Cell-free environment method for producing functional and physiologically relevant extracellular matrix from cellular supernatants

Abstract

The present invention relates to a method for producing an extracellular matrix (ECM) preparation, comprising the steps of providing at least one suitable cellular supernatant from a cell culture, mixing the supernatant with a suitable macromolecular crowding agent, and suitably incubating the mix to form ECM. The method according to the present invention does not involve a decellularization step of the ECM as formed. The invention further relates to a preparation comprising the ECM as produced according to the present invention and uses of the preparation, for example for tissue repair and regeneration, or for use in medicine and the treatment of diseases.

Description

[0001] Cell-free environment method for producing functional and physiologically relevant extracellular matrix from cellular supernatants

[0002] The present invention relates to a method for producing an extracellular matrix (ECM) preparation, comprising the steps of providing at least one suitable cellular supernatant from a cell culture, mixing the supernatant with a suitable macromolecular crowding agent, and suitably incubating the mix to form ECM. The method according to the present invention does not involve a decellularization step of the ECM as formed. The invention further relates to a preparation comprising the ECM as produced according to the present invention and uses of the preparation, for example for tissue repair and regeneration, or for use in medicine and the treatment of diseases.

[0003] Background of the invention

[0004] The extracellular matrix (ECM) is a complex, dynamic network of macromolecules that provides essential structural, mechanical, and biochemical support to cells and tissues. Comprising a variety of proteins and glycosaminoglycans, the ECM not only ensures structural integrity but also plays a pivotal role in transmitting biochemical signals that regulate cell behavior, differentiation, and tissue morphogenesis (1, 2). These multifunctional properties of the ECM have made it a cornerstone in tissue engineering and regenerative medicine, where ECM-derived biomaterials are widely used to promote cell adhesion, enhance cellular functions, and support tissue repair (3).

[0005] The synthesis of ECM-based biomaterials currently involves several established methods, each with its own advantages and limitations. Traditional approaches include tissue decellularization, hybrid biomaterials, and biomimetic biomaterials. Biomimetic biomaterials, while inspired by ECM structure, often fail to replicate the bioactive complexity of native ECM, limiting their effectiveness (4, 5).

[0006] Hybrid biomaterials attempt to address this gap by incorporating functional proteins or peptides, often derived from decellularized tissues or cell-based ECM, but their development requires intricate modifications to ensure compatibility with cell adhesion and growth (5). The widelyused ECM surrogate, Matrigel, derived from mouse sarcoma, has proven useful in research but is limited in clinical applications due to its origin and potential immunogenicity (5-7).

[0007] Tissue-derived ECM produced via organ decellularization is another key approach but faces significant challenges in scalability and clinical use. The scarcity of human donor tissues, the risks associated with non-human tissue sources — including incomplete decellularization, immunological rejection, and potential disease transmission — pose major obstacles (6, 7). Residual cellular nucleic acids from decellularization processes further exacerbate immunogenicity and cytotoxicity risks (7). Additionally, ECM derived from specific tissues can be difficult to source and lacks the flexibility required for diverse therapeutic applications (5).

[0008] Cell -derived ECM [CD-ECM or CDM (4, 5)] has emerged as an alternative, offering improved standardization and the ability to pre-screen ECM-producing cells (6). However, the decellularization process itself poses significant drawbacks, often compromising the structural and functional integrity of ECM through the use of detergents and chemical agents. This introduces artifacts, variability in essential soluble growth factors, and batch inconsistencies, which limit CD-ECM’ s utility. Large-scale production is further hindered by the significant surface area required for adherent cell cultures.

[0009] The ECM is the acellular mesh of proteins that provides the mechanical scaffold where multiple cell types in a tissue can home and give rise to the shape and size of the respective organ. Using bioinformatic and experimental knowledge, the mammalian ECM has been proposed to have around 274 proteins, with collagens, fibronectin and elastin being the top contributors as far the composition is concerned (Frantz C, Stewart KM, Weaver VM. The extracellular matrix at a glance. J Cell Sci. 2010 Dec 15;123(Pt 24):4195-200. doi: 10.1242 / jcs.023820. PMID: 21123617; PMCID: PMC2995612). ECM components have also been shown to be metabolised by multiple enzymes to generate small peptides (known as matrikines) with biochemical activity.

[0010] For its role in mechanical scaffolding, ECM is used as a biomaterial that enables cell adhesion, in in-vitro systems with applications in recreating cellular niches in-vitro, formation of scaffolds for 3D cell culture systems, in tissue regeneration for supporting cell patches, skin patches. Currently, the state of the art of preparing an ECM scaffold is to decellularize human tissues,decellularize mouse sarcomas (matrigel) or decellularize monolayers of cells cultured that have been subjected to long term cultures or macromolecular crowding agents. The major disadvantage of obtaining ECM from human tissues is the availability of cadaveric or diseased tissue (Assungao M, et al. Cell-Derived Extracellular Matrix for Tissue Engineering and Regenerative Medicine. Front Bioeng Biotechnol. 2020 Dec 3;8:602009. doi: 10.3389 / fbioe.2020.602009. PMID: 33344434; PMCID: PMC7744374), this has promoted the use of animal tissue-derived ECM as an alternative. However, all of the above methods carry a disadvantage of incomplete decellularization, giving rise to variable composition from batch to batch, carry over of nucleic acid contamination (DNA, RNA) and detergents. Also, such systems have no control or a prospect of synthesizing a customised ECM of defined composition and bioactivity.

[0011] Currently, ECM used for coating cell culture dishes, functionalizing hydrogels for 3D cell culture, and in organoid formation is derived from decellularizing mesenchymal cell cultures, complex tissues and mouse sarcomas (Matrigel). Major difficulties for the above-mentioned ECM are variable protein composition, interference from remains of ECM-producing cells (DNA, RNA fragments), and detergents from the methods used to generate ECM via decellularization.

[0012] Xu Y, et al. (in: Cell-Derived Matrix: Production, Decellularization, and Application of Wound Repair. Stem Cells Int. 2024 May 29;2024:7398473. doi: 10.1155 / 2024 / 7398473. PMID: 38882595; PMCID: PMC11178417) disclose that over the past few decades, tissue engineering materials have emerged as a viable option for wound healing, with cell-derived extracellular matrix (CDM) showing remarkable results. The CDM's compatibility and resemblance to the natural tissue microenvironment confer distinct advantages to tissue-engineered scaffolds in wound repair. Their review summarizes the current processes for CDM preparation, various cell decellularization protocols, and common characterization methods. Furthermore, it discusses the applications of CDM in wound healing, including skin defect and wound repair, angiogenesis, and engineered vessels, and offers perspectives on future developments.

[0013] Satyam A, et al. (in: Macromolecular crowding meets tissue engineering by self-assembly: a paradigm shift in regenerative medicine. Adv Mater. 2014 May 21;26(19):3024-34. doi: 10.1002 / adma.201304428) disclose that MMC, the addition of inert polydispersed macromolecules in the culture media, effectively emulates the dense in vivo extracellular space,resulting in amplified deposition of ECM in vitro and subsequent production of cohesive, ECM-rich living substitutes.

[0014] Kumar P, et al. (in: Macromolecularly crowded in vitro microenvironments accelerate the production of extracellular matrix-rich supramol ecul ar assemblies. Sci Rep. 2015 Mar 4;5:8729. doi: 10.1038 / srep08729. PMID: 25736020; PMCID: PMC4348624) assessed the influence of macromolecular crowding, a biophysical phenomenon that regulates intra- and extra-cellular activities in multicellular organisms, in human corneal fibroblast culture. In the presence of macromolecules, abundant extracellular matrix deposition was evidenced as fast as 48 hrs in culture, even at low serum concentration. Temperature responsive copolymers allowed the detachment of dense and cohesive supramolecularly assembled living substitutes within 6 days in culture. Morphological, histological, gene and protein analysis assays demonstrated maintenance of tissue-specific function. Macromolecular crowding opens new avenues for a more rational design in engineering of clinically relevant tissue modules in vitro.

[0015] WO 2015 / 143310A1 discloses methods for preparing sterilized, gelled, solubilized extracellular matrix (ECM) compositions useful as cell growth substrates. Also provided are compositions prepared according to the methods as well as uses for the compositions. In one embodiment a device, such as a prosthesis, is provided which comprises an inorganic matrix into which the gelled, solubilized ECM is dispersed to facilitate in-growth of cells into the ECM and thus adaptation and / or attachment of the device to a patient. The method of preparing an extracellular matrix-derived gel comprises (i) solubilizing extracellular matrix (ECM) that has not been dialyzed, by digestion with an acid protease in an acidic solution to produce a digest solution, (ii) drying the digest solution, and (iii) sterilizing the dried digest.

[0016] WO 2020 / 228733 Al relates to methods for producing ECM from mesenchymal stem cells (MSCs). It is shown that MMC drove deposition of all ECM components. The matrices were decellularized using sodium deoxycholate in combination with DNase.

[0017] Assungao M, et al. (in: Cell-Derived Extracellular Matrix for Tissue Engineering and Regenerative Medicine. Front Bioeng Biotechnol. 2020 Dec 3;8:602009. doi: 10.3389 / fbioe.2020.602009. PMID: 33344434) reviews methods to generate cell-derived ECM. It is discussed that MMC enhances ECM formation (p. 2, rc, paragraph 2; Fig. IB). Thedecellularization methods that aim at maintaining architecture and bioactivity of the formed ECM are also described. It is taught that 2D monolayer cultures with MMC results in dense ECM that is easy to decellularize.

[0018] Kumar P, et al. (in: Macromolecularly crowded in vitro microenvironments accelerate the production of extracellular matrix-rich supramol ecul ar assemblies. Sci Rep. 2015 Mar 4;5:8729. doi: 10.1038 / srep08729. PMID: 25736020) teaches that Ficoll acting as an MMC agent increases and accelerates ECM deposition in human corneal fibroblast cultures.

