Secretome-based biomaterial, method for its production and uses thereof

Abstract

The present invention relates to a secretome-based biomaterial, wherein the secretome is cell secretome selected from secreted soluble factors, apoptotic bodies, small extracellular vesicles or microvesicles that are released from cells of therapeutic interest to the cell culture medium or other fluids or combinations thereof. The invention further relates to a method for the production of the secretome-based biomaterial and to the use thereof for the treatment of diseases requiring tissue regeneration and / or immunomodulation.

Description

SECRETOME-BASED BIOMATERIAL, METHOD FOR ITS PRODUCTION AND USES THEREOF

[0001] The present application claims the benefit of priority to the Portuguese provisional patent application no. 119101, filed on December 6, 2023, and the Portuguese provisional patent application no. 119885, filed on December 5, 2024, the contents of which is incorporated herein by reference.

[0002] The present invention relates to the field of medicine, namely with the use of biomaterials in regenerative medicine strategies, and tissue engineering, mainly to treat diseases that require immunomodulation, regeneration of tissues, or modulation of fibrosis. Other biomedical and biological applications include cell expansion, organoid or 3D culture, disease modeling, induction of differentiation and drug delivery.Background of the invention

[0003] Biomaterials are substances engineered to interact with biological systems for therapeutic or diagnostic purposes. In the realm of tissue engineering and regenerative medicine, biomaterials are instrumental in restoring, maintaining, or improving tissue function.

[0004] Scaffolds and hydrogels are two primary classes of biomaterials extensively researched and utilized for tissue regeneration due to their ability to mimic the natural extracellular matrix (ECM) and provide a conducive environment for cell growth and tissue formation.

[0005] Hydrogels have found extensive use as fillers, drug or cell carriers, coatings, and substrates forin vitroculture of cells, tissues, or organoids1. Primarily, hydrogels sourced from synthetic or naturally derived animal materials have been created.

[0006] There is an increasing demand to develop naturally derived materials sourced directly from cells for numerous biomedical applications. This aims to enhance targeted bioactivity while maintaining biological safety, biocompatibility, and minimal immunogenicity.

[0007] By using different cells as sources of material to produce hydrogels, and also by previously modifying cell culture conditions to secretome collection, different products with high versatility and precision may be obtained. Furthermore, the ability to regulate degradation rate and mechanical stability emerges as a crucial prerequisite for novel materials.

[0008] In recent decades, cell secretomes have emerged as therapeutical candidates in tissue engineering and regeneration2. Secretomes have been described as a highly complex / heterogeneous and changing mixture of bioactive molecules (proteins, lipids, nucleic acids, and various signaling molecules) that cells release into the extracellular fluids.

[0009] These present a key role in cell-cell communication and can modulate the behavior of other cells and tissues. Cells release these molecules by different mechanisms, including exocytosis and shedding of extracellular vesicles of different origins (endosomal, plasmatic membrane, or apoptotic bodies).

[0010] The secretome effect on regeneration and tissue repair has been described in the past years as it can modulate immune responses, differentiation, and angiogenesis, among many others3,4.

[0011] Previous studies have attempted the incorporation of secretome or secreted extracellular vesicles into previously crosslinked or mixed with polymer-based hydrogels. Namely, silk protein as base material5,6, chitosan7, jellyfish collagen and polyurethane8, poly(ethylene glycol), or glycosaminoglycans9. Another study10used the solidified part of the secretome with dextran sulfate to form microparticles.

[0012] Despite significant progress, several challenges remain in the development and clinical translation of biomaterials for tissue regeneration, such as immunogenicity, good vascularization to promote rapid and adequate blood vessel formation within the biomaterial to supply nutrients and remove waste and optimal integration with the host tissue.

[0013] Another aim is to develop cost-effective, consistent, and reproducible methods which are suitable for large-scale production and regulatory approval.

