Polysaccharide- and collagen derivative-based microcarrier

Abstract

The current invention pertains to method for producing a microcarrier, and a microcarrier optionally obtainable by the method. The microcarrier comprises an ionically crosslinkable polysaccharide crosslinked with a collagen derivative, preferably characterized by covalent binding between the polysaccharide and the collagen derivative. The microcarrier is particularly suitable for large scale expansion of adherent cells in applications biologics manufacturing, regenerative medicine, cell therapy or cultivated meat or fish production.

Description

[0001] P36955PC00 / MCR

[0002] Title: Polysaccharide- and collagen derivative-based microcarrier

[0003] TECHNICAL FIELD

[0004] The present disclosure relates to a microcarrier suitable for culture of adherent cells, and a method by which the microcarrier is obtainable.

[0005] BACKGROUND OF THE INVENTION

[0006] In several industries such as biologies manufacturing, regenerative medicine, cell therapy, or cultivated meat or fish production, there is a growing need for efficient large-scale cell culture methods.

[0007] The microcarrier method is one particularly known method for large-scale production of cells. The microcarrier method involves growing adherent cells on the surface of small, spherical particles typically ranging from 100 to 500 micrometers in diameter. After initial cell attachment, the microcarriers are suspended in a 3D environment such as by stirring in a bioreactor or flask. Because microcarriers provide a high surface area-to-volume ratio, large numbers of cells can be cultured and expanded in a relatively small volume, which makes this method space-efficient.

[0008] Microcarriers sometimes comprise one, but typically two or more types of polymers (e.g. biomaterials, synthetic polymers, glass), which together better leverage the desirable properties of the microcarrier, such as mechanical strength, degradability, biocompatibility and bioactivity. Natural polymers have been favored for their biocompatibility and ability to mimic the extracellular matrix, for example, by presenting natural cell adhesive moieties such as arginyl-glycyl- aspartic acid (RGD) peptide sequences and / or due to their biodegradable nature, for example, via enzymatic degradation by enzymes such as matrix metalloproteinase and / or collagenase.

[0009] Alternatively, microparticle -shaped substrates that can bind biological entities are used in the field to capture, activate, purify, and release these biological entities on demand. Although it is possible for certain applications that the microcarriers including attached cells are together present in the final product (e.g., in an in body implantable or edible cell / microcarrier-based product), it is more common that the cells will need to be harvested when reaching a desired density atop the microcarrier substrate, followed by their reseeding on fresh microcarriers to allow further expansion or application of the harvested cells in the final microcarrier-free application. For cell harvesting, dissolvable (e.g., degradable) microcarriers are typically favored to facilitate the efficient release of cells without the need for harsh enzymatic treatments or mechanical disruption. Rather, it is preferred that the microcarrier can be easily dissolved using common cell culture agents such as a chelating agent like EDTA and / or an enzyme like trypsin.

[0010] EP3983481A1 discloses calcium-crosslinked polygalacturonic acid (PGA) microcarriers which have an improved stability and integrity under culture conditions. However, the improved stability and rigidity comes at the cost of a microcarrier which cannot be readily dissolved using commonly used cell dissociation agents. Rather, the method relies on the enzyme pectinase to allow breakdown of the microcarrier matrix. The method furthermore inevitably results in presence of galacturonic acid monomers and pectinase as by-products of PGA microcarrier digestion. The use of pectinase is considered to be more complex compared to use of common cell culture (chelating) agents, since it can be more difficult to regulate pectinase activity precisely. Moreover, treatment with pectinase may introduce more variability on cell functionality or viability. In addition, pectinase is not native to humans and is associated with regulatory hurdles to overcome, e.g. important in case of clinical translation.

[0011] EP4339283A1 discloses alginate-gelatin microcarriers obtained by immersing gel microparticles of alginate containing a divalent or higher-valent cation in a solvent containing a gelatin, allowing the gelatin to infiltrate inside the alginate gel microparticle, and finally removing excess and unnecessary gelatin outside the microcarrier. Because the chelating agent ethylenediaminetetraacetic acid (EDTA) is used to dissolve the alginate-gelatin microcarriers, EP4339283A1 furthermore discloses to avoid excessive crosslinking of the microcarrier components, as it otherwise becomes unlikely to be dissolved. The reduced dissolvability is presumably a downside of gelatin being substantially present inside the alginate microparticle.

[0012] Based on the previous, a major challenge in the field is to provide a microcarrier which has a high stability and mechanical properties, but without putting too much practical limitations on the ease of digestibility of the microcarrier with common cell culture reagents such as chelating agents (e.g. EDTA) and proteolytic enzymes, if needed.

[0013] In certain cases, microcarriers may be used in the manufacturing process for a therapeutic product (e.g. in expansion and differentiation of cells for in vivo use), or may even be part of the final therapeutic product (e.g. in encapsulated cell therapy). Accordingly, the microcarriers may be treated as components of an advanced therapy medicinal product (ATMP), combination products, and the like, under regulatory frameworks such that they typically should meet higher standards in terms of biocompatibility and safety.