[0019] Benny P, and Raghunath M. (in: Making microenvironments: A look into incorporating macromolecular crowding into in vitro experiments, to generate biomimetic microenvironments which are capable of directing cell function for tissue engineering applications. J Tissue Eng. 2017 Oct 6;8:2041731417730467. doi: 10.1177 / 2041731417730467. PMID: 29051808) disclose that MMC enhances ECM deposition in vitro.

[0020] None of the above documents disclose methods that use MMC to generate ECM from cellular supernatant or conditioned medium. Furthermore, none of the prior art documents would suggest or hint that ECM could be generated from cellular supernatant by use of MMC.

[0021] To address the limitations of the state of the art, an ideal solution would involve the creation of ECM scaffolds or bioinks without reliance on decellularization. A promising approach lies in synthesizing ECM from cell supernatants, using cells as a continuous source of ECM precursor materials such as the conditioned medium (CM) of cells cultured in vitro. In this method, ECM components are de novo synthesized from supernatants in a cell-free environment, eliminating the need for detergents and chemical treatments while improving batch-to-batch consistency. Despite its potential, the de novo synthesis of ECM from non-crosslinked raw components present in conditioned medium in a cell-free environment has not been attempted prior to this invention.

[0022] It is therefore an object of the present invention to provide a method for the production of functional ECM in a decellularization-independent manner from cell supernatants. The ECM can be used in biotechnology, medicine, environmental science, and chemical synthesis. Otherobjects and advantages will readily become apparent for the person of skill from studying the following more detailed description and examples.

[0023] In a first aspect thereof, the present invention solves the above object by providing a method, in particular an in vitro method, for producing an extracellular matrix (ECM) preparation, comprising the steps of: a) Providing at least one culture of suitable cells in a serum-free medium, b) Suitably harvesting the cellular supernatant of the at least one culture, and further removing cellular debris, for example using sterile filtration, c) Optionally, concentrating the supernatant of step b) in order to increase the concentration of milieu, for example using freeze-drying, d) Mixing the, optionally concentrated, supernatant with a suitable medium comprising a macromolecular crowding agent (MMC), and e) Suitably incubating the mix of step d) to form ECM.

[0024] Preferred is the method according to the present invention that does not involve or require a decellularization step, in particular a decellularization step of the ECM as formed.

[0025] In the context of the present invention, the inventors as a preferred embodiment established a synthesis of extracellular matrix (ECM) from fibroblast-conditioned medium. The protein assembly of ECM is de novo, and in a cell-free environment. The resulting ECM is devoid of detergents, and nucleic acid contaminants. The inventive technique implements a combined effect of exclusion-volume effect of macromolecular crowding and freeze-drying of cell-conditioned media supernatants. The system is called decellularization-free synthesized Extracellular Matrix (herein dcelfreeCEM, dCEM or CEM-ECM). dCEM has a wide variety of applications, including the functionalization of surfaces of non-biological materials (cell culture dishes, imaging plates, prosthetics) use as bio-ink and functionalization of hydrogel materials (used in tissue regeneration studies).

[0026] The inventors surprisingly found that a functional ECM can be produced only using the cell culture medium or supernatant from in-vitro culture fibroblasts, mesenchymal stem cells or other cells. The concept was implemented that ECM is composed of soluble ECM components (enzymes for crosslinking and main ECM components), that are polymerized by molecular crowding agents in order to form an acellular mesh of proteins. Such a mesh is devoid of any detergent treatment, does not contain DNA or RNA fragments, and also allows for synthesizing of a customized “modified” ECM of defined composition and bioactivity.In summary, the approach according to the present invention uses conditioned media collected from diverse cell sources, including fibroblasts and mesenchymal stem cells, combined with macromolecular crowding agents to facilitate ECM polymerization in vitro. CEM-ECM synthesis avoids the need for harsh detergents, ensures consistent batch quality and further enables scalable production. The inventor’s results demonstrate that CEM-ECM retains essential bioactivity, as shown by its capacity to influence cellular migration and interaction, validated through scanning electron microscopy and live-cell imaging (see below). Proteomic analyses confirmed the presence of core matrisome proteins and batch-to-batch consistency. High-speed atomic force microscopy further elucidated the physiological complexity of the precursor proteins in cell supernatants.

[0027] Advantageously, the CEM-ECM does not contain cell debris, no nucleic acid residual contaminants, and no detergents are used in the preparation thereof. When compared with existing production methods, due to a combination of freeze-drying of cell supernatants and macromolecular crowding, the time needed to synthesized ECM is shorter compared to conventional ECM manufacturing protocol currently used. The cells used as source for the supernatants used for CEM-ECM production can be manipulated genetically (± protein of interest), for example using siRNAs or CRISPR systems, in order to produce an ECM of choice. The CEM-ECM does not involve laborious decellularization protocols, such as using pumps and pipes for weeks. Finally, the CEM-ECM of the invention has a defined and consistent composition.

[0028] Another aspect of the present invention then relates to an in vitro method for producing an extracellular matrix (ECM) preparation according to the present invention, comprising the steps of: a) Providing at least one culture of suitable cells in an FCS-free medium, b) Suitably harvesting the cellular supernatant of the at least one culture in step a) 3 days after switching to the FCS free cell culture media and sterile-filtering using a 0.45pm syringe driven filter unit to remove cellular debris, c) Concentrating the supernatant of step b) by freeze-drying using a MiVac instrument at about 76 °C for 2 hrs, d) Mixing the concentrated supernatant with DMEM containing 6 x macromolecular crowder (MMC) Ficoll 70 / 400, and e) Suitably incubating the mix of step d) in a standard cell culture incubator at 37 °C with 5% CO2 for about 6 days to form ECM with 2 times medium replenishment.Another aspect of the present invention then relates to a preparation comprising the ECM as produced according to the present invention, together with an acceptable carrier or diluent. In some embodiments, the preparation comprises an aqueous formulation. Particularly preferred is a pharmaceutical preparation comprising the ECM as produced according to the present invention, together with a pharmaceutically acceptable carrier or diluent. In some embodiments, the pharmaceutical preparation is lyophilized. In some embodiment, the pharmaceutical preparation further comprises one or more additional therapeutic agents, e.g., second, third or fourth therapeutic agents.

[0029] In a further aspect, provided is a preparation according to the present invention, which is a plant-based ECM, or a mixture of an animal- and plant based ECM.

[0030] Another aspect of the present invention then relates to the use of the preparation according to the present invention for tissue repair and regeneration, for organoid formation, as bioink in tissue engineering, for a functionalization of surfaces of non-biological materials, as hybrid matrix with tunable properties when combined with synthetic polymers, as a cosmetic preparation, and delivery matrix in cell-based therapies. Preferably, the ECM generated can be used as a base and ingredient for generating cosmetic skin ointments, gels or creams. Other uses may be found in the respective literature, such as, for example, in Helena Vilaga-Faria, et al. (Extracellular matrix-derived materials for tissue engineering and regenerative medicine: A journey from isolation to characterization and application, Bioactive Materials, Volume 34, 2024, Pages 494-519, ISSN 2452-199X, https: / / doi.Org / 10.1016 / j.bioactmat.2024.01.004, incorporated by reference).

[0031] Another aspect of the present invention relates to the medical use of the pharmaceutical preparation according to the present invention in medicine and the treatment of diseases, in particular for use in tissue repair and regeneration, such as, for example, for cartilage, bone, and skin repair. Another aspect of the present invention then relates to a method for treating diseases related to an involving tissue repair and regeneration, such as for example, for cartilage, bone, and skin repair, comprising administering the pharmaceutical preparation according to the present invention to a patient or subject in need for such a treatment.

[0032] As mentioned, the problem of the present invention is solved by providing a method, such as an in vitro-method for producing an extracellular matrix (CEM-ECM) preparation.In a first step of the method according to the present invention, at least one culture of suitable cells in a serum-free medium is provided. Preferred is the method according to the present invention, wherein plant cells are cultured in this step, and further comprising the step of removing the cell wall of the plant cells, for example using enzymatic or chemical digestion.

[0033] In a preferred embodiment, the method according to the present invention further comprises a pre-culture of the cells before this step in a medium that is suitable for a sufficient growth of the cells. This medium may be a “rich” or “full supplemented” medium (see below) and may include serum or parts thereof, such as FCS or FBS.

[0034] The pre-culture is followed by culture in cultivated in a “starvation medium”, for example of DMEM in order to generate the at least one culture of suitable cells in a serum -free medium or step a). A preferred starvation medium of DMEM (Pyruvate, Ig / L D-Glucose) contains 0.1 ml NormoCure 100 mg. The cells were optionally treated with 5 ng / ml TGF-pi or 250 mM 2-0-a-Glucopyranosyl-L-ascorbic-acid (GAA).

[0035] There are many cell types that may be used for the production of various types of extracellular matrix, as the local components of ECM determine the properties of the connective tissue. In a preferred embodiment of the method according to the present invention the cells and tissues that are suitable for the production are selected from animal cells, such as vertebrates or invertebrates, in particular mammalian cells, individual cell types or mixtures of different cell types, cell lines, mesenchymal stem / stromal cells (MSCs) , primary cells, stem cells, mesenchymal stem cells, epithelial, endothelial, mesenchymal or immune cells, muscle cells, fibroblasts, such as skin fibroblasts, human embryonic fibroblasts, human bone marrow stromal cells, organoids, tissue samples, plant cells or mixtures thereof. Fibroblasts are the most common cell type in connective tissue ECM, in which they synthesize, maintain, and provide a structural framework; fibroblasts secrete the precursor components of the ECM. Preferred cells are selected from fibroblasts, adenocarcinomic human alveolar epithelial cell lines, hTERT-immortalized human skin-derived cells, such as BJ5Ta, HS-5 and the primary cells WI-38.

[0036] In a preferred embodiment, the method according to the present invention further comprises the use of suitable cells that are genetically modified or engineered, in order to provide a modified supernatant, i.e. includes the genetic manipulation of source cells / suitable cells to producemodified conditioned media. Preferably, the cells used as source for the supernatants used for the CEM-ECM production are manipulated genetically (± protein of interest), for example using siRNAs or CRISPR systems, in order to produce an ECM of choice.