[0014] In view of the above, the present invention proposes a secretome-based biomaterial, i.e., a biomaterial comprising secreted soluble factors and extracellular vesicles that are released to the cell culture medium or other fluids by cells of therapeutic interest. Thus, the biomaterial herein disclosed in completely autologous or derives from a compatible donor. It is also an object of the present invention the method for its production and uses thereof.Prior art

[0015] The state of the art reveals ongoing research on the biomaterial field, aiming to optimize their properties, address current challenges, and expand their applications, ultimately contributing to improved clinical outcomes in regenerative medicine.

[0016] The international application WO2018213795A1 relates to methods of delivering secreted factors from stem cells to tissues in order to immobilize and concentrate such secreted factors at or under the surface of damaged tissue to promote tissue regeneration.

[0017] US20220218873A1 patent application discloses the formulation of a bioink for application to the cornea comprising hyaluronic acid, gelatin, and exosomes derived from mesenchymal stem cells primed with a corneal stromal stem cell derived-conditioned medium.

[0018] US2022088274A1 relates to compositions and methods for repairing or regenerating damaged tissue, more specifically, methods of delivering secreted factors from stem cells to tissues in order to immobilize and concentrate such secreted factors at or under the surface of damaged tissue to promote tissue regeneration.

[0019] The international application WO2022159878A9 discloses a method for treating or preventing a cardiac injury in a subject comprising delivering a hydrogel-based composition into a portion of a pericardial cavity of a subject and improving at least one aspect of myocardial cells or tissue in the subject.

[0020] In all of the above cited prior art documents, the biomaterial includes other heterologous materials (such as polymers) which could induce an undesired physical and chemical / biological feature.

[0021] Thus, there is still a need to design and produce biomaterials based solely on cell secretomes, wherein the secretomes components can be further implemented, functionalized, and tailored in terms of mechanical properties, release profile, and composition depending on the requirements of the application.

[0022] The present invention relates to a secretome-based biomaterial, wherein the secretome is cell secretome selected from secreted soluble factors, apoptotic bodies, small extracellular vesicles or microvesicles that are released from cells of therapeutic interest to the cell culture medium or other fluids or combinations thereof.

[0023] The invention further relates to a method for the production of the secretome-based biomaterial and to the use thereof for the treatment of diseases requiring tissue regeneration and / or immunomodulation.

[0024] Current biomaterials used for immunomodulation and tissue regeneration, such as scaffolds and hydrogels, often face limitations in mimicking the natural extracellular matrix environment.

[0025] Due to this fact, existing biomaterials may lack the necessary biological cues required for optimal cell adhesion, recruitment, proliferation, differentiation, and phenotype modulation.

[0026] Furthermore, traditional methods for producing these materials may result in products with impurities due to insufficient purification processes. The presence of unwanted components from the cell culture medium or other fluids can compromise the material’s biocompatibility and effectiveness.

[0027] Additionally, the inability to tailor the material properties based on specific pre-conditioning or stimuli applied to parent cells limits the versatility and applicability of the biomaterials in diverse medical and research settings.

[0028] The present invention offers a comprehensive solution to the problems identified in the state of the art by introducing a novel biomaterial generated from secreted soluble factors, apoptotic bodies, small extracellular vesicles or microvesicles released by cells of therapeutic interest.

[0029] The invention proposes an optimized purification process that effectively discards other components of the cell culture medium or fluids, resulting in a highly pure product. By preserving the biological activity and integrity of the secreted factors and vesicles, the material maintains essential properties necessary for effective cell interaction and modulation, contrasting therefore with traditional methods that may compromise the functionality due to impurities or processing conditions.

[0030] Unlike synthetic scaffolds that may lack specific biological signals, the invention provides a material rich in natural factors that actively guide cell behavior.

[0031] By delivering a highly pure, biologically active, and versatile biomaterial that effectively supports immunomodulation and tissue regeneration, the present invention meets the needs unmet by current technologies.