[0014] To achieve such high standards, the microcarrier may be subjected to further purification and / or washing treatments to remove contaminants such natural contaminants in the source materials and / or contaminants introduced into the microcarriers during the manufacturing process. Such contaminants may include elemental impurities classified as Class 1, 2A, 2B or 3 elemental purities by the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) (Guideline for elemental impurities Q3D(R2), Adopted on 26 April 2022) and (toxic) heavy metals which may be naturally found in polysaccharides such as alginate (e.g. lead, cadmium, mercury, arsenic). Purification steps may for example include one or more of removing unbound salt and ions (e.g. excess from crosslinking), removing unwanted metal contaminants via chelation / ion exchange, removing residual reagents and reducing endotoxin and microbial burden.

[0015] Since the further purification may be performed on the final microcarrier, it is a further desire to achieve microcarriers which remain as stable as possible during said purification, preferably being more resistant towards chelation treatment.

[0016] It is an object of the present invention to provide for new and improved microcarriers suitable to efficiently cultivate adherent cells and / or that act as bodies that can capture and release biologic molecules such as cells, cytokines, proteins, peptides, and nucleotides. A particular object of the present invention is to provide a microcarrier which has a high stability and integrity, but can nevertheless be readily dissolved (e.g. by exposing to chelating agents, proteolytic enzymes) if required.

[0017] It is furthermore an object of the present invention to propose methods to produce such microcarriers as well as the uses of such microcarriers.

[0018] SUMMARY OF THE INVENTION

[0019] The object of the invention can be achieved by providing a microcarrier having a core-shell structure, wherein the ionically crosslinkable polysaccharide (e.g. alginate) is the predominant polymer in the core and wherein the collagen derivative (e.g. gelatin) forms a shell layer (i.e. coating). Said core-shell structure allows for a microcarrier with high stability and integrity (due to the core particle), provides excellent cell adherence and growth (due to the outer collagen derivative layer). Yet, the microcarrier is easily dissolvable if needed. Without being bound by theory, the inventors consider that the improved solubility is rendered by the fact that the dissolving agent can easily access the collagen derivative and / or does not have to reach the microparticle core by diffusion.

[0020] Among others, it was found that the core-shell structure can be achieved by initially providing a collagen derivative with a relatively high molecular weight (e.g. >80 kDa, preferably >100 kDa, more preferably >150 kDa) and / or positive charge (e.g. isoelectric point of 7-9). As such, the diffusion of the collagen derivative into the core particle can be controlled and a shell layer of collagen derivative is formed.

[0021] Further, or in addition, the object of the current invention can be achieved by heat-treating the microcarriers, thereby allowing a Maillard reaction between (functional) groups of an ionically crosslinkable polysaccharide (e.g. alginate) and a collagen derivative (e.g. gelatin). The heattreatment step may enhance non-covalent and / or covalent interactions in microcarrier functional groups which improves the functionality of the microcarrier.

[0022] Wet heating is advantageous over a dry heating method, as wet heating may lead to a more desirable Maillard reaction (and Maillard reaction products) and / or may allow for lower temperatures / time, in particular as compared to dry heating or sterilization methods. A lower temperature / time can also potentially protect proteins or other bioactive agents further present in the microcarrier.

[0023] Pressurized steam heating (e.g. autoclaving) of the microcarriers was also found to be particularly suitable. It was surprisingly found to reduce lipopolysaccharide (i.e. endotoxin) content / activity in the microcarriers, and furthermore, a sterile product is immediately also achieved, hence removing the need of additional sterilization step before start of cell culture.

[0024] In particular in the case of core-shell structure (e.g. polysaccharide core and collagen derivative coating) disclosed herein, it is found to be advantageous to provide for improved crosslinking between the core layer and the shell structure, as this leads to a stable shell which is yet easily dissolvable if needed. An improved crosslinking may be achieved by improved non-covalent and / or covalent interactions. As a result, the shell layer appears protected from deterioration under culture condition and / or by cells over time and better supports long-term cell culture and cell adhesion.

[0025] The heat-treatment, after a core-shell structure microcarrier is formed, may furthermore allow fragmentation of the collagen derivative into shorter (poly)peptides, thereby decreasing the molecular weight and / or otherwise changing the functional or structural properties thereof. In particular, the reduced molecular weight may allow for improved cell adhesion or growth, depending on the cell type or application. The reduced molecular weight may provide for a higher density of binding moieties (e.g. positive charges or RGD), potentially also presentation of more / different moieties that are otherwise ‘shielded’ in the larger (poly)peptides or non-hydrolyzed collagen derivative. The fragmentation may also provide for a change in surface roughness of the microcarrier, which can at least in part contribute to the aforementioned effects.

[0026] Irrespective of whether a core-shell structure is applied, it is found to be advantageous to chemically crosslink the collagen derivative (e.g. with glutaraldehyde), in particular when subsequently inducing the Maillard reaction. Without being bound by theory, the chemical crosslinking prior to inducing the Maillard reaction, may provide for a more desirable Maillard reaction (and Maillard reaction products) and / or may help prevent the collagen derivative from detaching or separating from the microcarrier, in particular during or after the Maillard reaction.

[0027] In a preferred embodiment, the crosslinked microcarrier of the present disclosure is hence characterised by covalent binding between (e.g. carbonyl) in the ionically crosslinkable polysaccharide (e.g. alginate) and amino groups in the collagen derivative (e.g. gelatin) leading to improved stability.

[0028] The further crosslinking under appropriate conditions provides a microcarrier which is stable yet highly digestible. The microcarrier of the present invention can be readily dissolved using a common chelating agent such as EDTA in combination with a protease such as trypsin and the like. In addition, the microcarrier of the present disclosure achieves an improved cellattachment and proliferation as a sign of excellent biocompatibility.

