NL2035750B1

NL2035750B1 - Glycogen for supporting tissue viability

Info

Publication number: NL2035750B1
Application number: NL2035750A
Authority: NL
Inventors: Eva Allijn Iris; Christianus Hermanus Leijten Jeroen; Robin Porsul Isa; Gurian Melvin; Godefridus Andreas Willemen Niels
Original assignee: Univ Twente
Priority date: 2023-09-06
Filing date: 2023-09-06
Publication date: 2025-03-13
Also published as: WO2025051833A1

Abstract

The present invention relates to use of glycogen for maintaining viability of non-perfused tissue or sub-optimally perfused tissue, for increasing survival time of a non-perfused or sub-optimally perfused tissue structure, for preventing necrotic core formation in a non-perfused or sub- 5 optimally perfused tissue structure, and/or for increasing production of angiogenic growth factors by non-perfused or sub-optimally perfused tissue. Also foreseen is the use of glycogen in the prevention of tissue implant rejection in a patient, or in the treatment of a tissue pathology in a patient wherein the glycogen may be in combination with replacement tissue and/or scaffold for tissue ingrowth.

Description

P36261NL00/MJO

Glycogen for supporting tissue viability

Technical field

The present invention relates to the field of tissue engineering and cell-based therapy, more specifically to means that can enhance and support cell/tissue/organ maintenance and/or survival, particularly under non-perfused, sub-optimally perfused, hypoxic and/or anoxic conditions.

Background of the invention

The creation of tissue engineered constructs offers a promising strategy to replace, restore and/or regenerate damaged tissue and/or organs. Unfortunately, despite remarkable progress, tissue engineered constructs are still limited to relatively thin and/or small tissues and/or organs, such as the skin and bladder. This can be explained by one of the major obstacles in the field, which is to provide an adequate supply of oxygen and nutrients to tissue engineered constructs. More specifically, as tissue engineered constructs become larger, oxygen and nutrients are not able to diffuse into the deeper layers of the tissue, resulting in hypoxia, which may lead to necrosis, inflammation, infection and ultimately even failure of the tissue engineered construct.

One approach to address the above-described issue involves the use of solid peroxides, which are used to generate oxygen in situ. However, as the generation of oxygen is mediated via the generation of toxic levels of hydrogen peroxide, such solid peroxides should be combined with hydrogen peroxide degrading enzymes, such as catalases (Aillemen, Niels GA, et al. "Enzyme-

Mediated Alleviation of Peroxide Toxicity in Self-Oxygsnating Biomaterials.” Advanced healthcare materials 11.13 {2022}. 2102697). Another approach to address the above- described issue is to use degradable starch-based hydrogels. Such hydrogels can, upon exposure to a starch-degrading enzyme, generate glucose in situ, thereby supporting cell survival and function (Zargarzadeh, Mehrzad, et al. "Self-glucose feeding hydrogels by enzyme empowered degradation for 3D cell cullure.” Materials Horizons 8.2 (2022): 694-707).

Whereas both the solid peroxides and degradable starch-based hydrogels may enhance oxygen and/or nutrient availability in tissue engineered constructs, both these approaches require the use of xenogeneic enzymes, which is not ideal. These type of enzymes do not only pose a challenge seen from an immunological point of view (i.e. immunogenicity), but also in terms of efficiency in the host organism. Xenogeneic enzymes are not produced by the host organism while still suffering functional depletion based on their half-life. Moreover, they may also have different stability profiles compared to endogenous enzymes, which may be linked to differences in the environment, such as differences in temperature and pH. Altogether, this may result in xenogeneic enzymes to not function properly in a host organism, directly adversely affecting the period that the cells are metabolically supported.

Therefore, there remains a need to adequately support tissue engineered constructs, particularly in terms of adequate oxygen and/or nutrient supply. It is an objective of the present invention to overcome one of the aforementioned or other problems.

Summary of the invention

The present inventors surprisingly found that glycogen can be degraded when exogenously added to cells and that the exogenous addition of glycogen has beneficial effects on cell behavior and viability, in particular in hypoxic or anoxic conditions.

Glycogen is a polysaccharide of glucose, which is typically present as a nanoparticle and generally is stored within a cell, where it serves as a form of energy storage. Specifically, glucose can be uptaken by cells and enzymatically converted into a glycogen nanoparticle, which acts as a metabolic storage that cannot pass through the cell's membranes. Glycogen is therefore assumed to be exclusively present within cells. Upon metabolic need, enzymes within a cell can release glucose from glycose via enzymatic degradation. Only specific cell types such as muscle cells and liver cells were known to convert glucose into glycogen by using such enzymes. These enzymes are not known to have function outside the cells, nor was it known that they were present outside cells. The inventors surprisingly found that a wide variety of cells produce and secrete glycogen degrading enzymes, which results in cell-mediated glycogen degradation outside of cells.

The current inventors have demonstrated that exogenously added glycogen can be used as an energy source in anoxia by human mesenchymal stem cells (hMSCs). It was not only shown that exogenous glycogen was able to maintain hMSC viability and metabolic activity for 28 days, but also that it resulted in enhanced viability and metabolic activity over a prolonged period of time compared to when using other metabolites, such as glucose. Furthermore, the current inventors showed that glycogen can maintain the pro-angiogenic potential of h(MSCs in anoxia, which was reflected on both the protein as gene level in a glycogen dose-dependent manner.

Compared to the currently available methods, i.e. solid peroxides and degradable starch-based hydrogels, which both rely on toxic intermediaries or xenogeneic enzymes, the current disclosure thus provides an method that enables metabolic support of tissue engineered constructs, particularly, which is cell-mediated and free of such enzymes. Moreover, the cell- mediated nutrient release allows for easy adaptation of the technique towards different systems as the nutrient availability is determined by the cells themselves. Glycogen is found to be less angiotoxic at higher concentrations than glucose, which is known to be angiotoxic at higher concentrations. Initial 2D Matrigel assay indeed showed less inhibition of endothelial tube formation at higher concentrations than glucose.

In addition, the inventors found that larger glycogen particles are particularly beneficial for tissue engineering applications, and it was surprisingly found that in case of incorporation of glycogen in hydrogels, such as Dex-TA, the glycogen is better retained and degradable for nearby cells.

Detailed description of the invention

The present invention relates to the use of glycogen - for maintaining tissue viability; - for increasing survival time of a tissue structure; - for preventing necrotic core formation in a tissue structure; and/or - for increasing production of angiogenic growth factor(s) by tissue.

These effects are particularly pronounced in case of a non-perfused or sub-optimally perfused tissue (structure) and/or in hypoxic or anoxic conditions, and can be seen relative to not using said glycogen.