[0032] The optimized purification method disclosed in the present invention ensures that other components of the medium or fluids are discarded, resulting in a highly pure product with conserved biological properties. This high level of purity enhances the material’s biocompatibility and effectiveness in clinical applications.

[0033] Also, the invention allows the biomaterial to be prepared in various forms, including particles, membranes, capsules, bioinks, foams, hydrogels, sponges, cream, ointments, patches, adhesives, fibers, 3D printed constructs, or discs. Each form contains biological cues essential for maintaining cell adhesion, promoting cell recruitment, supporting proliferation and differentiation, facilitating degradation, and modulating cell phenotype.

[0034] The ability to customize the material based on the pre-conditioning or stimuli applied to the parent cells adds a significant level of versatility. This adaptability enables the material to be tailored for specific applications such as cell expansion, organoid culture, disease modeling, and the induction of cell differentiation.

[0035] Overall, the invention offers a biomaterial that more closely mimics the natural extracellular matrix, with enhanced purity and customizable properties, overcoming the shortcomings of existing technologies in tissue engineering and regenerative medicine.

[0036] In order to facilitate an understanding of the principles according to the embodiments of this invention, reference will be made to the illustrated embodiments in the Figures and the language used to describe them.

[0037] It should also be understood that there is no intention to limit the scope of the invention to the content of the Figures and that modifications to the inventive features illustrated herein, as well as additional applications of the illustrated principles and embodiments, which would normally occur to a person skilled in the art having possession of this description, are considered within the scope of the claimed invention.Fig.1

[0038] graphically illustrates the post-purification characterization of the purified secretome by Dynamic Light Scattering, wherein (A) is the size measurements and (B) is the Z-potential measurements (B). Transmission electron microscopy of CD81-Dynabeads incubated with the purified secretome sample is also shown in (C) and (D).Fig.2

[0039] illustrates the obtained product after purification and freeze-drying (A). Macroscopic picture of the formed hydrogel with 10 % w / V after crosslinking with genipin 0.5% w / V is also shown (B).Fig.3

[0040] is a schematic representation of the genipin crosslinking reaction for secretome-based biomaterial production.Fig.4

[0041] graphically illustrates the rheological kinetics of the crosslinking process of secretome, wherein (A) is an schematic representation of the preparation of secretome samples and their crosslinking, (b) and (c) are the rheological analysis of the crosslinking process of secretome (obtained from naive ASCs) at a concentration of 10% (w / v) and 5% with genipin, respectively, (d) is the rheological analysis of the crosslinking process of secretome (obtained from IFN-gamma-modulated ASCs) at a concentration of 5% (w / v) with genipin and (e) is a summary data of the data obtained from three independent replicates from the rheology assays shown in (b) and (c). In all graphics, a timelapse analysis of the storage modulus (G’) and loss modulus (G’’) is shown.Fig.5

[0042] graphically illustrates the mouse endothelial cell (C166-GFP) viability evaluated by Alamar Blue after 24 and 72 hours of contact with the 10% w / V hydrogel (A), C166-GFP growing on the surface of the hydrogel after 7 days in contact (B and C), and mouse macrophages (RAW 264.7) adhered to the hydrogel after 48 h of culture, stained for nuclei (DAPI), actin and CD68 (D).Fig.6

[0043] shows secretome-based biomaterials produced by naive (control) or IFN-gamma-induced ASCs.

[0044] The present invention discloses, in a first aspect, a secretome-based biomaterial, wherein the secretome is cell secretome selected from secreted soluble factors, apoptotic bodies, small extracellular vesicles or microvesicles that are released from cells to the cell culture medium (adequate for animal cell culture) or other fluids or combinations thereof.

[0045] In a preferred embodiment of the present invention, the cell secretome is crosslinked via chemical crosslinking, physical crosslinking, guest-host interactions, enzymatic crosslinking via transglutaminase, photopolymerized or combinations thereof and is present in a concentration that ranges from 1-20 % (wsecretome / Vbiomaterial), preferably from 1-15% w / V, more preferably 5-10% w / V.