[0037] In a preferred embodiment, the method according to the present invention further comprises the modification of the ECM as produced directly, i.e. by at least one of the introducing of natural and / or synthetic peptides, the addition of chemical cross-linkers to enhance specific ECM properties, such as, for example stiffness and / or porosity, the incorporation of bioactive molecules, such as growth factors, cytokines, and / or small-molecule drugs into the ECM, the addition of recombinant proteins to the ECM during step d) or e), or the step of removing or depleting undesired proteins from the ECM during or after step d) or e). This achieves a “customization” of the CEM-ECM composition through adding recombinant ECM proteins to the concentrated milieu before (i.e. present during) matrix assembly. This includes spiking of single components or defined mixtures in order to tune composition and functional properties.

[0038] The carrier base for the CEM-ECM may also include hydrogels such as polyethylene glycol (PEG), alginate, plutonic acid etc. Therefore, an aspect of the invention relates to the CEM-ECM comprising Matrigel or PEG hydrogel functionalized with Matrigel. In an experiment, larger MDCK cysts were formed in a CEM-ECM+Matrigel+PEG mixture, compared to Matrigel or CEM-ECM alone, suggesting CEM-ECM as a supplement for better performance of Matrigel (Figure 7).

[0039] In another preferred embodiment, in the method according to the present invention the cells as cultured in step a) are immobilized, for example attached or seeded to a surface, such as the culture vessel, and / or a carrier matrix, such as microcarrier beads. This allows a permanent production method, for example comprising medium exchange, and / or an upscaling of the method in order to produce larger amounts of ECM. Suitable methods and materials to immobilize are known to the person of skill, and can be found in the state of the art.

[0040] In a second step of the method according to the present invention, the cellular supernatant of the at least one culture of a) is suitably harvested. Harvesting can be performed with any suitable method to obtain a cellular supernatant free of, or substantially free of cells, such as filtration or centrifugation. Subsequently, the supernatant is decanted or collected using a pipette or the like, without disturbing the cell pellet at the bottom of the culture vessel. The conditionedmedium from cells that are immobilized or adhered to cell culture dishes or other surfaces (e.g. microcarriers, see also above) may be collected using routine pipettes, leaving the adherent cells behind (on the cell culture dish or other surfaces). The adherent cell culture left behind is preferably provided with cell culture medium for further conditioning. Conditioned medium from suspension cell cultures can also be collected in this way. Other techniques, such as cell strainers or centrifugation or any other routine cell and conditioned media separation methods may be used as well.

[0041] In a preferred embodiment, in the method according to the present invention the supernatants from the cells in step b) are harvested at a time that is suitable to generate enough extracellular material to produce ECM, preferably at at least about 3 days, preferably at at least about 5 days, and more preferably at at least about 6 days after the start of culturing in the serum-free medium.

[0042] Furthermore, the step includes the removal of cellular debris, for example using sterile filtration or additional centrifugation, such as sterile-filtration using a 0.45pm syringe driven filter unit.

[0043] In a third step of the method according to the present invention, the cellular supernatant of step b) can be optionally concentrated in order to increase the concentration of milieu, for example using freeze-drying. Preferably, the concentrating step comprises size-exclusion centrifugation, for example using centrifugation through a membrane or filter, and / or freeze-drying in a suitable device, for example at 76 °C for about 2 hrs. Additional or alternative methods in order to concentrate the conditioned milieu can be selected from size based enrichment via filtration and targeting ECM proteins predominantly above 10-30 kDa, (for example MW-CO based concentration) or dialysis, and solvent removal approaches, such as vacuum assisted evaporation (vacuum centrifuge concentrator), again, optionally with freeze drying.

[0044] In a fourth step of the method according to the present invention, the optionally concentrated supernatant is mixed with a suitable medium comprising a macromolecular crowding agent (MMC).

[0045] In the context of the present invention the terms “macromolecular crowder” and “macromolecular crowding agent” are used interchangeably to designate a model “crowding agent” that mimics macromolecular crowding in vivo, such as poly(ethylene glycol), dextran,Ficoll, or inert proteins. Preferably, the macromolecular crowding agent (MMC) is selected from the group consisting of Ficoll 70, Ficoll 400, carrageenan, polyvinylpyrrolidone, dextran sulfate, polyethylene glycol (PEG), hyaluronic acid, Bovine pancreatic trypsin inhibitor (BPTI), ribonuclease A, lysozyme, P-Lactoglobulin, hemoglobin, Bovine serum albumin (BSA), Dextran 670, Poly(sodium 4-styrene sulfonate) (PSS), and mixtures thereof. See also Nilimesh Das, et al. (in: Macromolecular crowding effects on protein dynamics, International Journal of Biological Macromolecules, Volume 281, Part 3, 2024, 136248, ISSN 0141-8130, https: / / doi.Org / 10.1016 / j.ijbiomac.2024.136248, herewith incorporated by reference).

[0046] In a last step of the method according to the present invention, the mix of step d) is suitably incubated to form ECM. Incubating in is performed at standard temperature, e.g. in a standard cell culture incubator at 37 °C with 5% CO2 for a time suitable to form ECM; such as, for example, for about 4 to 10 days, preferably for about 4 to 6 days.

[0047] In one aspect, forming the ECM comprises polymerizing. Polymerization can be achieved using PBS or other solutions in addition to DMEM after collection. Polymerization can also be achieved at other temperatures or CO2 levels or humidity levels as above. Polymerization may also be performed outside of a cell culture incubator.

[0048] The ECM as produced may be used directly or used to produce preparations as described herein.

[0049] Preferred is the method according to the present invention without a decellularization step of the ECM as formed or the pre-formed ECM.

[0050] Preferred is the method according to the present invention, wherein plant cells are cultured in step a), and further comprising the step of removing the cell wall of the plant cells.

[0051] A particularly preferred embodiment of the method according to the present invention relates to an in vitro method for producing an extracellular matrix (ECM) preparation, comprising the steps of: a) Providing at least one culture of suitable cells in an FBS-free medium, b) Suitably harvesting the cellular supernatant of the at least one culture in step a) 3 days after switching to the FCS free cell culture media and sterile-filtering using a 0.45pm syringe driven filter unit to remove cellular debris, c) Concentrating the supernatant of step b) by freeze-drying using a MiVac instrument at about 76 °C for 2 hrs, d) Mixing the concentrated supernatant with DMEMcontaining 6 x macromolecular crowder (MMC) Ficoll 70 / 400, and e) Suitably incubating the mix of step d) in a standard cell culture incubator at 37 °C with 5% CO2 for about 6 days to form ECM with 2 times medium replenishment.

[0052] This aspect may also comprise the “customization” or spiking of the CEM-ECM composition through adding recombinant ECM proteins to the concentrated milieu before (i.e. present during) matrix assembly (see above).

[0053] Another aspect of the present invention relates to a preparation comprising the ECM as produced according to a method according to the present invention together with an acceptable carrier or diluent, in particular a pharmaceutical preparation together with a pharmaceutically acceptable carrier or diluent.

[0054] The term “pharmaceutically acceptable diluent or carrier” refers to a solid or liquid filler, diluent or encapsulating substance which does not interfere with the effectiveness or the biological activity of the active ingredients and which is not substantially toxic to the host, which may be either humans or animals, to which it is administered. Depending upon the particular route of administration, a variety of pharmaceutically acceptable carriers such as those well known in the art may be used. Non-limiting examples include sugars, starches, cellulose and its derivatives, malt, gelatin, talc, calcium sulfate, vegetable oils, synthetic oils, polyols, alginic acid, phosphate buffered solutions, emulsifiers, isotonic saline, and pyrogen-free water. Pharmaceutically acceptable carriers or excipients also include diluents (fillers, bulking agents, e.g. lactose, microcrystalline cellulose), disintegrants (e.g. sodium starch glycolate, croscarmellose sodium), binders (e.g. PVP, HPMC), lubricants (e.g. magnesium stearate), glidants (e.g. colloidal SiCh), sol vents / co- solvents (e.g. aqueous vehicle, Propylene glycol, glycerol), buffering agents (e.g. citrate, gluconates, lactates), preservatives (e.g. Na benzoate, parabens (Me, Pr and Bu), BKC), anti -oxidants (e.g. BHT, BHA, Ascorbic acid), wetting agents (e.g. polysorbates, sorbitan esters), thickening agents (e.g. methylcellulose or hydroxyethylcellulose), sweetening agents (e.g. sorbitol, saccharin, aspartame, acesulfame), flavoring agents (e.g. peppermint, lemon oils, butterscotch, etc.), humectants (e.g. propylene, glycol, glycerol, sorbitol). Other suitable pharmaceutically acceptable excipients are inter alia described in Remington's Pharmaceutical Sciences, 15thEd., Mack Publishing Co., New Jersey (1991) and Bauer et al., Pharmazeutische Technologic, 5thEd., Govi-Verlag Frankfurt (1997). The person skilled in the art knows suitable formulations for the compounds according to thepresent invention and will readily be able to choose suitable pharmaceutically acceptable carriers or diluents, depending, e.g., on the formulation and administration route of the pharmaceutical composition.

[0055] All suitable modes of administration are contemplated according to the invention. Administration of the composition as a medicament may be via oral, subcutaneous, direct intravenous, slow intravenous infusion, continuous intravenous infusion, intravenous or epidural patient controlled analgesia (PCA and PCEA), intramuscular, intrathecal, epidural, intracistemal, intraperitoneal, transdermal, topical, buccal, sublingual, transmucosal, inhalation, intra-atricular, intranasal, rectal or ocular routes, abuse deterrent and abuse resistant formulations, sterile solutions suspensions and depots for parenteral use, and the like, administered as immediate release, sustained release, delayed release, controlled release, extended release and the like. In the context of the present invention, preferred is an ECM-hydrogel-composition as mentioned herein.