In the present disclosure, the tissue may be for example bone tissue or organ (tissue), such as kidney (tissue), liver (tissue), heart (tissue), lung (tissue), pancreas (tissue), muscle (tissue), brain (tissue) or skin (tissue). The tissue may include blood (e.g. for infusion to traumatic brain injury patients) or body fluid. Preferably the tissue is mammalian or human. The tissue may be autologous, allogenic, or xenogeneic to a patient receiving the tissue. In addition or alternatively, the tissue may be devitalized and/or engineered (in vitro grown). Preferably the volume of the tissue is at least 1, 2, 3, 4, 5, 10, 25, 50 cm? and/or the tissue has a 3D shape with a smallest diameter of at least 0.1, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3,4, 5 cm (i.e. the narrowest width of the tissue (structure) or the shortest distance between any two opposite points on the surface of the tissue/tissue structure). As will be clear, the term “tissue (structure)” can refer to a group of structurally and functionally similar cells, and also encompasses engineered tissue structures. "Non-perfused" (or non-anastomosed) refers to lack of an active blood supply or circulation. In the initial stages of tissue engineering or tissue implantation, it is often difficult to provide adequate blood and/or nutrient/oxygen supply, and nutrient shortage,

hypoxia and even anoxia may occur, which affects overall celltissue health and may lead to necrotic core formation. Non-perfusion of tissue can also occur due to trauma, surgery, and/or pathology. Restoring/maintaining metabolic activity/nutrient supply can prevent deterioration of tissue function, disease onset/progression, and/or promote tissue survival. "Sub-optimally perfused tissue” may refer to tissue which has less perfusion than healthy tissue {of the same type under the same circumstances). The level of perfusion may be assessed by metabolic assays (e.g. lactate production, ATP levels), gene and expression analysis, perfusion imaging, tissue oxygen monitoring, ultrasound, clinical observation (e.g. skin color, temperature). In addition or alternatively, sub-optimally perfused tissue may refer to tissue that is characterized by higher lactic acid levels compared to healthy tissue (of the same type under the same circumstances). In addition or alternatively, sub-optimally perfused tissue may refer to tissue that is characterized by lower ATP levels than healthy tissue (of the same type under the same circumstances). In addition or alternatively, sub-optimally perfused tissue may refer to tissue that is has lower temperature than healthy tissue (of the same type under the same circumstances). Hypoxia or even anoxia thus refers to a condition where cells or tissues are deprived of (sufficient) oxygen. It occurs when there is an insufficient supply of oxygen to meet the metabolic demands of the cells. Anoxia can have detrimental effects on tissue function and viability, as oxygen is vital for cellular respiration and energy production. Hypoxia or anoxia can arise if the oxygen diffusion within the tissue (structure) is insufficient. Nutrients and oxygen are generally important to the cells within the tissue (structure) to support their survival and functionality. In case of a non-perfused or sub-optimally perfused tissue (structure), supply of nutrients and oxygen is limited or absent which can lead to anoxia, cell death, poor tissue development, and/or inadequate functionality. Hypoxia or anoxia can also occur during the transplantation of (engineered) tissues (or whole organs) into a recipient's body. The process of integrating the tissue with the host vasculature is crucial for oxygen and nutrient supply. If blood vessels fail to reestablish efficiently (poor angiogenesis), anoxia may affect the transplanted tissue, leading to poor integration, impaired functionality, or even failure (rejection). The present disclosure can be used to stimulate production of angiogenic factor(s), which are agent(s) that promote the growth and formation of new blood vessels. These factors play a critical role in various physiological processes, including tissue viability and tissue development. Examples of angiogenic factors are Vascular Endothelial Growth Factor (VEGF),

Fibroblast Growth Factor (FGF), Platelet-Derived Growth Factor (PDGF), Angiopoietin,

Transforming Growth Factor-beta (TGF-B) and Insulin-like Growth Factor (IGF).

Tissue viability or survival time can be determined through various methods, both ex vivo (outside the body such as in vitro) and in vivo (within the living organism).

Ex vivo methods for example include histological analysis. Tissue samples can be fixed, sectioned, and stained for examination under a microscope. Histological analysis allows the assessment of cellular morphology, tissue structure, and signs of cell death (such as apoptosis or necrosis). It provides valuable information on tissue health and viability. A lower proportion of dead cells in the tissue (structure), i.e. when using glycogen according to the present invention (relative to not using glycogen), for example after 1, 2, 3, 4, 5, 6, 7 days or after 1, 2, 3, 4, 5 weeks, indicates better maintaining of viability and/or increased survival time. In addition or alternatively, biochemical assay(s) can be performed on tissue samples to measure markers of cellular activity and viability. For example, assays for enzymatic activity {e.g., lactate dehydrogenase), ATP content, DNA fragmentation, or oxidative stress can indicate the metabolic state and health of the tissue. Also, cell viability assays may be performed, such as

MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) or calcein-AM/propidium iodide staining, in order to assess the viability of (samples of) tissue (structure(s)). These assays measure cell metabolic activity or membrane integrity and can provide an indication of tissue viability.

In addition or alternatively, in vivo methods may be used, such as imaging techniques including non-invasive imaging techniques like positron emission tomography (PET), magnetic resonance imaging (MRI), or near-infrared fluorescence imaging, in order to visualize and monitor transplanted or engineered tissues in real-time. These imaging modalities can provide information on tissue viability, functionality, and integration with the recipient. Measurement of blood flow within the tissue can also indicate its vascularization and viability. Techniques such as laser Doppler flowmetry or fluorescent microspheres can be used to assess tissue perfusion and blood flow rates, giving insights into tissue survival. In addition or alternatively, analysis of specific biomarkers in blood or tissue samples can provide information on tissue viability and function. For example, measuring oxygen or nutrient levels, metabolic waste products, or specific cellular markers can indicate the metabolic state and health of the tissue. Increasing the survival time of tissue in the context of the present disclosure may refer to the extension of the duration during which the tissue remains viable and functional ex vivo and/or in vivo such as within the recipient's body.

In accordance with the above, the present disclosure also encompasses a method for maintaining viability of (non-perfused, sub-optimally perfused, hypoxic or anoxic) tissue, for increasing survival time of a (non-perfused, sub-optimally perfused, hypoxic or anoxic) tissue structure, for preventing necrotic core formation in a (non-perfused, sub-optimally perfused, hypoxic or anoxic) tissue structure, and/or for increasing production of angiogenic growth factors by (non-perfused, sub-optimally perfused, hypoxic or anaxic) tissue, by using glycogen, in accordance with the above.

The uses or methods according to the present disclosure may be in vivo or ex vivo.

Glycogen can be defined as a branched polysaccharide. It normally serves as a storage form of glucose in animals and humans. It is sometimes referred to as animal starch. Glycogen is primarily found within liver and muscle cells, where it functions as an energy reserve. Glycogen particles, also known as glycogen granules or glycogen molecules, are the structures in which glycogen naturally occurs within cells (or mimicking synthetic structures). They are composed of multiple glucose units linked together in a branched configuration. The branching is typically achieved through a-1,4-glycosidic bonds that form the linear chains of glucose molecules, and a-1,6-glycosidic bonds that create branch points. Enzymes called glycogen phosphorylase and glycogen synthase break down or synthesize glycogen, respectively, to release or store glucose units. Naturally, glycogen particles are highly dynamic and subject to constant turnover and regulation within cells. Under a microscope, glycogen particles appear as granular structures that can be stained using e.g. periodic acid-Schiff (PAS) staining, which highlights the presence of glycogen in tissues.