[0046] In a preferred embodiment of the present invention, the secreted soluble factors, apoptotic bodies, small extracellular vesicles or microvesicles are chemically modified or reacted with other molecules to enable intermolecular click chemistry selected from azide-dibenzocyclooctyne (DBCO) (strain-promoted azide-alkyne cycloaddition, SPAAC) or tetrazine based (inverse electron-demand Diels–Alder reaction, IEDDA), Schiff base, Diels-Alder (normal and inverse electron-demand) reactions, photochemical crosslinking in the presence or absence of photo initiators, and metal coordination complexes formation.

[0047] In a preferred embodiment of the present invention, the secreted soluble factors are selected from growth factors, interleukins, cytokines, structural proteins and proteins from secreted vesicles.

[0048] In a preferred embodiment of the present invention, the cells are cells of animal origin selected from stem cells, immune cells, or other cells of therapeutic interest that can be in their native state, immortalized cell lines, cells primed with physical, chemical or biological stimuli, gene edited cells or a combination thereof.

[0049] In a preferred embodiment of the present invention, the stem cells are selected from induced pluripotent cells and mesenchymal stromal cells and the immune cells are selected from T lymphocytes, dendritic cells, macrophages and NK cells.

[0050] When the cell culture is stimulated during growth, its release profile is modulated and, consequently, the biological properties of the resulting biomaterial can be tailored.

[0051] In a preferred embodiment of the present invention, the physical stimuli are selected from hypoxia (in the range of 0.1-7% O2), low-level lasers, mechanical stretch, silica, pulsed electromagnetic fields, 3D culture, or combinations thereof.

[0052] In a preferred embodiment of the present invention, the chemical stimuli are selected from metabolic acidosis, BAY 11-708, LL-37, dimethyloxalylglycine, JI-34, sevoflurane, atorvastatin, oxytocin, cyclophosphamide or combinations thereof.

[0053] In a preferred embodiment of the present invention, the biological stimuli are selected from migration inhibitory factor, IFN-α, TGF-β1, interleukin (IL)-1α / β, IL-25, IL-6, TNF-α, IL-17, IFN-γ, stromal-derived factor 1, gene modification, polyinosinic-polycytidylic acid, lipopolysaccharide or combinations thereof.

[0054] In a preferred embodiment of the present invention, the cell culture medium is selected from Minimum Essential Medium, Dulbecco Modified Eagle Medium, RPMI 1640, DMEM / F12 and formulations supplemented with fetal bovine serum, platelet lysates, xenogeneic-free supplements for cell expansion.

[0055] In a preferred embodiment of the present invention, the fluids are selected from plasma, serum, urine, menstrual blood, amniotic fluid or combinations thereof.

[0056] In a preferred embodiment of the present invention, the cell secretome is chemically crosslinked via genipin, glutaraldehyde, formaldehyde, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide / N-hydroxy succinimide.

[0057] In a preferred embodiment of the present invention, the cell secretome is physically crosslinked via tripolyphosphate and calcium chloride.

[0058] In a preferred embodiment of the present invention, the secretome-based biomaterial further comprises a therapeutic or biologically active agent. The therapeutic agent is selected from a biomolecule, a diagnostic marker, a probe or a combination thereof and the biologically active agent is selected from a cell, a protein or a combination thereof.

[0059] The secretome-based biomaterial has a reconfigurable shape. In preferred embodiments of the present invention, the biomaterial is selected from the group consisting of particles, membranes, capsules, bioinks, foams, hydrogels, sponges, cream, ointments, patches, adhesives, fibers, 3D printed constructs, or discs.