[0056] Preferred is the preparation according to the present invention that is a plant-based ECM or a mixture of an animal- and plant-based ECM. For this preferred embodiment of the present invention, cell wall components are polymerized from the conditioned medium of cultured plant cells. For this, suitable plants cells are cultured in vitro. Then, using enzymatic or chemical digestion, the cell wall of the plant cells is removed, followed by harvesting the conditioned medium. Then, this conditioned medium is subjected to molecular crowding and the cell wall material and / or plant based extracellular biomaterial forms the ECM. In a preferred embodiment, the plant and animal cell culture conditioned medium is combined in order to create a hydbrid material. This material shows higher durability, and can be used for improved and safer medical and / or cosmetic applications

[0057] Another aspect of the present invention relates to non-medical uses of the preparation according to the present invention, including, but not limited to, tissue repair and regeneration, for organoid formation, as bioink in tissue engineering, for a functionalization of surfaces of non-biological materials, hybrid matrices with tunable properties when combined with synthetic polymers, and delivery matrix in cell-based therapies.Another aspect of the present invention relates to the preparation according to the present invention for use in medicine and the treatment of diseases, in particular for use in tissue repair and regeneration, such as, for example, for cartilage, bone, and skin repair.

[0058] Further provided are methods for treating diseases, such as conditions in tissue repair and regeneration, such as, for example, wound healing disorders, for cartilage, bone, and skin repair, comprising administering an effective amount of the pharmaceutical preparation according to the present invention a patient, e.g. human or animal patient, or even plants, or subject in need for such a treatment.

[0059] By “treatment” or “treating” is meant any treatment of a disease or disorder, in a mammal, including: preventing or protecting against the disease or disorder, that is, causing, the clinical symptoms of the disease not to develop; inhibiting the disease, that is, arresting or suppressing the development of clinical symptoms; and / or relieving the disease, that is, causing the regression of clinical symptoms. By “amelioration” is meant the prevention, reduction or palliation of a state, or improvement of the state of a subject; the amelioration of a stress is the counteracting of the negative aspects of a stress. Amelioration includes, but does not require complete recovery or complete prevention of a stress.

[0060] In the context of this invention, the inventors have successfully developed and demonstrated the formation of Cell-free Environment Synthesized ECM (CEM-ECM) using a novel method for producing an extracellular matrix that avoids traditional decellularization approaches. This invention offers transformative potential for tissue engineering, regenerative medicine, and fundamental ECM biology. CEM-ECM not only represents the first instance of ECM formation in a cell-free environment from non-cross linked constituent proteins present in the conditioned medium, CEM-ECM also provides several distinct advantages, including scalability, personalization, and modularity. These characteristics make it a highly versatile and adaptable platform for a wide range of applications.

[0061] Here, CEM-ECM was synthesized directly from conditioned media (CM) derived from various cell types, highlighting its adaptability and potential for personalization (Figure 2, Figure 5). Furthermore, CEM-ECM demonstrated functional bioactivity, as evidenced by its ability to influence cellular behavior. For example, epithelial cells interacting with CEM-ECM exhibited enhanced migration, a hallmark of mesenchymal transformation (Figure 5). This finding isconsistent with previous studies on fibroblast-derived ECM (17) and highlights the physiological relevance of CEM-ECM. The observed cellular responses validate the functional integrity of CEM-ECM and underscore its potential as a platform for studying cell-ECM interactions, wound healing, and tissue regeneration. This ability to generate functional ECM from different source cells can reflect the unique biochemical signatures of its source cells, thus also enabling precise control over its composition. Such control opens new avenues for investigating the specific contributions of individual cell types, or proteins to ECM formation, mechanical properties and their roles in tissue development, repair, and disease. By selecting specific cell types or pre-treating them to modify their secretome, or adding recombinant proteins during polymerization phase, the composition of CEM-ECM can be tuned for targeted applications.

[0062] Moreover, the CEM-ECM synthesis process avoids the inherent limitations of decellularization protocols, which are often labor-intensive, prone to variability, and carry risks such as incomplete removal of cellular debris, potential immunogenicity, and cytotoxicity (18). By utilizing CM as the raw material, CEM-ECM eliminates the need for detergent-based decellularization, which can damage ECM integrity and functionality. Additionally, this approach mitigates concerns associated with tissue sourcing, including the scarcity of human cadavers or the risks of immunological rejection and disease transmission associated with nonhuman tissues (5, 19).

[0063] One of the most exciting aspects of this invention is the opportunity to explore the fundamental principles of ECM assembly. By studying establishing the protocol for CEM-ECM formation, the inventors gained critical insights into how specific ECM proteins and macromolecules interact to form a functional matrix. High-speed atomic force microscopy (HS-AFM) allowed to visualize the presence of key ECM proteins, such as collagen and fibronectin, in their physiologically relevant states (Figure 1). Importantly, the inventors observed interactions between these proteins in the absence of cells (Figure 1), suggesting that the biochemical environment in CM is sufficient to support the initiation of ECM polymerization. This finding underscores the potential of CEM-ECM as a model system for investigating the molecular dynamics of ECM assembly, including the roles of individual proteins and external factors such as macromolecular crowding.Another key advantage of CEM-ECM is its scalability (5), which addresses one of the major challenges in ECM production for commercial and clinical applications. Traditional ECM production methods rely on adherent cell cultures that require large surface areas and intensive maintenance, making them impractical for large-scale production (20). In contrast, CEM-ECM leverages CM, which can be produced in bulk using bioreactors. This approach reduces the labor and infrastructure requirements associated with ECM production, making it more feasible for commercial preparation. By simply scaling up cell culture systems to produce CM, large quantities of ECM precursors can be generated, enabling the production of substantial amounts of CEM-ECM for use in research, therapeutics, and biomaterials development.

[0064] In summary, the successful synthesis of CEM-ECM represents an improvement in ECM research and biomaterial production. By providing a cell-free platform for ECM formation, this invention provides new approaches to studying ECM biology, designing biomaterials, and developing regenerative therapies. The ability to control and customize ECM composition, combined with the scalability of the synthesis process, makes CEM-ECM a transformative tool for advancing tissue engineering and regenerative medicine.

[0065] In conclusion, this invention not only demonstrates the feasibility of CEM-ECM synthesis but also highlights its significant advantages and potential applications. By addressing current limitations and expanding its capabilities, CEM-ECM can become a cornerstone technology for the next generation of ECM-based biomaterials and therapeutic interventions. This invention further establishes CEM-ECM as a versatile and scalable alternative to traditional ECM derivation methods. By leveraging the cell-free synthesis approach, CEM-ECM provides a reproducible and customizable platform for applications in tissue engineering, regenerative medicine, and beyond, offering a transformative tool for studying ECM biology and developing therapeutic scaffolds.

[0066] The present invention relates to the following items:

[0067] Item 1: An in vitro method for producing an extracellular matrix (ECM) preparation, comprising the steps of:

[0068] a) Providing at least one culture of suitable cells in a serum-free medium,

[0069] b) Suitably harvesting the cellular supernatant of the at least one culture, and further removing cellular debris, for example using sterile filtration,c) Optionally, concentrating the supernatant of step b) in order to increase the concentration of milieu, for example using freeze-drying,

[0070] d) Mixing the, optionally concentrated, supernatant with a suitable medium comprising a macromolecular crowding agent (MMC), and

[0071] e) Suitably incubating the mix of step d) to form ECM.

[0072] Item 2. The method according to Item 1, which does not involve a decellularization step of the ECM as formed.

[0073] Item 3. The method according to Item 1 or 2, wherein the cells as cultured in step a) are attached or seeded to a surface, such as the culture vessel, and / or a carrier matrix, such as microcarrier beads.

[0074] Item 4. The method according to any one of Items 1 to 3, further comprising at least one of the introduction of natural and / or synthetic peptides, addition of chemical cross-linkers to enhance specific ECM properties, such as, for example stiffness and / or porosity, the incorporation of bioactive molecules, such as growth factors, cytokines, and / or small-molecule drugs into the ECM, the addition of recombinant proteins to the ECM during step d) or e), or the step of removing undesired proteins from the ECM during or after step d) or e).

[0075] Item 5. The method according to any one of Items 1 to 4, further comprising the step depleting of an undesired protein before step d) or e).

[0076] Item 6. The method according to any one of Items 1 to 5, wherein the suitable cells are selected from animal cells, such as vertebrates or invertebrates, in particular mammalian cells, individual cell types or mixtures of different cell types, cell lines, MSCs, primary cells, stem cells, mesenchymal stem cells, epithelial, endothelial, mesenchymal or immune cells, muscle cells, fibroblasts, such as skin fibroblasts, human embryonic fibroblasts, human bone marrow stromal cells, organoids, tissue samples, plant cells or mixtures thereof.

[0077] Item 7. The method according to any one of Items 1 to 6, wherein the suitable cells are genetically engineered, in particular to provide a modified supernatant.Item 8. The method according to any one of Items 1 to 7, wherein the method further comprises a pre-culture of the cells before step a).

[0078] Item 9. The method according to any one of Items 1 to 8, wherein the serum-free medium is a fetal-calf-serum (FCS)-free medium, such as DMEM, optionally supplemented with 250pM 2-O-a-Glucopyranosyl-L-ascorbic-acid.

[0079] Item 10. The method according to any one of Items 1 to 9, wherein the supernatants from the cells in step b) are harvested at about 3 days after switching to the serum-free medium.

[0080] Item 11. The method according to any one of Items 1 to 10, wherein the removing cellular debris comprises centrifugation and / or sterile-filtration, for example using a 0.45pm syringe driven filter unit.

[0081] Item 12. The method according to any one of Items 1 to 11, wherein the concentrating comprises size-exclusion centrifugation and / or freeze-drying, for example at 76 °C for about 2 hrs.

[0082] Item 13. The method according to any one of Items 1 to 12, wherein the macromolecular crowding agent (MMC) is selected from the group consisting of Ficoll 70, Ficoll 400, carrageenan, polyvinylpyrrolidone, dextran sulfate, polyethylene glycol (PEG), hyaluronic acid, and mixtures thereof.

[0083] Item 14. The method according to any one of Items 1 to 13, wherein incubating in step e is performed at standard temperature, e.g. in a standard cell culture incubator at 37 °C with 5% CO2 for a time suitable to form ECM; such as, for example, for about 4 to 10 days, preferably 4 to 6 days.

