells, preferably a modified sialoglycoprotein.

4. The cell agglomerate material according to any of the previous claims wherein the surface glycoprotein comprises a non-natural sugar functionalized with the chemical moiety.

5. The cell agglomerate material according to any of the previous claim wherein the non-natural sugar is selected from a list comprising N-substituted mannosamine, O-substituted mannosamine, galactosamine, glucosamine, acetyl-glucosamine, trehalose, neuraminic acid, acylated sugars derivatives of neuraminic acid, tetraacylated sugars derivatives of neuraminic acid, tetraacylated mannosamine derivatives, or combinations thereof.

6. The cell agglomerate material according to any of the previous claims 4-5 wherein the non-natural sugar is tetraacylated mannosamine derivative functionalized with an azide chemical moiety.

7. The cell agglomerate material according to any of the previous claims wherein the modified cells are hematopoietic cells, stem cells, such as adipose stem cells, endothelial cells, fibroblasts, cancer cells, or mixtures thereof.

8. The cell agglomerate material according to any of the previous claims wherein the modified cells are hematopoietic cells, preferably of lymphoid or myeloid lineage.

9. The cell agglomerate material according to any of the previous claims wherein the cell is selected from a list comprising: natural killer cells, mast cells, myeloblasts, basophils, neutrophils, eosinophils, monocytes, megakaryocytes, thrombocytes, lymphoid progenitor cells, myeloid progenitor cells, multipotent hematopoietic stem cells B, T lymphocytes, macrophages, including all their subpopulation types and/or phenotypically differentiated derivatives, and combinations thereof.

10. The cell agglomerate material according to any of the previous claims wherein the chemically functionalized biomaterial is selected from a list comprising basement membrane extracts of animal or human origin, gelatin, poly(lactic acid), Poly (s- caprolactone), fucoidan, chitosan, laminarin, cellulose, glycogen, pectin, hyaluronic acid, chemically-modified hyaluronic acid, -poly-lactic acid-co- glycolic acid, decellularized extracellular matrix, fibronectin, collagen; or mixtures thereof.

11. The cell agglomerate material according to any of the previous claims wherein the chemically functionalized biomaterial is a decellularized extracellular matrix from human origin.

12. The cell agglomerate material according to any of the previous claims wherein the chemically functionalized biomaterial comprises a chemically reactive group that is orthogonally reactive to the chemical moiety comprised in the modified cell.

13. The cell agglomerate material according to any of the previous claims wherein the chemically functionalized biomaterial is functionalized with a reactive group selected from a list comprising azide, diazirine, alkyne, alkene, norbornene, furan, thiols, maleimide, tetrazine, dibenzocyclooctynol, dibenzocyclooctyne, strained alkyne, or combinations thereof.

14. The cell agglomerate material according to any of the previous claims wherein the chemically functionalized biomaterial is functionalized with dibenzocyclooctyne.

15. The cell agglomerate material according to any of the previous claims wherein the chemically functionalized biomaterial is hyaluronic acid functionalized with dibenzocyclooctyne.

16. The cell agglomerate material according to any of the previous claims wherein the chemically functionalized biomaterial is hyaluronic acid functionalized with dibenzocyclooctyne and the modified cells display at their surface an azide chemical moiety.

17. The cell agglomerate material according to any of the previous claims wherein the degree of functionalization of the chemically functionalized biomaterial is at least 10%, preferably 12 to 15%.

18. The cell agglomerate material according to any of the previous claims wherein the mass ratio between the modified cells and the chemically functionalized biomaterial is at least 1:0.2.

19. The cell agglomerate material according to any of the previous claims comprising at least 1 million cells per cubic centimetre, preferably at least 2 million cells per cubic centimetre of material.

20. The cell agglomerate material according to any of the previous claims wherein the material is implantable, injectable and/or mouldable.

21. The cell agglomerate material according to any of the previous claims wherein the covalent bound between the modified cells and the chemically functionalized biomaterial is degradable.

22. The cell agglomerate material according to any of the previous claims wherein the cell agglomerate is an immunoid.

23. A kit comprising a cell agglomerate material as described in any of the previous claims.

24. The kit according to the previous claim for use in tissue engineering, tissue repair, biomedical sensors, drug delivery systems, immunotherapy or diagnostics.

25. A cell agglomerate material as described in any of the previous claims 1-22 for use in medicine.

26. The cell agglomerate material according to the previous claim for use in the treatment of cancer, treatment of immune system diseases, immunotherapy, tissue engineering, cell delivery, wound healing, or tissue repair.

27. The cell agglomerate material according to the previous claims 25-26 for use as biomedical sensors, drug delivery systems, or diagnostics.

28. Use of a cell agglomerate material as described in any of the previous claims 1-22 as a drug screening platform, cell culture platform, or disease modelling platform.

29. A method for obtaining a cell agglomerate material as described in any of the previous claims the method comprising the following steps: incubating a pool of cells with a functionalized non-natural sugar to obtain a suspension of modified cells; mixing the suspension of modified cells with a chemically functionalized biomaterial for covalent bond formation, wherein the mass ratio between the modified cells and the chemically functionalized biomaterial ranges from 1:0.2 to 1:0.02; incubating the suspension of cells with the chemically functionalized biomaterial for at least 4 hours at 37 °C to obtain the cell agglomerate material. The method according to the previous claim wherein the functionalized non-natural sugar is incubated in the pool of cells for at least 24 h. The method according to any of the previous claims 28-29 wherein the suspension of cells is incubated with at least 0.1 pM of the non-natural sugar, preferably with 1 pM - 500 pM of the non-natural sugar. The use of the cell agglomerate material as described in any of the previous claims 1-22 for the manufacture of a medicament for the treatment of cancer and/or immune system diseases. A method for treating or preventing cancer and/or immune system diseases in a subject, the method comprising administering the cell agglomerate material of claim 1 to the subject.