[0060] In a second aspect of the present invention, it is disclosed a method for the production of the secretome-based biomaterial, comprising the steps of:

[0061] a) collecting the cell secretome from the cell culture medium or other fluid;

[0062] b) purifying the cell secretome by ultrafiltration or dialysis;

[0063] c) subjecting the product obtained in step (b) to size-exclusion chromatography;

[0064] d) subjecting the product obtained in step (c) to freeze-drying, and

[0065] e) dissolving and crosslinking the product obtained in step (d) to generate the secretome-based biomaterial.

[0066] In step (a), the cell secretome is collected from a patient fluid or after culturing the patient cellsin vitro, as the supernatant of cultured cells for at least 4 hours.

[0067] In step (b), the cell secretome is ultrafiltrated or dialyzed using a membrane having a value threshold (cut-off value) equal to or less than 3 kDa.

[0068] When the secreted soluble factors and small extracellular vesicles or microvesicles are chemically modified, or reacted with other molecules, this step takes places prior to the crosslinking step.

[0069] In a third aspect of the present invention, the secretome-based biomaterial is for use in tissue regeneration, immune-mediated diseases or conditions, transplantation and post-transplantation. Other biomedical and biological applications include cell expansion, organoid or 3D culture, disease modeling, induction of differentiation and drug delivery.

[0070] The secretome-based biomaterial is tailored by changing the percentage of weight used in the formulation, crosslinking degree, or crosslinking strategies.

[0071] Tuneable mechanical properties are defined as adjustable Young's modulus, brittleness, viscoelasticity, and stress relaxation. Tuneable degradation properties refer to the ability to control the stability of the biomaterial over time, through the tailoring of crosslinking extension, or the use of on-demand reversible crosslinking chemistries.Examples

[0072] The secretome-based biomaterial of the present invention must present tuneable mechanical properties and be non-cytotoxic, biocompatible, and suitable for cell culture of different cell types.

[0073] Due to these facts, several tests were performed.

[0074] Production of thesecretome-based biomaterial

[0075] A schematic representation of the preparation of secretome samples and their crosslinking is shown inA.

[0076] The secreted fraction (growth factors, interleukins, small extracellular vesicles and microvesicles) from a serum-deprived culture media is collected from human mesenchymal stem cells isolated from the adipose tissue.

[0077] The resulting medium is ultrafiltrated or dialyzed using a membrane having a value threshold (cut-off value) equal to or less than 3,000 Daltons, thus concentrating the sample at least 10x times.

[0078] The resulting purified medium is then passed through a size-exclusion column (desalting columns containing Sephadex G-25 resin) to remove the remaining components of the cell media.

[0079] Finally, the solution from the previous step is lyophilized to obtain a sponge-like product (A) with longer storage capability.

[0080] Dynamic light scattering tests

[0081] The purified product is evaluated by dynamic light scattering for size and z-potential measurements. The sample consists of different sizes with three predominant peaks around 10, 100, and 1000 nm (A). The zeta potential is negative (-3.52 mV) correlating with small extracellular vesicles’ negative charge and an overall negative charge of the protein content (B).

[0082] Small extracellular vesicles are CD81 positive, in order to confirm the maintenance of this marker after the purification and freeze-drying, the sample was put in contact with Dynabeads containing CD81 antibody (Exosome-Human CD81 Detection Reagent) and evaluated by TEM microscopy (Figures 1 C and 1 D), confirming the presence of the marker.

[0083] Crosslinking reaction

[0084] The freeze-dried product is weighted and dissolved in Dulbecco's Phosphate Buffered Saline (DPBS) to a final concentration of 5% and 10% w / V, and then genipin (0.5% w / V in deionized water (dH2O): dimethyl sulfoxide (DMSO), 4:1 mixture): is added to the solution (). The resulting hydrogel turns blue through the crosslinking reaction (B).

[0085] Rheological kinetics

[0086] The rheological kinetics was evaluated on biomaterials comprising cell secretome at concentrations of 5% and 10% w / V (in DPBS) to determine the maximum stiffness (G') of the biomaterial, as well as the time required to reach said stiffness (Figures 4 B and 4 C for secretome obtained from naive ASCs andD for secretome obtained from IFN-gamma-modulated ASCs).