[0084] Item 15. The method according to any one of claims 1 to 14, wherein plant cells are cultured in step a), and further comprising the step of removing the cell wall of the plant cells.

[0085] Item 16. An in vitro method for producing an extracellular matrix (ECM) preparation, according to any one of Items 1 to 15, comprising the steps of:

[0086] a) Providing at least one culture of suitable cells in an FCS-free medium,b) Suitably harvesting the cellular supernatant of the at least one culture in step a) 3 days after switching to the FCS free cell culture media and sterile-filtering using a 0.45pm syringe driven filter unit to remove cellular debris.

[0087] c) Concentrating the supernatant of step b) by freeze-drying using a MiVac instrument at about 76 °C for 2 hrs,

[0088] d) Mixing the concentrated supernatant with DMEM containing 6 x macromolecular crowder (MMC) Ficoll 70 / 400, and

[0089] e) Suitably incubating the mix of step d) in a standard cell culture incubator at 37 °C with 5% CO2 for about 6 days to form ECM with 2 times medium replenishment.

[0090] Item 17. A preparation, comprising the ECM as produced according to a method according to any one of Items 1 to 16, together with an acceptable carrier or diluent, in particular a pharmaceutical preparation together with a pharmaceutically acceptable carrier or diluent.

[0091] Item 18. The preparation according to Item 17, which is a plant-based ECM or a mixture of an animal- and plant based ECM.

[0092] Item 19. Use of the preparation according to Item 17 or 18 for tissue repair and regeneration, for organoid formation, as bioink in tissue engineering, for a functionalization of surfaces of non-biological materials, hybrid matrices with tunable properties when combined with synthetic polymers, as a cosmetic preparation, and delivery matrix in cell-based therapies.

[0093] Item 20. The preparation according to Item 17 or 18 for use in medicine and the treatment of diseases, in particular for use in tissue repair and regeneration (such as, for example, for cartilage, bone, and skin repair.

[0094] The present invention will now be described further in the following examples with reference to the accompanying figures, nevertheless, without being limited thereto. For the purposes of the present invention, all references as cited are incorporated by reference in their entireties.

[0095] Figure 1 shows that high speed atomic force (HS-AFM) microscopy highlights the physiological complexity of fibroblast cell supernatants. Fibroblast cell supernatants (conditioned medium [CM]) filtered with 0.45pm pore size PVDF membrane were analyzed. A, 1:50 diluted CM arrowheads show various proteins present in their physiological state. B,High speed AFM shows increased abundance of collagen and other protein CM derived from BJ-5ta cells treated with TGFbl. Zoom inset, Black arrow shows carboxy-terminal (NCI) domain, white arrow heads show the main polypeptide of collagen molecule. Green arrowhead shows potentially fibronectin molecules. Cyan / blue arrows show collagen molecules interacting with other proteins.

[0096] Figure 2 shows that macromolecular crowding enhances de novo polymerization of extracellular matrix from fibroblast conditioned medium. A: Schematic representation of CEM-ECM formation protocol. Al; Normal human lung fibroblast (WI-38) was cultured in DMEM. A2; after 48 hrs, conditioned media was filtered using a 0.45pm PVDF filter to remove cells and debris. A3, Conditioned medium was mixed with molecular crowding solution composed of a mixture of Ficoll 70 and 400 and A; placed the in-cell culture incubator at 37 °C and 5% CO2. for 48 hrs. B. Control; Representative images of 24 well glass bottom dishes incubated with WI-38 cell derived conditioned medium (CM), after removal of CM, the respective well was stained with CNA35-GFP probe, IX Ficoll 70 / 400; representative images of WI-38 cell-derived conditioned medium after incubating with a mix of molecular crowding agent for 48 hrs. After removal of CM, the respective well was stained with CNA35-GFP probe. C, the conditioned media was concentrated using a freeze-dryer. Control; Representative images of 24 well glass bottom dishes incubated with WI-38 cell derived conditioned medium (CM), after removal of CM, the respective well was stained with CNA35-GFP probe, IX Ficoll 70 / 400; representative images of WI-38 cell derived conditioned medium after incubating with a mix of molecular crowding agent for 48 hrs.). After removal of CM, the respective well was stained with CNA35-GFP probe. D, WI-38 derived conditioned medium (±TGFbl) was concentrated using a freeze-drying approach and mixed with different concentrations of molecular crowding agent mix. D, Control (no TGFbl treatment) and TGFbl, representative images of WI-38 cell derived concentrated conditioned medium after incubating with a mix of molecular crowding agent for 48 hrs.

[0097] Figure 3 shows that CEM-ECM can be synthesized from different fibroblast sources and shows ultrastructural similarities to physiological ECM. A, Conditioned cell culture medium was collected from cultured BJ-5ta (skin fibroblasts), WI-38 (human embryonic fibroblasts), HS-5 (human bone marrow stromal cell line) cells. CM was mixed with 3X Ficoll 70 / 400 solution, incubated for 48 hrs. Transmitted light images show formation of CEM-ECM, Matrigel coated dishes were used as a positive control. B-C, scanning electron microscopy (SEM) of CEM-ECM polymerized from WI-38 cell conditioned media. The self-assembled structures formed in presence of 3X Ficoll in a 10 cm2plastic dish. B, overview SEM images of CEM-ECM generated from WI-38 cells. (C) representative images at 4050 X magnification of CEM-ECM 3D structures formed after polymerization in presence of 3X Ficoll 70 / 400.

[0098] Figure 4 shows the proteomic analysis of BJ-5ta derived CEM-ECM: CEM-ECM derived from BJ-5ta cell conditioned medium was solubilized and subjected to mass spectrometry analysis. A, Shows batch to batch variation CEM-ECM. CEM-ECM 01 to 04. Each batch was synthesized from differentially passaged BJ-5ta. Intersection size shows the overlapping number of proteins detected between different batches consistently across 4 batches. No. of proteins detected shows the total number of proteins detected in respective CEM-ECM batch. B, Shows the relative abundance of common proteins in each CEM-ECM batch. C, Heat map, shows the core matrisome and core matrisome related proteins detected consistently across different batches. D, pie chart shows the number of proteins belonging to different classes of core matrisome and related proteins.

[0099] Figure 5 shows the functional implications of Cell-CEM-ECM interaction: A, scanning electron microscopy (SEM) of A549 cells cultured in the presence of polymerized CEM-ECM derived from WI-38 cell conditioned media. Zoom inset shows A549 cells interacting CEM-ECM. A549 cells are pseudo-colored in pink and CEM in green. B-C, A549 cells were plated on CEM-ECM functionalized 6 well cell culture dish. Cells were imaged live in a time lapse manner on a wide field microscope. B, representative transmitted light images of A549 cells cultured on differentially functionalized (Poly-L-Lysine [PLL], Matrigel andHS-5 derived) CEM-ECM. C, representation of tracks covered by A549 cells during migration on differentially functionalized cell cultured dishes, bar graph shows quantitative analysis of average speed of A549 cells on Glass, PLL, Matrigel and CEM-ECM. ****< 0.001, one-way ANOVA test was used for testing statistical significance.

[0100] Figure 6 shows tests of adding CM to previously MMC incubated CM in order to enhance the polymerisation of CEM-ECM (A, B). Indeed, the CNA35-GFP probe showed quantitatively increased presence of fibrillar collagen in wells where CM was added compared to replenishing with new CM. The CEM-ECM formation was highly enhanced so much so that it can be visualized without microscopic aid, the image shows extracellular matrix fibers formed after 4days of cell supernatant incubation with 6X macromolecular crowding (Ficoll 400 at 0.15g / ml and Ficoll 70 at 0.22g / ml concentration) agent (1:1 ratio).

[0101] Figure 7 shows a 3D cell culture with PEG. MDCK cells were cultured in Polyethylene glycol (PEG) hydrogel functionalized with Matrigel (A, 3mg / ml), CEM-ECM (B, 3mg / ml) and a mixture of CEM-ECM + Matrigel (C, 1.5mg / ml each) for 7 days. The images show 3D MDCK cysts. The images are taken with lOx 0.45 NA Air objective, Scale bar 500pm. D. Shows MDCK cysts stained with Calcein Am (For live cell signal) and Hoechst. Qualitative analysis shows that larger MDCK cysts are formed in CEM-ECM+Matrigel+PEG mixture compared to Matrigel or CEM-ECM alone, suggesting CEM-ECM as a supplement for better performance of Matrigel, the images are taken with 40x 1.4 NA oil objective.

[0102] Examples

[0103] MATERIALS AND METHODS

[0104] High Speed atomic force microscopy

[0105] MICA (Sigma Aldrich, AFM-71855-15-10) was freshly cleaved prior sample application, incubated with 10 pl 0.425 mM APTES (Sigma Aldric, 440140) 30 min at 23 °C and then rinsed with 1 ml bi-distilled water. Filtered CM supernatant (Millex®-HV Filter Unit, Millipore, SLHVM33RS) was diluted in medium and absorbed on APTES coated Muscovite MICA for 30 min at 23 °C. After sample incubation, the scanning chamber was filled with 1.5 ml medium.

[0106] All AFM and HS-AFM micrographs were acquired using a NanoRacer HS-AFM (JPK-Bruker) operated by JPK Scanning Probe Microscope control software. The acquisition was performed using photothermal activated on-resonance dynamic mode (AC feedback mode). The NanoRacer was operated in an acoustic isolated case on an active anti-vibration table (Bruker) to remove external noise vibrations. Sample scanning was performed in aqueous solution at 23 °C using ultra-short cantilevers (NanoWorld, USC-F1.2-k0.15) mounted on a HyperDrive holder (Bruker) and singularly calibrated prior scanning at spring force constant close to 0.15 N / m. The cantilevers were operated at frequency range of 400-1000 kHz, target amplitude 3-6 nm and gain 10-30k. The topographs were imaged maintaining a pixel size 1-1.5 nm and line rate 20-40 lines / s.Topographs were processed with the JPK data processing program as trace height is displayed. A 4 degrees polynomial fit was applied for both surface and single scan line and smoothed with low pass filter Gaussian smoothing (o = 1.0 px, in both X and Y). If not specified, the color scale range was set with an offset of - 1.0 nm in order to see the MICA surface and a range of 4.5 nm to have an ideal contrast for small molecules.