The present disclosure further provides for glycogen for use in treating a condition wherein tissue is sub-optimally perfused or non-perfused, e.g. wherein in the use the glycogen is combined or in combination with the tissue (for example extracellular addition of glycogen).

This may promote or rescue cell metabolism and help tissue survival. The present disclosure also provides for glycogen for use in the prevention or treatment of tissue implant failure (or tissue implant rejection) in a patient, e.g. wherein in the use the glycogen is in combination with the tissue implant. This embodiment may comprise administering the glycogen to the patient (for example to the implantation site) and/or to the tissue implant.

Implant failure can lead to implant rejection, also referred to as graft rejection. Implant rejection refers to the immune response mounted by the recipient's immune system against an implanted or transplanted material or tissue (structure). It occurs when the immune system recognizes the implanted material as foreign and activates an immune response to eliminate it. Implant rejection is a significant concern in organ transplantation, tissue engineering, and the use of scaffolds for tissue ingrowth. Implant failure may also encompass that implanted tissue is resorbed by the body, or the implant fails due to starvation, which may cause cellular autophagy, which continues until the cells have no other option than to commit apoptosis.

The present disclosure also provides for glycogen for use in the prevention or treatment of a condition characterized by (local) presence of non-perfused or sub-optimally perfused tissue, such as hypoxic and/or nutrient deficient tissue. For example, such condition may relate to (ischemic) stroke, (myocardial/cerebral) infarction, injured tissue/organ, or oxygen deprivation (e.g. due to drowning), and/or may be due to anesthesia. In this way local oxygen and/or nutrient deprivation can be treated or prevented. Local in this regard may refer to a volume of less than 20 vol.% of the body, or may refer to a specific tissue or specific organ, such as the brain or heart. The glycogen may be administered by bodily injection, preferably intravenous injection. Accordingly, the glycogen may be comprised in a liquid carrier suitable for such injection. In addition or alternatively, in the use the glycogen is combined (or in combination) with said tissue.

Also provided is glycogen for use in the prevention or treatment of a tissue pathology in a patient, e.g. wherein in the use the glycogen is in combination with (replacement) tissue and/or scaffold for tissue ingrowth. The use may comprise introducing the replacement tissue and/or scaffold into the patient. Alternatively, the (replacement) tissue and/or scaffold is already present into the patient and the use involves combining the glycogen with the (replacement) tissue and/or scaffold. Preferably, the glycogen relates to glycogen particles having an average diameter of 30-1000 nm, or 30-100 nm, or 40-60 nm, preferably oyster glycogen.

It is found that the glycogen can stimulate ingrowth of patient's cells into said tissue or scaffold, vascular ingrowth (angiogenesis) into said tissue or scaffold, and guide the immune response and immunological behavior of naive immune cells. The scaffold may comprise autologous/allogeneic cells or no cells at all. The (replacement) tissue can also refer to autologous/allogeneic tissue/organ (e.g. donor graft/tissue/organ). The use may involve treatment of autologous (sub-optimally/non-perfused) tissue (such as due to trauma, pathology or surgery), wherein in the use the glycogen is combined or in combination with said tissue.

A scaffold can provide a (temporary) three-dimensional framework for cell attachment, growth, and tissue formation. Scaffolds mimic the extracellular matrix (ECM) and provide mechanical support, guide tissue organization, and facilitate nutrient and oxygen diffusion. In the context of the present disclosure, several types of (porous) scaffold(s) may be used including: - Synthetic scaffold, typically made of biocompatible and biodegradable materials.

Examples include polymeric scaffolds comprising polymers like polylactic acid (PLA), polyglycolic acid (PGA), polycaprolactone (PCL), and/or their copolymers, due to their tunable properties and biocompatibility;

- Hydrogel, which are water-swollen networks of hydrophilic polymers, providing a hydrated environment similar to soft tissues. Examples include polyethylene glycol (PEG) hydrogels, alginate, dextran, fibrin gel, platelet-based gel, and hyaluronic acid hydrogels; - Ceramic scaffold like calcium phosphate and hydroxyapatite which can be used in scaffolds for bone tissue engineering due to their excellent biocompatibility and similarity to natural bone mineral; - Naturally derived scaffold, e.g. derived from biological sources, such as collagen which is the most abundant protein in the ECM and is commonly used as a scaffold material.

It provides a biocompatible and cell-adhesive environment. Collagen scaffolds can be derived from animal sources or produced through recombinant DNA technology; - Fibrin scaffold, e.g. formed from fibrinogen and thrombin, two components of the blood clotting cascade. Fibrin scaffolds mimic the natural wound healing process and support cell migration and tissue regeneration. - Decellularized Extracellular Matrix (ECM), e.g. natural tissues can be decellularized to remove cellular components while preserving the ECM architecture. The resulting ECM scaffolds provide a biomimetic environment with native tissue-specific biochemical and biomechanical cues; - Composite scaffold e.g. which combine different materials to take advantage of their synergistic properties. For example polymer-ceramic composites: Combining synthetic polymers with ceramic nanoparticles or fibers can enhance the mechanical strength and osteoconductivity of the scaffold, making it suitable for bone tissue engineering applications; - Polymer-hydrogel composite, e.g. incorporating hydrogels within polymer scaffolds can introduce a hydrated microenvironment, enhance cell encapsulation, and improve nutrient and oxygen transport; - 3D printed scaffold, e.g. which allows precise fabrication of complex scaffold structures.

It enables the design and fabrication of scaffolds with controlled pore size, geometry, and spatial distribution of bioactive cues; - Bioactive glass, e.g. surface reactive glass-ceramic biomaterials.

It may be important to stimulate tissue ingrowth into the scaffold, and enhance the integration of the scaffold with the surrounding tissue. Accordingly, the glycogen and/or the scaffold may be combined with agent(s) that promote cell migration, proliferation, differentiation, and extracellular matrix (ECM) production. Preferred agent(s) include growth Factor(s) such as

Transforming Growth Factor-beta (TGF-B), Platelet-Derived Growth Factor (PDGF), Vascular

Endothelial Growth Factor (VEGF), Bone Morphogenetic Proteins (BMPs), Cell Adhesion

Molecules such as Arg-Gly-Asp (RGD) peptide(s), laminin and fibronectin, integrin-binding peptide(s), proteoglycan(s) and/or glycosaminoglycan(s), mineralization agent(s), anti- inflammatory agent(s) such as non-steroidal anti-inflammatory drug(s) (NSAIDs) or anti- inflammatory cytokine(s).