[0087] For that purpose, a time sweep test was performed at 37 ºC, 0.01% strain (fixed), 1 Hz frequency (fixed), and a 0.2 mm gap with or without genipin (crosslinker). This test also allowed the assessment of the influence of concentration on the final hydrogel stiffness (G’) (E shows a summary of the data obtained from three independent replicates from the rheology assays shown in 4 B and 4 C).

[0088] 10% w / V hydrogels showed a more linear kinetics and a higher maximum stiffness (G’) (A).

[0089] Cellular metabolic activity assay

[0090] The quantification of the cellular metabolic activity (Alamar Blue assay) was performed in endothelial mouse cells (C166-GFP) and mouse macrophages (RAW 264.7).

[0091] The cytocompatibility result showed maintained biocompatibility of more than 90% after the culture of a monolayer of C166-GFP cell (20000 cells / cm2seeding density) in contact with the biomaterial for 1 day and more than 80% after 3 days without significant differences compared to control cells (unpaired t-test, p value = 0,2206) (A). The assay was designed following the ISO 10993-5:2009.

[0092] Endothelial mouse cells (C166-GFP) are able to grow and proliferate after 7 days of contact with the biomaterials (B and 5 C). Mouse macrophages (RAW 264.7) are able to attach, grow, and proliferate after 48 h of seeding (20.000 cells / cm2) into the biomaterials (D).

[0093] As shown in, the secretome-derived biomaterials produced by naive (control) or IFN-gamma-induced ASCs lead to the organization of human umbilical vein endothelial cells, indicating theirin vitropro-angiogenic properties. This is consistent with their potential biocompatibility and effectiveness in clinical applications.

[0094] The subject matter described above is provided as an illustration of the present invention and, therefore, should not be construed to limit it. The terminology employed to describe preferred embodiments of the present invention should not be restricted to them.

[0095] As used in the description, definite and indefinite articles, in their singular form, are intended for interpretation to also include plural forms, unless the context of the description explicitly indicates otherwise.

[0096] The term “one”, as well as the articles “a” and “an”, should generally be interpreted as “one or more” unless specified otherwise or clear from the context to be directed to a singular form.

[0097] It will be understood that the terms “comprise” and “include” when used in the present description, specify the presence of features, elements, components, steps, and related operations, but do not exclude the possibility of other features, elements, components, steps, and operations as well contemplated.

[0098] As used throughout the present patent application, the term “or” is used in an inclusive sense rather than an exclusive sense, unless the exclusive meaning is clearly defined in a specific situation. In this context, a phrase of the type “X uses A or B” should be interpreted as including all relevant inclusive combinations, for example, “X uses A”, “X uses B” and “X uses A and B”.

[0099] All modifications, provided they do not change the essential features of the following claims, must be considered within the scope of the protection of the present invention.

[0100] The citation list is as follows:

[0101] WO2018213795A1

[0102] US20220218873A1

[0103] US2022088274A1

[0104] WO2022159878A9Non-Patent Literature

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[0106] L. Daneshmandi, S. Shah, T. Jafari, M. Bhattacharjee, D. Momah, N. Saveh-Shemshaki, K.W. Lo, C.T. Laurencin, Emergence of the Stem Cell Secretome in Regenerative Engineering, Trends in biotechnology, 38 (2020) 1373-1384.

[0107] S. Bruno, M.C. Deregibus, G. Camussi, The secretome of mesenchymal stromal cells: Role of extracellular vesicles in immunomodulation, Immunology Letters, 168 (2015) 154-158.

[0108] V.B.R. Konala, M.K. Mamidi, R. Bhonde, A.K. Das, R. Pochampally, R. Pal, The current landscape of the mesenchymal stromal cell secretome: A new paradigm for cell-free regeneration, Cytotherapy, 18 (2016) 13-24.