[0029] The present findings are surprising, considering that the art teaches that excessive crosslinking or covalent binding of the microcarrier constituents is to be avoided in view of otherwise reduced dissolvability (e.g. with a chelating agent) and that therefore excessive further crosslinking is not preferable. Moreover, the art typically teaches to provide the collagen derivative in the core of the microcarrier in conjunction with the ionically crosslinkable polysaccharide, however present findings show that this may come at the cost of reduced dissolvability and otherwise reduced functionality since the collagen derivative is not readily accessible to cells.

[0030] The microcarrier of the invention not only surprisingly combines high stability and integrity with controlled digestibility, but if necessary can be produced to meet further biocompatibility and safety requirements such as applicable to ATMPs or combination products, or other in vivo use. In particular, it was found that an improved stability and integrity of the microcarrier renders it more stable during further purification treatments, even when involving chelating agents such as EDTA. Accordingly, the final microcarrier of the invention may be simply subjected to one or more purification steps (e.g. with a chelating agent) and washing after their preparation. For example, purification of the microcarrier after its preparation may circumvent the need for (more complex and time-consuming) ultrafiltration and / or dialysis treatments of the raw materials.

[0031] The present inventors surprisingly found that the crosslinking method disclosed herein leads to overall improved performed performance of cells when cultured on the microcarrier. Without being bound by theory, the present inventors consider that the specific crosslinking between the ionically crosslinkable polysaccharide and the collagen derivative herein may provide for more physiological conditions for cell growth and function. In particular, the formation of Maillard reaction products appears associated with a beneficial anti-inflammatory and / or anti-oxidant effect of the microcarriers.

[0032] The microcarrier of the invention is particularly suitable for use in culturing cells but in addition or alternatively can be suitable for e.g. for drug delivery, immobilization or encapsulation of active ingredients, as biosensor, for cryopreservation (i.e. cell freeze storage), and the like.

[0033] In an aspect, the current invention pertains to a method for producing a microcarrier, comprising a) providing a microparticle comprising ionically crosslinkable polysaccharide in a solvent containing a collagen derivative to obtain a composite microparticle, and b) preferably providing further crosslinking, more preferably heat-treating the composite microparticle such as to allow a Maillard reaction between groups of the ionically crosslinkable polysaccharide and the collagen derivative, and preferably obtaining a crosslinked microcarrier characterized by covalent binding between the ionically crosslinkable polysaccharide and the collagen derivative. In an aspect, the current invention pertains to a microcarrier, obtainable by the method disclosed herein.

[0034] In an aspect, the current invention pertains to a microcarrier, optionally obtainable by the method disclosed herein, wherein the microcarrier comprises ionically crosslinkable polysaccharide crosslinked with a collagen derivative, and preferably characterized by covalent binding between the ionically crosslinkable polysaccharide and the collagen derivative.

[0035] In an aspect, the current invention pertains to a plurality of microcarriers as disclosed herein, provided as a dry powder or in a liquid suspension and / or a slurry.

[0036] In an aspect, the current invention pertains to a cell-laden microcarrier, comprising the microcarrier disclosed herein and one or more adherent cells on the surface of the microcarrier.

[0037] In an aspect, the current invention pertains to a use of the microcarrier, plurality of microcarriers, or the cell-laden microcarrier disclosed herein, in an application selected from the group consisting of pharmaceutical production, vaccine production, gene therapy, regenerative medicine, cell therapy, biologies manufacturing such as vaccine, antibody or recombinant protein production, viral vector production, cosmetic testing, toxicology testing, 3D cell culture, extracellular vesicle and / or exosome production, bioreactor culture and cultivated meat or fish production.

[0038] In an aspect, the present invention pertains to a method for culturing of adherent cells, comprising contacting one or more adherent cells with the microcarrier disclosed herein in an aqueous medium, thereby allowing / inducing cell adhesion and culture on the microcarrier. DETAILLED DESCRIPTION OF THE INVENTION

[0039] Method for producing microcarrier and microcarrier obtainable thereby

[0040] The present disclosure pertains to a method for producing a microcarrier, e.g. for culture of adherent cells, comprising a) providing a microparticle comprising ionically crosslinkable polysaccharide in a solvent containing a collagen derivative to obtain a composite microparticle, and b) preferably providing further crosslinking, more preferably heat-treating the composite microparticle such as to allow a Maillard reaction between groups of the ionically crosslinkable polysaccharide and the collagen derivative, wherein the heat-treating (i.e. heat-treatment) thereby preferably results in a crosslinked microcarrier characterized by covalent binding between the ionically crosslinkable polysaccharide and the collagen derivative.

[0041] Ionically crosslinkable polysaccharide, collagen derivative

[0042] The term “ionically crosslinkable polysaccharide” in the context of the present invention (herein also referred to interchangeably as “polysaccharide microparticle”) means a polysaccharide which can be crosslinked by a divalent or higher-valent cation (e.g. trivalent cation), preferably a divalent cation. The “ionically crosslinkable polysaccharide” encompasses polysaccharides which can gel via an egg-box mechanism and / or via helixhelix aggregation. The ionically crosslinkable polysaccharide in the context of the present invention is preferably one or more of alginate, pectin, galacturonic acid and carrageenan. The ionically crosslinkable polysaccharide is preferably a polysaccharide capable of providing reducing sugars and / or form carbonyl groups (C=O) upon degradation, thereby allowing a Maillard reaction with amino groups (e.g. in gelatin). The ionically crosslinkable polysaccharide is preferably alginate and / or pectin. The pectin disclosed herein is preferably low methoxyl pectin. The ionically crosslinkable polysaccharide is most preferably alginate. The term “crosslinkable” can refer to polymer (e.g. polysaccharide) either in crosslinked or non-crosslinked state (i.e. before actual crosslinking). Hence, after the crosslinking, the term “crosslinkable” can also be interchanged with the term “crosslinked”.