The use and/or treatment according to the disclosure may involve implantation of the (replacement) tissue and/or scaffold, e.g. introducing the tissue and/or scaffold into the recipient (patient), wherein the cells/tissue and/or grown tissue (in the scaffold) is capable of treating e.g. a tissue pathology. The tissue pathology may refer to tissue loss, tissue damage, tissue injury or combination thereof (such as due to surgery). The treatment may involve tissue repair, regrowth, resurfacing, regeneration, or combination.

The tissue in the present disclosure may be for example bone tissue or organ tissue, such as kidney tissue, liver tissue, heart tissue, lung tissue, pancreas tissue, muscle tissue, brain tissue, or skin tissue. Preferably mammalian tissue and particularly human tissue. The tissue may be autologous, allogenic, or xenogeneic to a patient receiving the tissue. In addition or alternatively, the tissue may be devitalized and/or engineered (in vitro grown). In the present disclosure, it may be excluded that the tissue is periodontal tissue, tooth embryo tissue, or (tooth) bone tissue.

In the context of the present disclosure, the glycogen may be natural glycogen and/or synthetic glycogen. Synthetic glycogen can be obtained through chemical synthesis or enzymatic methods. Chemical synthesis may start with a core molecule that serves as a template for glycogen branching. Monomer units (such as glucose or glucose derivatives) are sequentially added, and branching points are introduced using appropriate linkers. Various protecting groups are used to selectively react at specific positions, resulting in the desired glycogen structure. Alternatively, enzymatic methods utilize specific enzymes involved in glycogen synthesis, such as glycogen synthase and branching enzyme, to build glycogen. For example, sucrose phosphorylase (EC 2.4.1.7) and a-glucan phosphorylase (EC 2.4.1.1) and branching enzyme (EC 2.4.1.18) are added to sugar and primer molecules. The enzymes catalyze the addition of glucose units and the formation of branches, mimicking the natural glycogen synthesis process. This approach may result in glycogen structures closer to natural glycogen.

In the current disclosure, the glycogen may be present, e.g. in a composition or liquid (e.g. aqueous medium), in an amount of between 0.1-100 g/L, 10-100 g/L, 0.1-10 g/L or 0.1-5 g/L, preferably 2-10 g/L or 3-7 g/L, or 4-6 g/L, or alternatively between 0.5-1.5 g/L, more preferably between 0.8-1.2 g/L. In the context of the present disclosure, the glycogen preferably relates to glycogen particles having an average diameter of 30-1000 nm, 30-100 nm, 35-80, or 40-60 nm, and preferably relates to oyster glycogen (particles). In addition or alternatively, the glycogen is encapsulated by a (porous) matrix material e.g. having a network with an average mesh size that prevents diffusion of the glycogen, e.g. prevents diffusion of spherical particles having a diameter of 20, 25, 30, 35, 40, 45 nm or more.

The matrix material may be a (porous) hydrogel, e.g. a water-swollen network of hydrophilic polymers, providing a hydrated environment similar to soft tissues. Examples include polyethylene glycol (PEG) hydrogels, alginate, and hyaluronic acid hydrogels. Particularly preferred is a Dex-TA gel composition. The matrix material may also be fibrin glue, in particular in the context of treating bone defects. Fibrin glue, also known as fibrin sealant or fibrin adhesive, is a biological adhesive used in various medical procedures. It is derived from human or animal blood plasma and consists primarily of fibrinogen and thrombin.

Preferably the glycogen is comprised in (aqueous medium in) cavities in the matrix material, wherein the cavities have an average (substantially spherical) volume of at least 0.1 x 108, 0.2 x 108, 0.3 x 108, 0.4 x 10°, 0.5 x 108, 0.6 x 108, 0.7 x 10%, 0.8 x 105, 0.9 x 10°, 1 x 108, 2 x 105, 3 x 108 4 x 108, or within 1 x 108 and 10 x 108, or between 2 x 105 and 6 x 10° um3. The matrix material may comprise at least 1 x 103, 2 x 103, 3 x 103, 4 x 103, 5 x 103, 6 x 103, 7 x 10%, 8 x 10% 9x 103, 10 x 10% 20 x 103, 30 x 103, 40 x 103, 50 x 103, 75 x 103, 100 x 103 or at least 10%, 102, 103, 10%, 105, 108, 107, 108 10° 10' cavities (that may comprise glycogen) per cm? preferably (substantially) evenly distributed. In addition or alternatively, between 0.1-75 vol®%, 1-20 vol%, 10-95 vol%, or 50-90 vol%, more preferably 60-80 vol%, even more preferably 1-20 vol®% of the matrix material relates to glycogen containing cavities. The cavities may for example be formed by core-shell particles. In a preferred embodiment, the glycogen is comprised in a liquid phase in a core-shell particle, preferably liposome or (two) phase separated system. The concentration of glycogen in the (liquid within the) cavities, e.g. within the a core-shell particle, preferably liposome or (two) phase separated system, may be at least 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150 g/L. In the present disclosure, the (porous) matrix material and (porous) scaffold may be the same or different.

The present disclosure also provides for a method for forming tissue (e.g. method for growing tissue or method for forming/growing self-feeding tissue), the method comprising a) seeding a scaffold with cells capable of forming tissue, wherein the scaffold is in combination with glycogen and preferably wherein the glycogen relates to glycogen particles having an average diameter of 30-1000, 30-100, 35-80 or 40-60 nm, preferably oyster glycogen; b) culturing the cells under conditions suitable to grow tissue on and/or in the scaffold.

The method may be ex vivo or in vivo, preferably ex vivo.

In a), the cells can be seeded or delivered in a liquid or gel medium, wherein the total concentration of cells in the medium is optionally between 1x10° to 1x10" cells/mL. Following seeding of the cells in or onto the scaffold, the cells can be allowed to adhere to the (surface of the) scaffold for a period of time prior to placing the seeded scaffold in culture medium.

In the method, e.g. with respect to conditions suitable to grow tissue on and/or in the scaffold, the initial cell seeding density within the scaffold may be varied to promote cell attachment, proliferation, and tissue formation. The optimal density depends on the specific cell type and scaffold characteristics. Sufficient nutrient supply may be considered for cell metabolism and tissue growth. Adequate nutrient diffusion within the scaffold can be facilitated by designing scaffolds with interconnected pore networks and optimizing the culture medium composition and flow conditions. The skilled person may additionally consider oxygen diffusion within the scaffold to avoid, as much as possible, hypoxic conditions that could impair cell viability and tissue formation. Strategies such as scaffold porosity, oxygen-permeable materials, and appropriate perfusion systems can enhance oxygen availability. Maintaining a proper pH level and removing waste products may also be conducive to cell viability and functionality. The culture medium can be refreshed periodically to maintain optimal pH and remove metabolic waste products. In addition, maintaining a suitable temperature and humidity level can be considered for maintaining cell viability and activity. Typically, incubators or bioreactors are used to provide a controlled environment with stable temperature and humidity conditions. For some tissue types, mechanical forces play a role in tissue development and functionality.

Applying appropriate mechanical stimulation, such as mechanical stretching, compression, or shear stress, can help promote tissue maturation and improve mechanical properties.