[0107] Cell lines and primary cells

[0108] Normal human lung fibroblasts WI-38 (#CAT) was acquired from ATCC. The adenocarcinomic human alveolar epithelial cell line (A549) (#CAT) was purchased from ATCC, while hTERT-immortalized human skin-derived BJ5Ta cells (#CAT) were obtained from manufacturer. The HS-5 bone stoma-derived fibroblasts were a generous gift from Dr. Judith Zaugg, European Molecular Biology Laboratory (EMBL) Heidelberg.

[0109] The WI-38 cells were cultivated in Dulbecco’s Modified Eagle Medium (DMEM, Pyruvate, Ig / L D-Glucose) purchased from Gibco supplemented with 0.1 ml NormoCure 100 mg (InvivoGen) and the contents of the FGM-2 SingleQuots Supplement Pack (0.5 ml rhFGF-B, 0.5 ml Insulin) (CC-3132) from Lonza.

[0110] The cell lines HS-5, A549 and BJ5Ta were grown in DMEM ([+] Pyruvate, Ig / L D-Glucose) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (#CAT, Gibco), 0.1 ml NormoCure 100 mg and 1% L-Glutamine.

[0111] Cell-free environment synthesized extracellular matrix (CEM-ECM) formation and widefield microscopy (3)

[0112] The cell lines BJ5Ta, HS-5 and the primary cells WI-38 were cultivated in starvation medium of DMEM (Pyruvate, Ig / L D-Glucose) containing 0.1 ml NormoCure 100 mg for the time indicated in each experimental setup. The cells were optionally treated with 5 ng / ml TGF-pi (11409-BH, RD Systems) or 250 mM 2-O-a-Glucopyranosyl-L-ascorbic-acid (GAA) (SMB00390) obtained from Merck. 3 days after the media change from full supplemented to starvation medium, the conditioned medium (native) was harvested and sterile-filtered using HV PVDF 0.45 pm Filter Units (SLHVM33RS, Millex). In combination with a mixture of IX (3.75% w / v Ficoll 70 (#CAT, Merck) 2.5% w / v Ficoll 400 (#CAT, Merck)), 1.3X or 3X Ficoll PM 70 and 400 (1:1) resuspended in DMEM indicated in the respective experimental setup. Optionally the conditioned medium was vacuum concentrated. The miVac QuattroConcentrator (GeneVac) was pre-heated at 70 °C for 30 min and the medium in a 6 well plate reduced by the factor 3.75 from 4.5 ml to 1.2 ml at 46 °C. The conditioned medium was either inNunclon Delta Surface 10 cm2(150318, Thermo Fisher) or a 24-well glass bottom plate (P24-0-N, CellVis) kept in a humidified incubator (37 °C, 5% CO2). The mixture of conditioned medium and macromolecular crowder was replenished every 3 days, while every second time fresh Ficoll 70 / 400 was added alternate times. Widefield images of CEM-ECM of different origins were acquired with an Olympus CKX41 microscope equipped with a 20X objective.

[0113] Immunofluorescence staining and confocal microscopy

[0114] CEM-ECM was grown from native and vacuum concentrated cell conditioned medium for minimum 2 days. The CEM-ECM was fixed in pre-chilled 4% PFA (Al 1313-22) (10 min) on ice and washed 3 times with IX PBS. The fluorescent probe CNA35-GFP (1 :250) (#CAT) was incubated for 1 h 30 min at room temperature. Images were acquired using the Nikon AIR HD25 confocal microscope with a 40X objective.

[0115] Scanning electron microscopy of CEM-ECM

[0116] CEM-ECM was grown from 500 pM GAA-induced WI-38 conditioned medium in presence of 6X Ficoll 70 / 400 on Indium tin oxide-coated (ITO) coverslips. The CEM-ECM was grown for 21 days and then 459 cells were seeded and allowed to grow for 2 days. The following sample preparation for scanning electron microscopy was completed at Electron Microscopy Core Facility (EMCF) EMBL, Heidelberg. The samples were rinsed with 0.1 M PHEM (#CAT) buffer (pH 6.9) and fixed in 4 % PFA (#CAT) diluted in 0.1 M PHEM for 1 h at room temperature. Again, the sample was rinsed 3 times with 0.1 M PHEM and additionally 2 times with ddH2O. A dehydrated series was performed with increasing ethanol (#CAT) concentration (20%, 40%, 60%, 70%, 80%, 90%, 95%, 3x 100%) with rapid exchange and 5 min incubation each. Following, the samples were incubated in an increasing Hexamethyldisalazane (HDMS) (#CAT) concentration series (25% 25 min, 50% 20 min, 75% 20 min, 100% 2x15 min). The excessive HDMS was removed by dry blotting and left for drying at room temperature for 2 hrs. The samples were mounted on SEM stubs with conductive double sided carbon tabs (#CAT). Next, a gold sputtering coating was applied with the Quorum Q150RS (Quorum Technologies) for 180 s at 60 mA. Images of the samples were acquired using the Crossbeam 540 (Zeiss) scanning electron microscope and pseudo colored with Adobe Photoshop CS4.

[0117] Mass spectrometry analysis of CEM-ECMCEM-ECM was produced as described above from WI-38 and BJ5-Ta cells, which were GAA (500 pM)-induced. The native conditioned medium was grown in combination with 6X Ficoll 70 / 400 for 21 days. The CEM-ECM was harvested into 4mM HEPES (#CAT) with IX Proteinase inhibitor (#CAT) diluted in ddEEO. The samples were pelleted at 16 000 rpm / rcf? For 30 min at 4 °C and the supernatant discarded. The proteins were digested overnight in 200 ml Lysis buffer (100 mM Tris-HCl (#CAT) pH 8.5, 2M Urea (#CAT), 6 M, GuanidinHCl (#CAT), 10 mM DTT (#CAT), IX Protease inhibitor (#CAT)) at constant agitation of 14 000 rpm (37 °C). The following sample preparation for LC-MS / MS was completed at Proteomics Core Facility (PCF), EMBL Heidelberg.

[0118] To prepare the samples for a LC-MS / MS analysis, the protein lysates were in-solution degraded with a tryptic digest according to a modified Single-Pot Solid-Phase-enhanced Sample Preparation (SP3) protocol (PMID: 25358341, PMID: 29565595). In order to achieve this, 10 pg protein lysates were combined with Sera-Mag Beads (Thermo Fisher Scientific, #4515-2105-050250, 6515-2105-050250) 10 pl 15% formic acid and 30 pl ethanol. The binding of proteins to Sera-Mag Beads was facilitated using constant shaking for 15 min at room temperature. Following, the SDS was removed from the solution by 4 subsequent washes with 200 pl 70% ethanol and the samples digested over night at room temperature in presence of 0.4 pg sequencing grade modified trypsin (Promega, #V5111), 40 pl HEPES / NaOH (pH 8.4), 1.25 mM TCEP and 5mM chloroacetamide (Sigma-Aldrich, #C0267). To remove the beads, the samples were washed with 10 pl of an aqueous solution containing 2% DMSO and the eluates dried down.

[0119] The LC-MS / MS analysis was conducted on an Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific). First, the samples were separated with an Ultimate 3000 nano RSLC system (Dionex) equipped with a trapping cartridge (Precolumn Cl 8 PepMap 100, 5 mm, 300 pm i.d., 5 pm, 100 A) and an analytical column (Acclaim PepMap 100, 75 x 50 cm C18, 3 mm, 100 A) connected to a nanospray -Flex ion source. Then, the generated peptides were transferred onto the trap by using a gradient from 2% to 85% Solvent B (0.1% formic acid in acetonitrile) for 60 min at 0.3 pl per min. After loading the samples, the Orbitrap Fusion Lumos was operated in positive ion mode with a spray voltage range of 2.4 kV and capillary temperature of 275 °C. The full scan MS spectra were flowingly acquired with a mass range of 300-150 m / z in profile mode and a resolution of 120.00. The AGC target was set to 50% and the max injection time to 250 ms. Precursors were isolated using the quadrupole with a window of 1.4 m / z and collisionenergy of 30%. The MS2 spectra were generated using a 15.000 resolution in the Orbitrap, the AGC was defined 200% and the max injection time set to 32 ms.

[0120] For data analysis, the obtained raw files were converted to mzML format in MSConvert by ProteoWizard taking into account peak picking, 64-encoding, lib compression and filtering for the 1000 peaks of highest intensity. Raw data were assigned to the Uniprot Homo sapiens FASTA database (UP000005640, ID9606, 20.594 entries, date: 26.10.2022, downloaded: 11.01.2023) including common contaminants and reversed sequences using the database search tool MSFragger (v3.8) in FragPipe (20.0). For filtering and intensity -based and label-free quantification, the tolls philosopher (v5.1.0) and lonQuant (1.9.8) were utilised. Following search parameters were adjusted: Carbamidomethylation (C, 57.0215) as fixed modifications; Oxidation (M, 15.9949), Acetylation (protein N-terminus, 42.0106) as variable modifications. Both, the full scan (MSI) and MS / MS (MS2) allowed a mass error tolerance of 20 PPM. The MSFragger used for the search per default the ‘stricttrypsin’ (Trypsin / P) proteolytic enzyme which allows a maximum of 2 missed cleavages with a minimum peptide length of 7 amino acids. The false discovery rate on peptide and protein level was adjusted to 0.01. Further, standard settings of the FragPipe workflow were utilized apart from following adjustments: ionquant.maxlfq: 0, ionquant.mbr: 0, ionquant.mbrtoprun: 100000, ionquant.normalization: 0, ionquant.uniqueness: 1, msfragger.add topN complementary: 0, msfragger.output_report_topN_dia2: 3, phi-report.filter: —sequential —picked — prot 0.01 — razor, quantitation. run-label-free-quant: true (PMID: 25987413).