[0043] Alginate is preferred in the context of the present invention, since it can among others, efficiently crosslink with collagen derivatives, such as gelatin through the Maillard reaction (as further described below). Alginate has other advantages such as e.g. high biocompatibility and tuneable crosslinking and mechanical properties. Pectin contains sugars (e.g. galactose, rhamnose, arabinose), which upon degradation may however also provide reducing sugar residues which can react with amino groups in a Maillard-type reaction.

[0044] Alginic acid is the linear polysaccharide composed of mannuronic acid and guluronic acid and which is the natural form of the polysaccharide found in the cell walls of brown algae. “Alginate” refers to the salts and esters of alginic acid and therefore essentially is the ionic form of alginic acid. The alginate as disclosed herein is preferably one or more of sodium alginate, calcium alginate, potassium alginate and magnesium alginate, preferably sodium alginate or calcium alginate.

[0045] The alginate disclosed herein is preferably ionically crosslinked with a divalent or higher cation. The present inventors found that the ionic crosslinking may protect the alginate network during the heat-treatment process and provide a further enhanced microcarrier. The divalent or higher-valent cation can be for instance one or more selected from the group consisting of a calcium ion, a magnesium ion, a divalent iron ion, a trivalent iron ion, a divalent silver ion, a trivalent silver ion, a copper ion, a zinc ion, a selenium ion, and other trace element ions essential for humans, and preferably a calcium ion. In a preferred embodiment, the alginate is ionically crosslinked with one or more of Ca2+, Mg2+, Sr2+and Ba2+.

[0046] The term “collagen derivative” as used in the context of the present invention means a product obtained from collagen through one or more degrees of processing, in particular comprises hydrolysis The collagen derivative is preferably a hydrolyzed or partially hydrolyzed form of collagen. The collagen derivative is most preferably gelatin or hydrolyzed gelatin. In addition or alternatively, the collagen derivative may be collagen hydrolysate.

[0047] The collagen derivative, e.g. as provided in step a) of the method, is most preferably gelatin (i.e. partially hydrolysed collagen), more preferably high molecular weight gelatin. The present inventors consider that the advantage of the larger molecular weight of gelatin compared to hydrolysed collagen derivatives is that the gelatin can be deposited substantially at the outside of the polysaccharide microparticle (i.e. as core-shell structure or coating), as further elaborated on in present disclosure.

[0048] The term “collagen” in the context of the current invention means an amino acid sequence comprising a repeating (Gly-X-Y) sequence, preferably comprising at least 2, 3, 4, 5, 10, 20, 50, 100, 200, 300, or 400 sequences containing the sequence Gly-X-Y, where X and Y are an amino acid residue independently chosen from each other, but X and / or Y are more preferably proline. The “collagen” preferably has a sequence found in native collagen in one or more animal species. In addition or alternatively, the “collagen” can mean a full-length sequence or fragment or subunit thereof of (native) collagen, preferably one or more of collagen types I - XXVII, more preferably one or even more of type I, II, III, V, or X collagen, even more preferably one or more of type I, II or III collagen. For example, the term “collagen” may refer to an alpha-1 (I), alpha-2(l), alpha-1 (II) or alpha-1 (I II) chain, or a fragment thereof. The term “collagen” encompasses the triple helix structure as formed by three subunits as present in native collagen.

[0049] The term “gelatin” in the context of the current invention means a mixture of water-soluble proteins derived from partial hydrolysis of collagen. The gelatin of the invention may be type A gelatin, type B gelatin or a combination thereof. The hydrolysis may be performed by any acid or alkali condition, or by enzymatic hydrolysis, as known in the art. Depending on the physical and chemical methods of the partial hydrolysis, the molecular weight of the peptides can fall within a broad range. The partial hydrolysis provides the gelatin the ability to hold water and its gelling capacity, typically distinguishing it from hydrolysed collagen, i.e. product obtained by further hydrolysis of collagen. The term "gelatin” in the context of the current invention encompasses a modified gelatin, e.g. a chemically modified gelatin. The term “modified gelatin” (also referred to as “functionalized gelatin”) in the context of the current invention encompasses gelatin modified with a chemical group or moiety (e.g. a methacryloyl group or any other chemical moiety as disclosed herein in the context of functionalised gelatins) attached to at least one amine group, at least one hydroxyl group, at least one carboxyl group and / or at least one phenol group of the gelatin. In a preferred embodiment, the gelatin is gelatin methacryloyl (i.e. GelMA) or gelatin desaminotyrosine (i.e. GelDAT), or a combination thereof.

[0050] In an embodiment, the collagen derivative is a ’’type A” collagen derivative, i.e. a material obtained by a process involving an acid-extraction process. In an embodiment, the collagen derivative is a ’’type B” collagen derivative, i.e. a material obtained by a method involving an alkaline-extraction process.