Biochemical cues, including growth factors, cytokines, and signaling molecules, can be incorporated into the scaffold or delivered through the culture medium to guide cell behavior, differentiation, and tissue formation. Of course, maintaining sterility and preventing contamination is to be considered to avoid adverse effects. Strict aseptic techniques, proper sterilization of scaffolds and culture materials, and regular monitoring of cultures for potential contamination can be considered. Also, the degradation rate (if applicable) of the scaffold should preferably match the tissue formation rate to avoid hindering tissue formation or causing an inflammatory response. The scaffold should ideally degrade in a controlled manner, allowing the gradual replacement with newly formed tissue. Regular monitoring of the tissue culture is important to assess cell viability, proliferation, and tissue development. Techniques such as microscopy, biochemical assays, gene expression analysis, and histological evaluation can be used to monitor the tissue culture and cell viability, proliferation, and tissue formation, growth and maturation.

The present disclosure also provides for preparing a (self-feeding) tissue for implantation into a patient, the method comprising a) providing a (self-feeding) tissue (e.g. autologous, engineered or allogeneic/donor tissue); b) combining the tissue with glycogen, preferably wherein the glycogen relates to glycogen particles having an average diameter of 10-1000 nm or 30-1000 nm, 35-80 nm or 40-60 nm, preferably oyster glycogen. The method preferably is ex vivo.

Also provided is a (self-feeding) tissue structure and/or scaffold for tissue ingrowth, in combination with glycogen according to the present disclosure. For example, the glycogen is preferably present in composition or liquid in an amount of between 0.1-10 g/L or 0.1-5 g/L, preferably between 0.5-1.5 g/L. In addition or alternatively, the glycogen relates to glycogen particles having an average diameter of 30-1000 nm, or 30-100 nm, or 40-60 nm, preferably oyster glycogen. The scaffold may or may not comprise (pre-seeded) cells.

Clauses 1. Use of glycogen for maintaining viability of non-perfused or sub-optimally perfused tissue, the use comprising combining the tissue and the glycogen, wherein the use is ex vivo. 2. Use of glycogen for increasing survival time of a non-perfused or sub-optimally perfused tissue structure, the use comprising combining the tissue structure and the glycogen, wherein the use is ex vivo. 3. Use of glycogen for preventing necrotic core formation in a non-perfused or sub-optimally perfused tissue structure, the use comprising combining the tissue structure and the glycogen, wherein the use is ex vivo. 4. Use of glycogen for increasing production of angiogenic growth factors by non-perfused or sub-optimally perfused tissue, the use comprising combining the tissue and the glycogen, wherein the use is ex vivo. 5. Glycogen for use in the prevention or treatment of tissue implant failure in a patient, wherein in the use the glycogen is in combination with the tissue implant. 6. Glycogen for use in the prevention or treatment of a condition characterized by presence of non-perfused or sub-optimally perfused tissue, preferably hypoxic and/or nutrient deficient tissue.

7. Glycogen for use according to clause 6, wherein the glycogen is administered by bodily injection, preferably intravenous injection, and/or wherein in the use the glycogen is in combination with said tissue.

8. Glycogen for use in the treatment of a tissue pathology in a patient, wherein in the use the glycogen is in combination with replacement tissue and/or scaffold for tissue ingrowth, and wherein the use comprises introducing the replacement tissue and/or scaffold into the patient, and wherein the glycogen relates to glycogen particles having an average diameter of 30-1000 nm, preferably oyster glycogen.

9. Glycogen for use according to any one of clauses 5-8, wherein the tissue is bone tissue, organ tissue, kidney tissue, liver tissue, heart tissue, lung tissue, pancreas tissue, muscle tissue, brain tissue, or skin tissue and/or wherein the tissue is autologous, allogenic, or xenogeneic to the patient.

10. Glycogen for use according to any one of clauses 5-9, wherein the glycogen is encapsulated by a matrix material having a network with an average mesh size that prevents diffusion of the glycogen, preferably the glycogen is comprised in cavities in said matrix material, wherein the cavities have an average volume of at least 100 nm?.

11. Glycogen for use according to any one of clauses 5-10, wherein the glycogen is comprised in a liquid phase in a core-shell particle. 12. Glycogen for use according to any one of clauses 5-11, wherein the glycogen is present in a composition or liquid in an amount of between 0.1-100 g/L, preferably between 1-20 g/L . 13. Ex vivo method for forming tissue, the method comprising a) seeding a scaffold with cells capable of forming tissue, wherein the scaffold is in combination with glycogen and wherein the glycogen relates to glycogen particles having an average diameter of 30-1000 nm, preferably oyster glycogen; b) culturing the cells under conditions suitable to grow tissue on and/or in the scaffold. 14. Method according to clause 13, wherein the glycogen is encapsulated by a matrix material having a network with an average mesh size that prevents diffusion of the glycogen, preferably the glycogen is comprised in cavities in said matrix material, wherein the cavities have an average volume of at least 100 nm?.

15. Method according to any one of clauses 13-14, wherein the glycogen is comprised in a liquid phase in a core-shell particle.

16. Method according to any one of clauses 13-15, wherein the glycogen is present in a composition or liquid in an amount of between 0.1-100 g/L, preferably between 1-20 g/L.

17. Method according to any one of clauses 13-16, wherein the tissue relates to bone tissue, organ tissue, kidney tissue, liver tissue, heart tissue, lung tissue, pancreas tissue, muscle tissue, brain tissue, or skin tissue.

18. Ex vivo method for preparing a tissue for implantation into a patient, the method comprising a) providing a tissue;

b) combining the tissue with glycogen and wherein the glycogen relates to glycogen particles having an average diameter of 30-1000 nm, preferably oyster glycogen.

19. Method according to clause 18, wherein the tissue is bone tissue, organ tissue, kidney tissue, liver tissue, heart tissue, lung tissue, pancreas tissue, muscle tissue, brain tissue, skin tissue and/or wherein the tissue is autologous, allogenic, or xenogeneic to the patient.

20. Method according to any one of clauses 18-19, wherein the glycogen is encapsulated by a matrix material having a network with an average mesh size that prevents diffusion of the glycogen, preferably the glycogen is comprised in cavities in said matrix material, wherein the cavities have an average volume of at least 100 nm?.

21. Method according to any one of clauses 18-20, wherein the glycogen is comprised in a liquid phase in a core-shell particle.

22. Method according to any one of clauses 18-21, wherein the glycogen is present in a liquid in an amount of between 0.1-100 g/L, preferably between 1-20 g/L.

23. Ex vivo tissue structure and/or scaffold for tissue ingrowth, in combination with glycogen, wherein - the glycogen is present in composition or liquid in an amount of between 0.1-100 g/L, preferably between 1-20 g/L; and/or

- the glycogen relates to glycogen particles having an average diameter of 30-1000 nm, preferably oyster glycogen.