[0121] After data processing in FragPipe, the output files were analyzed with the programming language R (ISBN 3-900051-07-0). For quality control, the contaminants and reverse proteins were excluded from the data set so that only proteins with a minimum of two unique peptides (Razor.Peptides >= 2) remained. The obtained 650 protein intensities within the Log2 transformed raw Razor MSI files were cleaned for batch effects using the ‘removeBatchEffect’ function of the Lima package (PMID: 25605792). Moreover, the data points were normalized with the ‘normaliseVSN’ function of the Lima package (VSN - variance stabilization normalization - PMID: 12169536). Missing values within the dataset were imputed with the ‘knn’ method utilizing the ‘impute’ function of the Msnbase package (PMID: 22113085). The differential expression of the proteins was identified using the moderated t-test of the limma package (TmFif and 'eBayes' functions). The replicate information of the dataset was implemented in the analysis as an argument to the ‘ImFot’ function of limma. Moreover, theimputed values were weighed at 0.01 while the quantified values had the weight 1 in the ‘ImFif function. Finally, the proteins were annotated as a hit if the false discovery rate was less than 0.05 and the absolute fold-change greater than 2. In contrast, a protein candidate was assigned if the false discovery rate was less than 0.2 and an absolute fold-change of minimum 1.5.

[0122] Time-lapse microscopy

[0123] CEM-ECM was grown on sterile glass coverslips derived from GAA-induced (500 pM) HS-5 cell conditioned medium for 18 days in presence of 6X Ficoll 70 / 400 and stored at -80 °C. CEM-ECM was compared to 0.265 mg / ml Matrigel Matrix (#2073001) (Corning) diluted in IX PBS and 1 mg / ml Poly-L-lysine (#RNBJ8041) (Sigma). The PLL coating was applied by incubating the solution for 5 min at room temperature on the glass coverslips, rinsing with ddEEO and drying for 2 hrs.

[0124] 15 000 A549 cells were cultivated on CEM-ECM, PLL or Matrigel -coated surfaces in growth medium for 24 hrs in a humidified incubator (37 °C, 5% CO2). The A549 cells were washed 3 times in IX PBS before a supplemented CCh-independent culture medium (Gibco) was applied. The widefield images were acquired every 30 min over 7 hrs with a high-throughput ImageXpress Micro XLS (Molecular devices) microscope at ALMF (EMBL Heidelberg) equipped with a 4X objective.

[0125] The 8-bit images were converted to a stack and the background subtracted. The cells were selected by applying a threshold and binary mask, which was divided by 255. The created stack was multiplied with the original stack. Cells were automated detection and tracked by the TrackMate Plugin available in Fiji. The individual cell trajectories and mean velocity were calculated within TrackMate. The obtained data was analyzed and visualized as barplots with GraphPad Prism 10 (Version 10.1.1 (270), USA). A one-way ANOVA with Dunnett’s multiple comparison test was conducted to determine statistical difference. *, p-value < 0.05.

[0126] Results

[0127] HS-AFM confirms presence of precursor proteins needed for ECM mesh formation: Fibroblasts are the master synthesizers of the ECM (2, 8). The secretome of fibroblasts is known to contain a range of ECM components and enzymes that are essential for ECM assembly (9). The studies as disclosed are mass spectrometry based and do not provide any insights into the physiological state, and their capability of protein / protein interaction. This knowledge couldprovide a basis for exploring the possibility if ECM proteins can interact and form ECM in the absence of cells that produce these proteins. To this end, the inventors used high HS-AFM to understand the structural / compositional complexity of fibroblast conditioned media (CM) supernatants, furthermore to check if proteins such collagens or fibronectin are present in their physiological state and can potentially interact. Here, the inventors used the conditioned medium from the culture of human dermal fibroblast cell line, BJ-5ta. Cells were grown in an FCS free medium, and sieved through a 0.45 pm pore size filter membrane. The CM (in various dilutions) was allowed to settle on an APTES coated Muscovite (MICA) surface for 15 min, subsequently the HS-AFM scanning was performed. The resulting topograph (Figure 1A) showed a presence of collagen molecules (Figure 1A, black and white arrowheads) with prominent C-terminal non-collagenous domain of collagen [NCI] (black arrowheads) (10) and collagen polypeptide backbone (Figure 1 A, white arrowheads).

[0128] HS-AFM imaging showed a wide variety of other molecules present as well, some similar to fibronectin (green arrowheads), identified based on previous literature studies of HS-AFM of recombinant fibronectin. Studies have shown that fibronectin can be a nucleator for collagen 1 polymerization (Graham J, Raghunath M, Vogel V. Fibrillar fibronectin plays a key role as nucleator of collagen I polymerization during macromolecular crowding-enhanced matrix assembly. Biomater Sci. 2019 Nov 1;7(11):4519-4535. doi: 10.1039 / c9bm00868c. Epub 2019 Aug 22. PMID: 31436263; PMCID: PMC6810780). Interestingly, the inventor’s HS-AFM images show molecules similar to collagen and fibronectin interacting (Figure 1A, cyan arrowheads). These observations suggest that the molecules present in the conditioned medium of cells, are capable of interacting and potentially capable of performing physiological activity as well. Since TGFbl is widely used to increase the synthesis ECM proteins, particularly collagens, fibronectin and related enzymes, here, as a proof of concept, the inventors want to analyze if HS-AFM imaging of the CM of fibroblasts treated as ±TGFbl (Figure IB) would reflect this increased synthesis. Indeed, as expected, the CM of fibroblasts treated with TGFbl showed high presence of collagen and fibronectin like molecules (zoom inset Figure IB). Keeping in view the possibility that the molecular interactions that the inventors observe in HS-AFM can happen stochastically as well, it was needed to analyze further if indeed such interactions occur and can result in an ECM mesh formation.

[0129] Combining fibroblast conditioned medium and macromolecular crowding facilitates protein self-assembly of the Cell-free Environment synthesized ECM (CEM-ECM)Previous studies have shown that ECM assembly in vitro is slow and macromolecular agents (MMC agents) such as a Ficoll, carrageenan, polyvinylpyrrolidone and dextran sulfate have been used to accelerate the polymerization of ECM (11-13). MMC agents mimic the crowded conditions present in vivo, occupying space in cell culture media, therefore, pushing molecules in close quarters to facilitate interactions, enzymatic reactions, which subsequently accelerate the formation or polymerization of ECM (14). However, to date, macromolecular crowding has only been used on cells and the respective cell culture media in the same dish, if macromolecular crowding would enhance formation of ECM from CM in a separate dish where no cells a present remains to be tested. To this end, the inventors cultured WI-38 cells in 10cm cell culture dishes (Figure 2A) in FCS containing DMEM media. 18hrs after seeding, the cells were washed with PBS (3X) and the cells were incubated in DMEM in the presence of 500pm 2OGAA without FCS. After 48 hrs of culture, the CM was removed and sieved with 0.45pm PVDF low protein biding filter. The sieving step was necessary in removing cells or debris from the CM. The filtered media was transferred to a glass bottom dish ± MMC agent (a mixture of Ficoll 70 and Ficoll 400). The CM was placed in the cell culture incubator for the next 48 hrs. Post incubation, CNA35-GFP probe was used to visualize if a fibrillar collagen network formed. The wide field imaging of the glass bottom showed no visible signal of CNA35-GFP probe (Figure 2B, Control), however, strikingly, the well where CM was mixed with MMC agent (Figure 2B, IX Ficoll 70 / 400), a CNA35-GFP positive signal was detected. These data suggest that indeed, CM can be used for de novo synthesis of ECM in cell free environment. Next, the inventors tested if the amount of fibrillar collagen fibers formed could be enhanced by further enriching the CM prior to molecular crowding using freeze-drying approach. Here, the volume of the CM from WI-38 cells was reduced by half before incubating with MMC media. Building on the observation of increased presence of ECM proteins upon TGFb (Figure IB), the inventors also tested the CM of WI-38 cells after TGFbl treatment (Figure 2C, TGFbl). Both untreated and TGFbl treated WI-38 CM showed presence of thicker and larger fibrils (Figure 2C), and the fibrillar collagen network was more pronounced in CM coming from WI-38 cells treated with TGbl. The network of fibrillar collagen was further enhanced by mixing the CM with different concentration of MMC agent (Figure 2D) in addition to freeze drying approach of enriching the CM.

[0130] Ultrastructural analysis of CEM-ECM shows preservation of ECM structural integrity ECM used in tissue engineering applications have diverse sources. To this end, the inventors wanted to test if CEM-ECM can be polymerized from CM of different cell types. Here, theinventors used CM from BJ-5ta (skin fibroblast), primary lung fibroblasts (WI-38) and HS-5 cells (bone marrow derived fibroblast like cell line). The CM were not subjected to freeze drying but mixed with IX Ficoll for 48 hrs (Figure 3 A). For comparison of ECM mesh, 6 well cell culture dish was also coated with Matrigel (Figure 3A, Matrigel). The CEM-ECM mesh was analyzed using transmitted light imaging on a wide field microscope (Figure 3 A). The data analysis showed that indeed CEM-ECM can be polymerized from a diverse set of cell types (Figure 3A, Zoom insets), furthermore, the mesh resembles in morphology the standard Matrigel ECM network. To prepare more quantities of CEM-ECM, the inventors also tested if adding CM to previously MMC incubated CM would enhance the polymerization of CEM-ECM (Figure 6A-B). Indeed, the CNA35-GFP probe showed quantitatively increased presence of fibrillar collagen in wells where CM was added compared to replenishing with new CM.

[0131] In order to test if the CEM-ECM production can be further enhanced, the inventors also tested CM media from BJ-5ta cells cultured in 15cm dishes, the CM was subjected to enrichment via freeze drying and then incubated with 6X Ficoll mixture. Using this protocol, the CEM-ECM formation was highly enhanced so much that it can be visualized without microscopic aid (Figure 6C).

[0132] However, the complexity of ECM mesh cannot be visualized just using light microscopic or macroscopic imaging, to this end, WI-38 cell culture derived CM was allowed to polymerize in presence of MMC crowding agent on Indium tin oxide-coated (ITO) coverslips. Subsequently, the CEM-ECM was subjected to scanning electron microscopy (Figure 3C). The image analysis showed that the CEM-ECM has characteristic features of matrisome network. This includes the D banding pattern of fibrillar collagen (15) (Figure 3C, zoom inset, magenta arrowhead). The SEM images also show the integration of Collage VI beaded (16) molecules (HS-AFM, Figure 3C, zoom inset, black arrowhead) into the CEM-ECM mesh.