[0051] In an embodiment, the collagen derivative, preferably gelatin, is high molecular weight. In an embodiment, the collagen derivative, preferably gelatin, provided in step a) of the method disclosed herein has a weight-average molecular weight of more than 100 kilodalton (kDa), preferably more than 150 kDa, even more preferably more than 200 kDa, e.g. 100-300 kDa, preferably 150-200 kDa. In an embodiment, the collagen derivative, preferably gelatin, is low molecular weight.ln a preferred embodiment, the collagen derivative, preferably gelatin, has a weight-average molecular weight ranging from 50 - 200 kDa, preferably 75-150 kDa, more preferably 1 DO- 125 kDa.

[0052] In embodiments of the partially hydrolysed collagen (e.g. gelatin), it has a molecular weight of at least 40 kDa, 50 kDa, 60 kDa, 70 kDa, 80 kDa, 90 kDa, 95 kDa, 100 kDa, 110 kDa, 120 kDa, 130 kDa, 140 kDa, 150 kDa or 160 kDa and / or less than 240 kDa, 230 kDa, 220 kDa, 210 kDa, 200 kDa, 190 kDa, 180 kDa, 170 kDa, 160 kDa, 150 kDa, 140 kDa, 130 kDa, 120 kDa, 110 kDa, 100 kDa, 90 kDa, 80 kDa or 70 kDa.

[0053] Gelatin does not constitute a uniform protein molecule, but comprises a variable amount of protein molecules of variable length. The molecular weight (i.e. MW) in the context of the current invention is preferably the weight-average molecular weight.

[0054] A preferred method of determining the molecular weight of the collagen derivative is by a size exclusion high performance liquid chromatography (HPLC) technique known in the art (e.g. see Olijve et.al. 2000. Journal of Colloid and Interface Science 243: 476-482). In addition or alternatively, a way of measuring the (average) molecular weight of gelatin is by high performance size exclusion chromatography (HPSEC). The following protocol is a preferred HPSEC protocol:

[0055] The Agilent HPLC, 1260 Infinity series (G1316A, G1329B, G1311C, G1315D) with a TSKgel SWXL precolumn und a G2000SWXL column (Tosoh Bioscience) is used. Analysis is performed with the WinGPC software (PSS). The eluent is 100 mM phosphate buffer pH 5.3. Samples are eluted from the column (e.g. 0.5 mL / min, isocractic) and monitored with UV detection (e.g. 214 nm, analysis time: 40 min per injection + 180 min equilibration). Calibration is performed with the Narrow Calibration Standard (Low FILK).

[0056] In an embodiment, the gelatin has a high bloom, e.g. defined by a bloom value 200-370 grams, such as 250-300 grams.

[0057] The present inventors found that is the microcarrier is further improved if the collagen derivative is in a positively charged state and / or has an isoelectric point reflecting of 7-9 (i.e. having a net charge of zero at pH 7-9), e.g. in the range 7.5-8.5. In embodiments, the collagen derivative has an isoelectric point of at least 6.0. In embodiments, the collagen derivative has an isoelectric point of at least 6.5. In embodiments, the collagen derivative has an isoelectric point of at least 7.0. In embodiments, the collagen derivative has an isoelectric point of no more than 10.0. In embodiments, the collagen derivative has an isoelectric point of no more than 9.5. In embodiments, the collagen derivative has an isoelectric point of no more than 9.0. In addition or alternatively, the collagen derivative is preferably of type A.

[0058] Preferably, when obtaining the composite microparticle, the ionically crosslinkable polysaccharide and the collagen derivative have an opposite net charge when contacted, i.e. the polysaccharide has a net positive charge and the collagen derivative has a net negative charge, or vice versa. More preferably, when obtaining the composite microparticle, the ionically crosslinkable polysaccharide has a net negative charge and the collagen derivative preferably has a net positive charge when contacted. In an embodiment, the ionically crosslinkable polysaccharide has a net positive charge when contacted with the collagen derivative. In an embodiment, the ionically crosslinkable polysaccharide has a net neutral charge when contacted with the collagen derivative. In an embodiment, the ionically crosslinkable polysaccharide has a net negative charge when contacted with the collagen derivative. In an embodiment, the collagen derivative has a net positive charge when contacted with the ionically crosslinkable polysaccharide. In an embodiment, the collagen derivative has a net neutral charge when contacted with the ionically crosslinkable polysaccharide. In an embodiment, the collagen derivative has a net negative charge when contacted with the ionically crosslinkable polysaccharide.

[0059] The ionically crosslinkable polysaccharide and the collagen derivative can interact with each other via multivalent electrostatic interactions, which can be described by a charge ratio (Z) defined as:

[0060] [positive charges on the collagen derivative] [negative charges on the polysaccharide] wherein:

[0061] Z = 1 means a stoichiometric neutral complex, which typically leads to coacervate formation, Z > 1 means excess positive complex, Z < 1 means excess negative complex.

[0062] For example, gelatin Type A (i.e. acid hydrolyzed gelatin) typically has an isoelectric point of 7-9. At a pH below said isoelectric point 7-9 (e.g. at pH 6 or lower), most basic residues are fully protonated and the gelatin is positively charged. At pH above the isoelectric point 7-9 (e.g. at pH 10 or higher), the carboxyl groups in gelatin dominate, the fraction of protonated basic residues is very small, and the gelatin is negatively charged. In comparison, alginate is a polysaccharide of mannuronic acid and guluronic acid, wherein each monomer has a carboxyl group (-COOH). Since carboxyl groups are weak acids, alginate is almost always negative under physiological and typical experimental pH ranges. Accordingly, below pH 3 -

[0063] 3.5, alginate is mostly neutral; above pH 4, alginate becomes increasingly negatively charged; at pH 6-11, alginate is strongly negatively charged.