24. Ex vivo tissue structure according to clause 23, wherein the tissue relates to bone tissue, organ tissue, kidney tissue, liver tissue, heart tissue, lung tissue, pancreas tissue, muscle tissue, brain tissue, or skin tissue. 25. Ex vivo tissue structure and/or scaffold according to any one of clauses 23-24, wherein the glycogen is encapsulated by a matrix material having a network with an average mesh size that prevents diffusion of the glycogen, preferably the glycogen is comprised in cavities in said matrix material, wherein the cavities have an average volume of at least 100 nm?. 26. Ex vivo tissue structure according to any one of clauses 23-25, wherein the glycogen is comprised in a liquid phase in a core-shell particle. 27. Ex vivo tissue structure according to any one of clauses 23-26, wherein the glycogen is present in a liquid in an amount of between 0.1-100 g/L, preferably between 1-20 g/L.

Brief description of the figures

Figure 1: Sugars, glycogen in particular, maintain hMSC viability and metabolic activity in anoxia the best a) Viability of (MSCs exposed to different metabolites ati) 1 g/L, ij) 0.1 g/L, iii) 0.01 g/L b) and the corresponding metabolic activity.

Figure 2: Exogenous glycogen degradation towards glucose is cell mediated and dose dependent a) Schematic representation of glycogen degradation b) hMSC viability after 72h while GP activity is being blocked representative live dead images c) GP secretion of hMSCs being exposed to different glycogen concentrations after 24h d) GP release over time of (MSCs initially exposed to 0.1 g/L glycogen e) GP secretion from different cell amounts after 24h.

Figure 3: Pro-angiogenic factor secretion of hMSCs after 7 days in anoxia in chemically defined medium containing only a single metabolite a) pro-angiogenic factor secretion of hMSCs exposed to glycogen compared to glucose at different initial concentrations b) fold change of hMSC pro-angiogenic factor secretion compared to 1 g/L initial glycogen concentration c)

VEGF expression of hMSCs exposed to different glycogen concentrations

Figure 4: Glycogen allows for endothelial tube formation at high concentrations a) representative brightfield images of tube-like networks after six hours which were then analyzed for the b) total network length, c) the number of junctions and d) the total segments length using imagej

Figure 5: Glycogen for glucose storage in tissue engineering a) representation of glycogen fate in 1. large pored hydrogels such as GelMA, where it readily diffuses out 2. Small pored hydrogels such as Dex-TA, where the enzymes cannot degrade the glycogen 3. Core-shell particles, where glycogen remains inside a liquid phase and is contained by a small pore hydrogel shell. b) Glycogen retention in hydrogels with large (GelMA) and small (Dex-TA) hydrodynamic radius c) Representative fluorescent images of viable (calcein-AM stained) cells entrapped in Dex-TA gels after seven days of anoxic culture d) Glycogen retention in Dex-TA core-shell particles with different glycogen concentrations and representative fluorescent image of glycogen loaded core-shell particles. Crosslinked Dex-TA stained with ethidium homodimer- 1 (grey) e) i) hMSC viability in presence of glycogen free and loaded core-shell particles in a transwell system ii) and their respective metabolic activity after seven days of anoxic culture

In this document and in its claims, the verb "to comprise" and its conjugations is used in its non- limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one".

EXAMPLES

The following Examples illustrate the different embodiments of the invention.

Example 1. The effect of exogenously added glycogen on hMSC viability and metabolic activity

The purpose of this experiment was to evaluate the effect of exogenous addition of metabolites, particularly sugars e.g. glycogen, on cell behavior.

Methods

Human mesenchymal stem cells (hMSCs) were cultured in aMEM medium containing 10% viv

FBS, 1% v/v, 100 U ml-1 Penicillin, 100 ug mL-1 Streptomycin, 1% v/v 20 mM ASAP, and 1% 100X GlutaMAX (hereafter referred to as ‘proliferation medium’). For the anoxic culture, hMSCs were seeded in proliferation medium at 10000 cells/cm in 24 wells plates. After overnight incubation at 37 °C, the medium was exchanged with nutrient-free DMEM containing 1% v/v 100 U ml’ Penicillin and 100 ug mL™" Streptomycin and the respective metabolite (hereafter referred to as ‘chemically defined medium’). Afterwards, the cells were placed in a hypoxic chamber (XVIVO) containing 0.1% oxygen. Cell viability was assessed using live dead staining i.e. incubation with 1.5 uM calcein AM (live) and 6 pM EthD-1 (dead) and imaged using fluorescent microscopy. Metabolic activity was assessed using PrestoBlue according to the manufacturer's protocol and measured using a plate reader.

Results

As can be seen in Figure 1a-1b, among the tested metabolites, sugars, particularly glycogen, maintained the highest levels of hMSC viability (Figure 1a i-iii) and metabolic activity (Figure 1b iii) in anoxia. Whereas this trend was observed when evaluating higher concentrations (i.e. 1g/L) of the tested metabolites, the positive effect of especially glycogen on hMSC viability and metabolic activity was even more pronounced when using lower concentrations (i.e. 0.1 and 0.01 g/L) of the tested metabolites. The effect of glycogen on hMSC viability is dose dependent.

In addition, it was seen that glycogen (0.5-1.5 g/L particularly 1 g/L) was able to maintain hMSC viability and metabolic activity for a period of 28 days, with enhanced viability and metabolic activity compared to cells exposed to the same amount of glucose.

Conclusion

This example shows that glycogen is surprisingly effective in supporting cell viability and metabolic activity in anoxia.

Example 2. Degradation of exogenously added glycogen

The purpose of this experiment was to assess how exogenously added glycogen is degraded.

Methods hMSCs were seeded in 24 wells plates as described in Example 1. 100 uM of glycogen phosphorylase (GP) inhibitor KB228 was added to the chemically defined medium and refreshed daily. Cell viability was assessed as described in Example 1. Protein secretion of hMSCs into the supernatant was quantified using ELISA kits according to manufacturer's instructions.

Results

As can be seen in Figure 2b, compared to the control (e.g. no glycogen, no GP inhibitor), cell viability decreased drastically upon exposure to a GP inhibitor. However, when combining a

GP inhibitor with more exogenous glycogen, cell viability returned to control levels. Moreover, further analysis of the supernatant showed that the secretion of GP increases with increased glycogen concentration or amount of cells (Figure 2c, 2e). In addition, it was shown that GP releases over time i.e. at least 14 days (Figure 2d).

Conclusion

This example shows that the degradation of exogenous glycogen is cell-mediated and shows that the GP concentration is dependent on the glycogen concentration and amount of cells.

Example 3. Expression of pro-angiogenic factors in anoxia.