[0133] Mass spectrometry analysis of different batches of CEM-ECM confirms presence of diverse core matrisome and associated proteins

[0134] While SEM imaging of the CEM-ECM confirmed the physiologically relevant mesh formation (Figure 3, the complexity of the diverse molecular components polymerizing in the CEM-ECM needed further analysis. To this end, CEM-ECM polymerized from CM derived from BJ-5ta cells was subjected to mass spec analysis. More importantly, the inventors also compared different CEM-ECM batches prepared from differentially passaged BJ-5ta cells. In total, 4CEM-ECM were prepared. For each, fresh vial of B J-5ta was thawed and expanded. Each batch was prepared at least 1 week apart from each other. The CEM-ECM batches were flash frozen till all the batched were ready, and subsequently subjected to mass spec analysis (Figure 4A-D). The data analysis showed the presence of a diverse set of proteins. Across 4 batches, the analysis showed that 823 proteins were consistently detected with similar levels of abundance (Figure 4A-B). Figure 4A shows the comparative analysis of different batches, and Figure 4B shows the relative abundance of different proteins present across different batches. The analysis further shows a consistent presence of 138 core matrisome and related protein (Figure 4C-D). The data shows that COL1, FN1, DCN, FBLN1, SPARC are among the most abundant protein present in CEM-ECM. Overall, from the core matrisome CEM-ECM consistently contained 18 Collagens, 47 ECM glycoproteins, 5 proteoglycans. Notably, 41 ECM regulators and 14 ECM-affiliated proteins were found as well. These data suggest that de novo synthesized CEM-ECM is complex, capturing the structural and functional aspects of physiological ECM.

[0135] Epithelial cells interact with CEM-ECM and enhance mesenchymal cells like migration While SEM and mass spec analysis of CEM-ECM confirms the physiological complexity of CEM-ECM, the idea that the proteins in CEM-ECM are also physiologically functional remains to be tested. Previous studies (17) have shown that epithelial cells acquire a mesenchymal phenotype upon interaction with fibroblast derived ECM (prepared using decellularization protocol) To test if CEM-ECM exhibits similar features, CEM-ECM was synthesized from HS-5 cells. A549 cells were plated on dishes coated with polymerized CEM-ECM. Subsequently, scanning electron microscopy was performed (Figure 5A). The SEM micrographs show that CEM-ECM indeed interacts with A549 cells. Furthermore, the inventors also performed live imaging of A549 cells seeded on HS-5 derived CEM-ECM (Figure 5B). Consistent with previous literature, A549 cells showed enhanced migration (Figure 5B bar graph) compared to glass, poly-lysine or matrigel coated surfaces. These data further confirm the functional relevance of CEM-ECM.

[0136] References

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[0138] 4. R. Kaukonen, G. Jacquemet, H. Hamidi, J. Ivaska, Cell-derived matrices for studying cell proliferation and directional migration in a complex 3D microenvironment. Nat Protoc 12, 2376-2390 (2017).

[0139] 5. M. Assungao, et al. Cell-Derived Extracellular Matrix for Tissue Engineering and Regenerative Medicine. Frontiers Media S.A. [Preprint] (2020). https: / / doi.org / 10.3389 / fbioe.2020.602009.

[0140] 6. J. E. W. Ahlfors, K. L. Billiar, Biomechanical and biochemical characteristics of a human fibroblast-produced and remodeled matrix. Biomaterials 28, 2183-2191 (2007).

[0141] 7. J. M. Aamodt, D. W. Grainger, Extracellular matrix-based biomaterial scaffolds and the host response. Elsevier Ltd [Preprint] (2016). https: / / doi.Org / 10.1016 / j.biomaterials.2016.02.003.

[0142] 8. A. Naba, Mechanisms of assembly and remodelling of the extracellular matrix. Nature Research [Preprint] (2024). https: / / doi.org / 10.1038 / s41580-024-00767-3.

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[0146] 12. R. Rashid, et al., Novel use for polyvinylpyrrolidone as a macromolecular crowder for enhanced extracellular matrix deposition and cell proliferation. Tissue Eng Part C Methods 20, 994-1002 (2014).

[0147] 13. D. Gaspar, K. P. Fuller, D. I. Zeugolis, Poly dispersity and negative charge are key modulators of extracellular matrix deposition under macromolecular crowding conditions. Acta Biomater 88, 197-210 (2019).

[0148] 14. C. Chen, et al., Applying macromolecular crowding to enhance extracellular matrix deposition and its remodeling in vitro for tissue engineering and cell-based therapies. Adv Drug Deliv Rev 63, 277-290 (2011).

[0149] 15. M. Fang, M. M. B. Holl, Variation in type I collagen fibril nanomorphology: the significance and origin. Bonekey Rep 2 (2013).

[0150] 16. Z. Lansky, et al. 3D mapping of native extracellular matrix reveals cellular responses to the microenvironment. J Struct Biol X 1 (2019).17. M. T. Scherzer, et al., Fibroblast-derived extracellular matrices: An alternative cell culture system that increases metastatic cellular properties. PLoS One 10 (2015).

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Claims

Claims1. An in vitro method for producing an extracellular matrix (ECM) preparation, comprising the steps of:a) Providing at least one culture of suitable cells in a serum-free medium,b) Suitably harvesting the cellular supernatant of the at least one culture, and further removing cellular debris, for example using sterile filtration,c) Optionally, concentrating the supernatant of step b) in order to increase the concentration of milieu, for example using freeze-drying,d) Mixing the, optionally concentrated, supernatant with a suitable medium comprising a macromolecular crowding agent (MMC), ande) Suitably incubating the mix of step d) to form ECM,that does preferably not involve a decellularization step of the ECM as formed.

2. The method according to claim 1, wherein the cells as cultured in step a) are attached or seeded to a surface, such as the culture vessel, and / or a carrier matrix, such as microcarrier beads.

3. The method according to claim 1 or 2, further comprising at least one of the introduction of natural and / or synthetic peptides, addition of chemical cross-linkers to enhance specific ECM properties, such as, for example stiffness and / or porosity, the incorporation of bioactive molecules, such as growth factors, cytokines, and / or small-molecule drugs into the ECM, the addition of recombinant proteins to the ECM during step d) or e), or the step of removing undesired proteins from the ECM during or after step d) or e).

4. The method according to any one of claims 1 to 3, wherein the suitable cells are selected from animal cells, such as vertebrates or invertebrates, in particular mammalian cells, individual cell types or mixtures of different cell types, cell lines, MSCs, primary cells, stem cells, mesenchymal stem cells, epithelial, endothelial, mesenchymal or immune cells, muscle cells, fibroblasts, such as skin fibroblasts, human embryonic fibroblasts, human bone marrow stromal cells, organoids, tissue samples, plant cells or mixtures thereof.

5. The method according to any one of claims 1 to 4, wherein the suitable cells are genetically engineered, in particular to provide a modified supernatant.

6. The method according to any one of claims 1 to 5, wherein the serum-free medium is a fetal-calf-serum (FCS)-free medium, such as DMEM, optionally supplemented with 250pM 2-O-a-Glucopyranosyl-L-ascorbic-acid, and / or wherein the supernatants from the cells in step b) are harvested at about 3 days after switching to the serum-free medium.

7. The method according to any one of claims 1 to 6, wherein the removing cellular debris comprises centrifugation and / or sterile-filtration, for example using a 0.45 pm syringe driven filter unit.

8. The method according to any one of claims 1 to 7, wherein the concentrating comprises size-exclusion centrifugation and / or freeze-drying, for example at 76 °C for about 2 hrs.

9. The method according to any one of claims 1 to 8, wherein the macromolecular crowding agent (MMC) is selected from the group consisting of Ficoll 70, Ficoll 400, carrageenan, polyvinylpyrrolidone, dextran sulfate, polyethylene glycol (PEG), hyaluronic acid, and mixtures thereof.

10. The method according to any one of claims 1 to 9, wherein incubating in step e is performed at standard temperature, e.g. in a standard cell culture incubator at 37 °C with 5% CO2 for a time suitable to form ECM; such as, for example, for about 4 to 10 days, preferably for about 4 to 6 days.

11. The method according to any one of claims 1 to 10, wherein plant cells are cultured in step a), and further comprising the step of removing the cell wall of the plant cells.

12. An in vitro-method for producing an extracellular matrix (ECM) preparation, according to any one of claims 1 to 11, comprising the steps of:a) Providing at least one culture of suitable cells in an FCS-free medium,b) Suitably harvesting the cellular supernatant of the at least one culture in step a) 3 days after switching to the FCS free cell culture media and sterile-filtering using a 0.45pm syringe driven filter unit to remove cellular debris.c) Concentrating the supernatant of step b) by freeze-drying using a MiVac instrument at about 76 °C for 2 hrs,d) Mixing the concentrated supernatant with DMEM containing 6 x macromolecular crowder (MMC) Ficoll 70 / 400, ande) Suitably incubating the mix of step d) in a standard cell culture incubator at 37 °C with 5% CO2 for about 6 days to form ECM with 2 times medium replenishment.

13. A preparation, comprising the ECM as produced according to a method according to any one of claims 1 to 12 together with an acceptable carrier or diluent, in particular a pharmaceutical preparation together with a pharmaceutically acceptable carrier or diluent, wherein the preparation preferably is a plant-based ECM or a mixture of an animal- and plant based ECM.

14. Use of the preparation according to claim 13 for tissue repair and regeneration, for organoid formation, as bioink in tissue engineering, for a functionalization of surfaces of non-biological materials, as hybrid matrix with tunable properties when combined with synthetic polymers, as a cosmetic preparation, and delivery matrix in cell-based therapies.

15. The preparation according to claim 13 for use in medicine and the treatment of diseases, in particular for use in tissue repair and regeneration (such as, for example, for cartilage, bone, and skin repair.

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