[0064] In view of the previous, at pH below the isoelectric point of the collagen derivative, the charge ratio Z is greater than 0. The opposite charges of the polysaccharide and collagen derivative allow for electrostatic attraction and complex / coacervate formation. At pH above the isoelectric point of the collagen derivative, the charge ratio Z is smaller than 0 and the negative charges allow for electrostatic repulsion or weak gelatin-alginate interaction.

[0065] In preferred embodiments, the ionically crosslinkable polysaccharide and the collagen derivative are contacted in a solution such that the charge ratio Z is equal or larger than 0, when forming the microcarrier.

[0066] In preferred embodiments, the ionically crosslinkable polysaccharide and the collagen derivative are contacted in a solution with a pH below the isoelectric point of the collagen derivative. In an embodiment, the pH of the solution is at least 4.0. In an embodiment, the pH of the solution is at least 4.5. In an embodiment, the pH of the solution is at least 5.0. In an embodiment, the pH of the solution is at least 5.5. In an embodiment, the pH of the solution is at least 6.0. In an embodiment, the pH of the solution is at least 6.5. In an embodiment, the pH of the solution is at least 7.0. In an embodiment, the pH of the solution is no more than 8.0. In an embodiment, the pH of the solution is no more than 7.5. In an embodiment, the pH of the solution is no more than 7.0. In an embodiment, the pH of the solution is no more than

[0067] 6.5. In an embodiment, the pH of the solution is no more than 6.0.

[0068] Without being bound by theory, the present inventors consider that when the collagen derivative is positively charged, it forms a more stable microcarrier but which can nevertheless be easily degraded with a chelating agent (e.g. using EDTA in combination with a protease such as trypsin, TrypLE and the like). It appears that cell culture of the microcarrier is promoted when using said positively charged particles. Moreover, the positive charge may lead to improved cell culturing efficiency because of e.g. improved cell attachment and reduced aggregation and clumping of particles.

[0069] The term “collagen hydrolysate” in the context of the current invention means a mix of short chains of amino acids (di-, tri, oligopeptides, polypeptides) derived from hydrolysis from native (full-length) collagen, such as by enzymatic hydrolysis. The degree of hydrolysis has an impact on the average molecular weight (expressed in Dalton, Da) of the final product. The “collagen hydrolysate” in the context of the current invention may be produced from a collagen-containing material in a one-step process or via an intermediate gelatin stage (i.e. thus “hydrolysed gelatin” is obtained). The term “collagen hydrolysate” may be used interchangeably and synonymous with the terms “hydrolysed collagen” or “collagen peptide”. The “collagen hydrolysate” in the context of the current invention encompasses collagen which is subjected to hydrolysis or partial hydrolysis. The collagen hydrolysate can be one or more of enzyme-hydrolysed, alkali hydrolysed and acid-hydrolysed collagen hydrolysate.

[0070] If the collagen derivative is collagen hydrolysate or gelatin hydrolysate, it may have an average molecular weight in the range of 500 Da - 25,000 Da, e.g. 1000 Da to 15000 Da or 2000 Da to 10000 Da. The collagen hydrolysate may have an average molecular weight of at least 500 Da, or 600 Da, or 700 Da or 800 Da, or 900 Da, or 1000 Da or 1100 Da, or 1200 Da, or 1300 Da, or 1400 Da, or 1500 Da, or 2000 Da, or 2500 Da, or 3000 Da, or 3500 Da, or 4000 Da, or 4500 Da, or 5000 Da, or 5500 Da. In addition or alternatively, the collagen hydrolysate may have an average molecular weight of no more than 10000 Da, or 9500 Da, or 9000 Da, or 8750 Da, or 8500 Da, or 8250 Da, or 8000 Da, or 7500 Da, or 7000 Da, or 6500 Da, or 6000 Da, or 5500 Da, or 5000 Da, or 4500 Da, or 4000 Da. In a preferred embodiment, the collagen hydrolysate has an average molecular weight of 500-10000 Da, preferably 1000-8000 Da, more preferably 2000 - 7000 Da, even more preferably 3000 - 6000 Da.

[0071] The collagen derivative, e.g. gelatin, may be derived from collagen from one more animals or species of animals, such as collagen of bovine (species), pig (species), chicken and fish (species), or combinations thereof. The collagen can be or comprise collagen from a single source or a variety of different sources, such as collagen originating from one or more multiple animal species. In addition or alternatively, the collagen-containing material as disclosed herein preferably may be, or comprise one, two, or more collagens selected from the group consisting of skin-, cartilage-, bone-, and / or connective tissue- derived collagen. The term “skin” as used herein encompasses “hide” (i.e. the outer covering of large animals such as from the bovine group or any other large animals).