[0109] B.M. Bakadia, A.A. Qaed Ahmed, L. Lamboni, Z. Shi, B. Mutu Mukole, R. Zheng, M. Pierre Mbang, B. Zhang, M. Gauthier, G. Yang, Engineering homologous platelet-rich plasma, platelet-rich plasma-derived exosomes, and mesenchymal stem cell-derived exosomes-based dual-crosslinked hydrogels as bioactive diabetic wound dressings, Bioactive Materials, 28 (2023) 74-94.

[0110] K. Rui, X. Tang, Z. Shen, C. Jiang, Q. Zhu, S. Liu, N. Che, J. Tian, J. Ling, Y. Yang, Exosome inspired photo-triggered gelation hydrogel composite on modulating immune pathogenesis for treating rheumatoid arthritis, Journal of Nanobiotechnology, 21 (2023) 111.

[0111] V.A. Kudinov, R.I. Artyushev, I.M. Zurina, R.D. Lapshin, L.B. Snopova, I.V. Mukhina, O.S. Grinakovskaya, I.N. Saburina, Antimicrobial and Regenerative Effects of Placental Multipotent Mesenchymal Stromal Cell Secretome-Based Chitosan Gel on Infected Burns in Rats, Pharmaceuticals, 2021.

[0112] M.I. León-Campos, N. Rodríguez-Fuentes, J.A. Claudio-Rizo, D.A. Cabrera-Munguía, J.J. Becerra-Rodríguez, A. Herrera-Guerrero, F. Soriano-Corral, L.E. Alcántara-Quintana, Development and in vitro evaluation of a polymeric matrix of jellyfish collagen-human stem cell secretome-polyurethane for wound healing, Journal of Materials Science, 58 (2023) 8047-8060.

[0113] D. Silva, L. Schirmer, T.S. Pinho, P. Atallah, J.R. Cibrão, R. Lima, J. Afonso, S. B-Antunes, C.R. Marques, J. Dourado, U. Freudenberg, R.A. Sousa, C. Werner, A.J. Salgado, Sustained Release of Human Adipose Tissue Stem Cell Secretome from Star-Shaped Poly(ethylene glycol) Glycosaminoglycan Hydrogels Promotes Motor Improvements after Complete Transection in Spinal Cord Injury Rat Model, 12 (2023) 2202803.

[0114] T. Später, M. Assunção, K.K. Lit, G. Gong, X. Wang, Y.-Y. Chen, Y. Rao, Y. Li, C.H.K. Yiu, M.W. Laschke, M.D. Menger, D. Wang, R.S. Tuan, K.-H. Khoo, M. Raghunath, J. Guo, A. Blocki, Engineering microparticles based on solidified stem cell secretome with an augmented pro-angiogenic factor portfolio for therapeutic angiogenesis, Bioactive Materials, 17 (2022) 526-541.