Methods hMSCs were seeded in 24 wells plates and protein levels of selected pro-angiogenesis-related factors were determined using ELISA kits, both as previously described. The data was plotted as a fold-change compared to exposure to glucose or a certain glycogen concentration (i.e. 5.5 mM). For VEGF expression, total RNA was isolated using an RNeasy micro kit (Qiagen) and measured using a Nanodrop ND2000 (Thermo Scientific). Complementary DNA (cDNA) was synthesized using the RevertAid H Minus First Strand cDNA Synthesis Kit (Thermo Scientific) and 500 ng of non-amplified total RNA. For each condition a total of 20ng of cDNA was amplified using a Fast Sybr green master mix (Applied Biosciences) and a Corbett rotor gene

QPCR (Qiagen). All steps were performed according to their respective manufacturer’s instructions. Gene expression was normalized on beta-actin (ACTB) expression. ACTB was validated to act as a stable reference gene within the experiments dataset.

Results

As can be seen in Figure 3a, when exposing the cells to glycogen, the secretion of several pro- angiogenic factors was enhanced (when compared to exposure to glucose). Whereas this trend was seen at all evaluated concentrations (i.e. 0.01 g/L, 0.1 g/L and 1 g/L), this trend was even more pronounced for the lower concentrations (i.e. 0.01 g/L, 0.1 g/L) of glycogen (Figure 3b).

As shown in Figure 3c, VEGF expression is increased as the concentration of glycogen is increased.

Conclusions

This example shows that glycogen enhances pro-angiogenic potential of hMSCs in anoxia, which is reflected on both the protein and gene level.

Example 4. The effect of glycogen on endothelial tube formation

Methods

Human umbilical vein endothelial cells (HUVECs) were cultured in EBM-2 medium supplemented with 2% supplement mix (denoted as EGM-2). Matrigel was mixed with EBM-2 medium (1:1 ratio) and 50 pL/ well was added to a 96 wells plate on ice. The gel mixture was crosslinked by incubation at 37 °C for 30 minutes. Subsequently, 15000 HUVECs/ well were seeded on top of the Matrigel by suspending the cells in EGM-2 medium supplemented with glycogen or glucose (between 1 g/L or 10 g/L). After 6 hours, pictures were taken with an inverted microscope and tube formation was analyzed using the Angiogenesis Analyzer plugin in ImageJ.

Results

As can be seen in Figure 4a, glycogen results in increased endothelial tube formation when compared to glucose. Further image analysis showed that glycogen results in tubes with increased total length, amount of junctions and total segment lengths than when exposing the cells to glucose. Whereas this effect was seen at both the lowest and highest tested concentrations (i.e. 1, 4.5 and 10 g/L), the lowest concentration (i.e. 1 g/L), resulted in a significant increase on all mentioned aspects compared to the control.

Conclusion

This example shows that glycogen supports endothelial cell tube formation and that tube formation is enhanced compared to when using glucose.

Example 5. Glycogen retention in hydrogels

Methods

Dex-TA hydrogels were prepared with 10% (w/v) of 15% degree of substitution (DoS) tyramine conjugated dextran (Dex-TA), 3U/mL HRP, 0.03% H202 and 1mg/mL glycogen or glucose as final concentrations. 10 pL of glycogen loaded hydrogels were submerged in 500 pL PBS and incubated at 37 °C. Each timepoint, 250 pL of this mixture was taken and frozen at -20 °C until further analysis. To compensate for the reduction in volume after sampling, 250 uL of fresh

PBS was added back. Glycogen retention was quantified using a glycogen assay kit (abcam) following manufacturer's instructions.

Results

As can be seen in Figure 5b, when glycogen was incorporated in a hydrogel with large hydrodynamic radius (i.e. GelMA), glycogen got released quicker than when glycogen was incorporated in a hydrogel with smaller hydrodynamic radius (i.e. Dex-TA), for which the glycogen concentration in the hydrogel remained close to constant over a period of 14 days.

Conclusion

In hydrogels with small hydrodynamic radius (e.g. Dex-TA), glycogen can be better retained than in hydrogels with larger hydrodynamic radius (e.g. GelMA).

Example 6. Effect of core-shell microparticles comprising glycogen on hMSC viability and metabolic activity

Methods

Core-shell particles were prepared using a microfluidic droplet generator, of which the latter was produced using standard soft-lithography techniques. The microfluidic droplet generator was perfused with aquapel to ensure hydrophobicity of all components. Microdroplets were created by emulsifying a hydrogel precursor solution containing 10% w/v of 15% DoS tyramine conjugated dextran (Dex-TA), 100 g/L glycogen and 80 U/mL horseradish peroxidase (HRP) in

Novec 7500 Engineered Fluid oil containing 2% (w/w) Pico-Surf 1 surfactant at a 10:80 pL min 1 (hydrogel:oil) flow ratio. Flowing the microdroplets through a semipermeable silicone tubing that was submerged in a hydrogen peroxide bath allowed for an outside-in crosslink of the microdroplets. Ultimately, this resulted in the formation of a crosslinked hydrogel shell surrounding non-crosslinked hydrogel pre-cursor solution.

To evaluate the effect of glycogen loaded core-shell particles, transwell experiments were performed. hMSCs were seeded in 24 wells plates as previously described. Instead of adding nutrients directly to the chemically defined medium, 10uL of core-shell microparticles were added to a transwell insert that was added to the respective wells. Cell viability and metabolic activity were assessed as previously described.

Results

As can be seen in Figure 5e i and ii, in general, the addition of glycogen resulted in both enhanced cell viability and metabolic activity. No clear differences in cell viability were observed between cells exposed to glycogen via glycogen loaded core-shell particles and direct supplementation in the medium. Interestingly, metabolic activity was enhanced for cells exposed to glycogen loaded core-shell particles.

Conclusion

Providing glycogen in cavities such as core-shell particles may allow better degradation of the glycogen.

Example 7. Effect of glycogen on tissue implants

Methods

Tissue viability is assessed using live dead staining i.e. incubation with 1.5 uM calcein AM (live) and 6 uM EthD-1 (dead) and imaged using fluorescent microscopy. Necrotic core formation is assessed by visual inspection. Implant acceptance is assessed by biomarker analysis of biomarkers related to inflammation, tissue healing, and integration in the vicinity of the implant (cytokines, growth factors, cellular markers that indicate the local tissue response and the level of implant acceptance, fa. IL1, IL6, TNF-a and MMPs). In vivo angiogenesis is determined by in vivo imaging.

Results

As can be seen in Table 1 below, the addition of glycogen (in particular larger particles and/or in concentration of 0.5-1.5 g/L) results in enhanced survival time, better in vivo implant acceptance, and better in vivo angiogenesis.