[0072] In an embodiment, the collagen derivative may be obtained recombinantly or by chemical synthesis. Recombinant synthesis encompasses that a protein or peptides is encoded by recombinant DNA that is expressed in an expression system. The expression system for the recombinant peptide can be cells such as a bacterial cell (e.g., Escherichia coli, Bacillus subtilis species), yeast cell [e.g. Saccharomyces cerevisiae, Pichia pastoris or Ogataea angusta (Hansenula polymorpha), Candida bodini], fungal cell (e.g. Aspergillus oryzae, Aspergillus niger, Trichoderma reesei), mammalian cell (e.g. a CHO cell, a HeLa cell, a HEK293 cell, NSO, Sp2 / 0), insect cell and plant cell (e.g. tobacco, cereal, legume, fruit, vegetable).

[0073] Formation of composite microparticles

[0074] In an embodiment, a microparticle comprising ionically crosslinkable polysaccharide is provided. The term “microparticle comprising ionically crosslinkable polysaccharide” does not exclude that other polymers may be present in the microparticle, other than ionically crosslinkable polysaccharide. However, in the context of the present invention, it may be preferred that the microparticle comprising ionically crosslinkable polysaccharide consists of ionically crosslinkable polysaccharide as only structural or functional polymer. The microparticle comprising ionically crosslinkable polysaccharide is preferably a gel microparticle. The term “gel” in the context of the present invention means a semi-solid material comprising a network of cross-linked polymers able to swell by a liquid, e.g. water. The microparticle comprising ionically crosslinkable polysaccharide is preferably produced by ionic gelation. The ionic gelation in the context of the present invention preferably involves dissolving a powder of the ionically crosslinkable polysaccharide (e.g. in case of alginate, sodium salt of alginic acid) in water or any other suitable buffer able to dissolve the polysaccharide and forming a polysaccharide solution. Formation of the polysaccharide solution is preferably done under stirring and heating to form a homogenous solution. The polysaccharide solution may comprise the polysaccharide in an amount of 0.1-10 wt.%, preferably 0.2 - 5 wt.%, more preferably 0.3 - 2.5 wt.%, even more preferably 0.5-1.5wt.%.

[0075] Microparticle comprising ionically crosslinkable polysaccharide may be produced by formation of polysaccharide droplets, e.g. by a method based on one or more selected from (in- air)microfluidics, emulsification, droplet generation, extrusion, electrospraying, 3d printing or a phase inversion method. The microparticles are most preferably produced by microfluidics, even more preferably in-air microfluidics. The in-air microfluidics method advantageously achieves high monodispersity in microcarrier size and shape. In-air microfluidics is for example described in WO2017167798A1.

[0076] For non-spherical particles, the Feret diameter can be used to provide more detailed information about the actual dimensions and shape variations of the microparticle or microcarrier The Feret diameter method allows measuring the distance between parallel tangents on opposite sides of a particle. It allows the determination of an equivalent diameter representing a spherical diameter that corresponds to the particle's observed dimensions and helps relate the non-spherical shape back to a single characteristic size. The skilled person is familiar with the procedure.

[0077] Preferably, the polysaccharide droplets are deposited into a solution, preferably a solution containing crosslinking agent for the ionically crosslinkable polysaccharide, such as providing divalent or higher cations suitable to crosslink (e.g. calcium chloride, CaCh). The crosslinking agent (e.g. calcium chloride, CaCh) may be provided in the solution in a concentration of 0.1- 10 wt.%, preferably 0.2 - 5 wt.%, more preferably 0.5 - 2.5 wt.%, even more preferably 1.0- 2.0 wt.%. In a preferred embodiment, the crosslinking solution comprises 0.01 - 1 M CaCh. In a preferred embodiment, the crosslinking solution comprises 2-20 wt.%, preferably 5-15 wt. EtOH. The solvent for the crosslinking solution is preferably water or demineralized water.

[0078] After gel formation, the polysaccharide microparticles may be optionally washed with water or other aqueous solution. A preferred washing solution for this purpose comprises CaCI2, e.g. in an amount of 0.05 - 1 M, preferably 0.1 -0.5 M. Preferably, any consecutive washing steps are ended in (demineralized) water.

[0079] In an embodiment, the microparticles are dried to produce a dry powder of microparticles. The washing step may for example remove excess crosslinking agent (e.g. calcium chloride) or unreacted polysaccharide. In an embodiment, the polysaccharide microparticles are collected and immersed in a solvent containing collagen derivative such as to form the composite microparticles, without prior drying. The term “dry powder” in the context of the present invention means a finely divided solid substance in particulate form, with a moisture content low enough to prevent clumping, dissolution, or degradation of its components under standard storage conditions, preferably a moisture content of <8 wt%, e.g. <5 wt.%, < 2 wt.% or < 1 wt.%.

[0080] The collagen derivative, in particular in case of a non-hydrolysed or partially hydrolysed collagen derivative (e.g. gelatin), before or during step a) of the method is preferably not subjected to a sterilization method and / or heat-treatment (e.g. autoclaving) and / or other time / pressure combination which may reduce the molecular weight of the collagen derivative. For example, autoclaving of gelatin or otherwise wet heattreatment is preferably avoided before and during preparation of the composite microparticle (i.e. during or before step b of the method disclosed herein) because high temperature and pressure in an autoclave cause hydrolytic cleavage of peptide bonds in the collagen derivative. This process fragments the long polypeptide chains into shorter ones, decreasing the molecular weight. The reduced molecular weight may allow the collagen derivative to (too) easily infiltrate the polysaccharide core. Excessive infiltration of the collagen derivative in the core particle may be undesirable in certain scenarios. For instance, it may not achieve the appropriate ionically crosslinkable polysaccharide:collagen derivative weight ratio (i.e. excessive amounts of collagen derivative relative to polysaccharide). In addition or alternatively, excessive infiltration of the collagen derivative in the core particle may be undesirable if the goal is to obtain a core-shell structure wherein the collagen derivative is only or mostly present in the shell, i.e. as coating.