Claims

Secretome-based biomaterial characterized by the fact that:the secretome is cell secretome selected from secreted soluble factors, apoptotic bodies, small extracellular vesicles or microvesicles that are released from cells to the cell culture medium or other fluids or combinations thereof;wherein the cell secretome is crosslinked; andwherein the cell secretome is present in a concentration that ranges from 1-20 % w / V.Secretome-based biomaterial according to claim 1 wherein the secreted soluble factors are selected from growth factors, interleukins, cytokines, structural proteins and proteins from secreted vesicles.Secretome-based biomaterial according to claim 1 or 2 wherein the cells are cells of animal origin selected from stem cells, immune cells, or other cells of therapeutic interest that can be in their native state, immortalized cell lines, cells primed with physical, chemical or biological stimuli, gene edited cells or a combination thereof.Secretome-based biomaterial according to any of claims 1-3 wherein the stem cells are selected from induced pluripotent cells and mesenchymal stromal cells and the immune cells are selected from T lymphocytes, dendritic cells, macrophages and NK cells.Secretome-based biomaterial according to claim 3 wherein the physical stimuli are selected from hypoxia, low-level lasers, mechanical stretch, silica, pulsed electromagnetic fields, 3D culture or combinations thereof.Secretome-based biomaterial according to claim 3 wherein the chemical stimuli are selected from metabolic acidosis, BAY 11-708, LL-37, dimethyloxalylglycine, JI-34, sevoflurane, atorvastatin, oxytocin, cyclophosphamide or combinations thereof.Secretome-based biomaterial according to claim 3 wherein the biological stimuli are selected from migration inhibitory factor, IFN-α, TGF-β1, interleukin (IL)-1α / β, IL-25, IL-6, TNF-α, IL-17, IFN-γ, stromal-derived factor 1, gene modification, polyinosinic-polycytidylic acid, lipopolysaccharide or combinations thereof.Secretome-based biomaterial according to any of claims 1-7 wherein the cell culture medium is selected from Minimum Essential Medium, Dulbecco Modified Eagle Medium, RPMI 1640, DMEM / F12 and formulations supplemented with fetal bovine serum, platelet lysates, xenogeneic-free supplements for cell expansion.Secretome-based biomaterial according to any of claims 1-8 wherein the fluids are selected from plasma, serum, urine, menstrual blood, amniotic fluid or combinations thereof.Secretome-based biomaterial according to any of claims 1-9 wherein the cell secretome is crosslinked via chemical crosslinking, physical crosslinking, guest-host interactions, enzymatic crosslinking via transglutaminase, photopolymerized or combinations thereof.Secretome-based biomaterial according to claim 10 wherein the chemical crosslinking is selected from genipin, glutaraldehyde, formaldehyde, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide / N-hydroxy succinimide.Secretome-based biomaterial according to claim 10 wherein the physical crosslinking is selected from tripolyphosphate and calcium chloride.Secretome-based biomaterial according to any of claims 1-12 wherein the secreted soluble factors and small extracellular vesicles or microvesicles are chemically modified or reacted with other molecules to enable intermolecular click chemistry.Secretome-based biomaterial according to any of claims 1-13 wherein it further comprises a therapeutic or biologically active agent, wherein the therapeutic agent is selected from a biomolecule, a diagnostic marker, a probe or a combination thereof and the biologically active agent is selected from a cell, a protein or a combination thereof.Secretome-based biomaterial according to any of claims 1-14 wherein it is selected from the group consisting of particles, membranes, capsules, bioinks, foams, hydrogels, sponges, cream, ointments, patches, adhesives, fibers, 3D printed constructs, or discs.Method for the production of the secretome-based biomaterial as disclosed in any of claims 1-15, characterized by comprising the steps of:a) collecting the cell secretome from the cell culture medium or other fluid;b) purifying the cell secretome by ultrafiltration or dialysis; andc) subjecting the product obtained in step (b) to size-exclusion chromatography;d) subjecting the product obtained in step (c) to freeze-drying, ande) dissolving and crosslinking the product obtained in step (d) to generate the secretome-based biomaterial.Method, according to claim 16, wherein, in step (a), the cell secretome is collected from a patient fluid or after culturing the patient cellsin vitro, as the supernatant of cultured cells for at least 4 hours.Method, according to claims 16 or 17, wherein, in step (b), the cell secretome is ultrafiltrated or dialyzed using a membrane having a value threshold (cut-off value) equal to or less than 3 kDa.Method, according to any of claims 16-18, when the secreted soluble factors and small extracellular vesicles or microvesicles are chemically modified or reacted with other molecules, this step takes places prior to the crosslinking step.Secretome-based biomaterial, as defined in any of claims 1-15, for use in tissue regeneration, immune-mediated diseases or conditions, transplantation and post-transplantation.Secretome-based biomaterial, as defined in any of claims 1-15, for use in the promotion of cell expansion, organoid or 3D culture, disease modeling, induction of differentiation and drug delivery.

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