Table 1. Effect of glycogen on tissue implants

Type of | Survival over prolonged | In vivo implant | Presence of in vivo implant necrotic core formation)

Bone Tissue in combination with Tissue in combination with Tissue in combination with tissue Solid peroxide - - Solid peroxide - - Solid peroxide - -

Starch based hydrogel - - Starch based hydrogel - - Starch based hydrogel - -

Glycogen > 30 nm ++ Glycogen > 30 nm ++ Glycogen > 30 nm ++

Glycogen < 30 nm 0.25 g/L + | Glycogen < 30 nm 0.25 g/L + | Glycogen < 30 nm 0.25 g/L +

Glycogen < 30 nm 1 g/L ++ Glycogen < 30 nm 1 g/L ++ Glycogen < 30 nm 1 g/L ++

Glycogen > 30 nm in coreshell | Glycogen > 30 nm in coreshell | Glycogen > 30 nm in coreshell particles +++ particles +++ particles +++

Glycogen > 30 nm in coreshell | Glycogen > 30 nm in coreshell | Glycogen > 30 nm in coreshell ee:

Kidney When in combination with When in combination with When in combination with tissue Solid peroxide - - Solid peroxide - - Solid peroxide - -

Starch based hydrogel - - Starch based hydrogel - - Starch based hydrogel - -

Glycogen > 30 nm ++ Glycogen > 30 nm ++ Glycogen > 30 nm ++

Glycogen < 30 nm 0.25 g/L + | Glycogen <30 nm 0.25 g/L + | Glycogen < 30 nm 0.25 g/L +

Glycogen < 30 nm 1 g/L ++ Glycogen < 30 nm 1 g/L ++ Glycogen < 30 nm 1 g/L ++

Glycogen > 30 nm in coreshelf | Glycogen > 30 nm in coreshell | Glycogen > 30 nm in coreshell ==

Conclusion

Providing glycogen allows for enhanced survival time, better in vivo implant acceptance, and better in vivo angiogenesis, in particular when the glycogen is provided as larger particles (< 30 nm) and/or in a concentration of 0.5-1.5 g/L. Providing the glycogen in cavities such as in core-shell particles and preferably in a hydrogel (so as to prevent diffusion) further enhances survival time, in vivo implant acceptance, and in vivo angiogenesis.

Claims

Conclusions

1. Use of glycogen to maintain viability of non-perfused or sub-optimally perfused tissue, the use comprising combining the tissue and the glycogen, the use being ex vivo.

2. Use of glycogen to prolong survival time of a non-perfused or sub-optimally perfused tissue structure, the use comprising combining the tissue structure and the glycogen, the use being ex vivo.

3. Use of glycogen to prevent necrotic core formation in a non-perfused or sub-optimally perfused tissue structure, the use comprising combining the tissue structure and the glycogen, the use being ex vivo.

4. Use of glycogen to enhance production of angiogenic growth factors by non-perfused tissue, the use comprising combining the tissue and the glycogen, the use being ex vivo.

5. Glycogen for use in the prevention or treatment of tissue implant failure in a patient, wherein the glycogen is in association with the tissue implant.

6. Glycogen for use in the prevention or treatment of a condition characterised by the presence of non-perfused or sub-optimally perfused tissue, preferably hypoxic and/or nutrient-deficient tissue.

7. Glycogen for use according to claim 6, wherein the glycogen is administered by body injection, preferably intravenous injection, and/or wherein the glycogen is in combination with said tissue when used.

9. Glycogen for use according to any one of claims 5 to 8, wherein the tissue is bone tissue, organ tissue, kidney tissue, liver tissue, heart tissue, lung tissue, pancreatic tissue, muscle tissue, brain tissue, or skin tissue and/or wherein the tissue is autologous, allogeneic or xenogeneic to the patient.

10. Glycogen for use according to any one of claims 5 to 9, wherein the glycogen is encapsulated by a matrix material having a network with an average mesh size which prevents diffusion of the glycogen, preferably the glycogen is comprised in cavities in said matrix material, the cavities having an average volume of at least 100 nm?.

11. Glycogen for use according to any one of claims 5 to 10, wherein the glycogen is comprised in a liquid phase in a core-shell particle.

12. Glycogen for use according to any one of claims 5 to 11, wherein the glycogen is present in a composition or liquid in an amount between 0.1-100 g/L, preferably between 1-20 g/L.

13. Ex vivo method for forming tissue, the method comprising a) seeding a scaffold with cells capable of forming tissue, the scaffold being in combination with glycogen and the glycogen being glycogen particles having an average diameter of 30-1000 nm, preferably oyster glycogen; b) culturing the cells under conditions suitable for growing tissue on and/or in the scaffold.

14. A method according to claim 13, wherein the glycogen is encapsulated by a matrix material having a network with an average mesh size which prevents diffusion of the glycogen, preferably the glycogen is comprised in cavities in said matrix material, the cavities having an average volume of at least 100 nm?.

15. The method of any one of claims 13 to 14, wherein the glycogen is contained in a liquid phase in a core-shell particle.

16. A method according to any one of claims 13-15, wherein the glycogen is present in a composition or liquid in an amount between 0.1-100 g/L, preferably between 1-20 g/L.

17. The method of any one of claims 13 to 18, wherein the tissue is bone tissue, organ tissue, kidney tissue, liver tissue, heart tissue, lung tissue, pancreatic tissue, muscle tissue, brain tissue, or skin tissue.

18. An ex vivo method of preparing a tissue for implantation in a patient, the method comprising a) providing a tissue; b) combining the tissue with glycogen and wherein the glycogen refers to glycogen particles having an average diameter of 30-1000 nm, preferably oyster glycogen.

19. The method of claim 18, wherein the tissue is bone tissue, organ tissue, kidney tissue, liver tissue, heart tissue, lung tissue, pancreatic tissue, muscle tissue, brain tissue, skin tissue and/or wherein the tissue is autologous, allogeneic or xenogeneic to the patient.

20. A method according to any one of claims 18 to 19, wherein the glycogen is encapsulated by a matrix material having a network with an average mesh size that prevents diffusion of the glycogen, preferably the glycogen is comprised in cavities in said matrix material, the cavities having an average volume of at least 100 nm?.

21. The method of any one of claims 18 to 20, wherein the glycogen is contained in a liquid phase in a core-shell particle.

22. A method according to any one of claims 18 to 21, wherein the glycogen is present in a liquid in an amount between 0.1-100 g/L, preferably between 1-20 g/L.

23. Ex vivo tissue structure and/or scaffold for tissue ingrowth, in combination with glycogen, wherein - the glycogen is present in composition or liquid in an amount between 0.1-100 g/L, preferably between 1-20 g/L; or - the glycogen relates to glycogen particles with an average diameter of 30-1000 nm, preferably oyster glycogen.

24. The ex vivo tissue structure of claim 23, wherein the tissue relates to bone tissue, organ tissue, kidney tissue, liver tissue, heart tissue, lung tissue, pancreatic tissue, muscle tissue, brain tissue, or skin tissue.

25. Ex vivo tissue structure and/or scaffold according to any of claims 23-24, wherein the glycogen is encapsulated by a matrix material having a network with an average mesh size that prevents diffusion of the glycogen, preferably the glycogen is comprised in cavities in said matrix material, the cavities having an average volume of at least 100 nm3.

26. Ex vivo tissue structure according to any one of claims 23 to 25, wherein the glycogen is contained in a fluid phase in a core-shell particle.

27. Ex vivo tissue structure according to any one of claims 23 to 28, wherein the glycogen is present in a fluid in an amount between 0.1-100 g/L, preferably between 1-20 g/L.