[0081] In an embodiment, the collagen derivative, preferably gelatin, is not subjected to wet heattreatment and / or autoclaving before or during step a), preferably before step b) of the method disclosed herein.

[0082] In an embodiment, the composite microparticle or microcarrier is not dried (e.g. to dried powder) before the heat-treatment in step b) disclosed herein.

[0083] In an embodiment, composite microparticles are formed by embedding the collagen derivative into the polysaccharide microparticle and / or by coating the collagen derivative onto the polysaccharide microparticle. Embedding and / or coating the collagen derivative may be done by providing the polysaccharide microparticles in a solution comprising dissolved collagen derivative.

[0084] In embodiments of the invention, in step a) of the method the composite microparticle is obtained by providing the collagen derivative dissolved in an appropriate solvent and directly coating the ionically crosslinkable polysaccharide microparticle, as an alternative to immersing the polysaccharide microparticles in a collagen derivative solution. In the embodiment wherein the polysaccharide microparticle is directly coated, any appropriate technique may be used for the coating such as (in air)microfluidics, extrusion, electrospraying, 3d printing or the like.

[0085] Most preferably, the solvent for the collagen derivative is water or demineralized water, although other good solvents for collagen derivatives disclosed herein may be used. Preferably, the solvent for the collagen derivative is heated to 40-75°C, preferably 45-65°C, more preferably 50-60° to prevent it from prematurely solidifying.

[0086] Preferably, the collagen derivative (e.g. gelatin) is provided in the solvent in a concentration of 0.1-15 wt.%, preferably 0.5 - 10 wt.%, more preferably 1 - 5 wt.%, even more preferably 2-4 wt.%. In embodiments, the collagen derivative (e.g. gelatin) is provided in the solvent in a concentration of at least 0.01 wt.%, 0.05 wt.%, 0.1 wt.%, 0.5 wt.%, 1 wt.%, 2 wt.%, 3 wt.%, 4 wt.% or 5 wt.% and / or in a concentration no more than 15 wt.%, 10 wt.%, 5 wt.%, 4 wt.%, 3 wt.%, 2 wt.%, 1 wt.%, 0.5 wt.% or 0.1 wt.%.

[0087] Embedding and / or coating the collagen derivative may be done by collecting the polysaccharide microparticles and immersing the particles into a solution comprising collagen derivative dissolved in a appropriate solvent. Alternatively, embedding and / or coating the collagen derivative may be done by adding the polysaccharide microparticles into a solvent containing a collagen derivative to obtain a composite microparticle. Alternatively, embedding and / or coating the collagen may be done by adding the collagen derivative (powder) into the solution containing ionically crosslinkable polysaccharide and crosslinking agent (e.g. calcium chloride). In a preferred embodiment, embedding and / or coating the collagen derivative is by immersing the polysaccharide microparticle in a solvent containing the collagen derivative, thereby allowing to obtain the composite microparticle. Preferably the solvent is agitated or stirred when reacting the ionically crosslinkable polysaccharide and collagen derivative. The collagen derivative and ionically crosslinkable polysaccharide are preferably reacted in step a) of the method at 40-70 °C. The collagen derivative and ionically crosslinkable polysaccharide are preferably reacted in step a) of the method for at least 1 min, preferably at least 15 min, more preferably at least 30 min and / or no more than 4 h, preferably no more than 2 h. In an embodiment, the collagen derivative and ionically crosslinkable polysaccharide are reacted for 30 min (minutes) - 4 h (hours), preferably 1-2 h.

[0088] Optionally, the collagen derivative can be further stabilized in the composite microparticle by using further chemical agents capable of crosslinking the collagen derivative.

[0089] The present inventors found that a further improved microcarrier is achieved if the constituents, in particular the collagen derivative present in the composite microparticle is subjected to chemical crosslinking, in particular if thereafter heat-treated as disclosed herein. Without being bound to theory, present inventors consider that chemical crosslinking the collagen derivative helps prevent (excessive amount) of the derivative being lost from the microcarrier, in particular during and / or after the heat-treatment step. To this end, the collagen derivative can be chemically crosslinked by subjecting the microcarriers to a chemical crosslinking agent, such as one or more selected from the group consisting of glutaraldehyde, genipin, a carbodiimide, an epoxy compound and a transglutaminase, preferably glutaraldehyde. In addition or alternatively, embodiments of the invention include providing a microcarrier wherein the collagen derivative is crosslinked and characterized by one or more linkages selected from the group consisting of a Schiff base, a secondary amine linkage [e.g. in case of glutaraldehyde], primary amine [e.g. in case of genipin], amide bond [e.g. in case of carbodiimides] ether linkage, hydroxyl linkages [e.g. in case of epoxy] and amide linkages [e.g. in case of transglutaminase], preferably a Schiff bases and a secondary amine linkage. In a preferred embodiment, the microcarrier is chemically crosslinked with glutaraldehyde after preparing the composite microparticle. It is found to be particularly advantageous to use glutaraldehyde because it is considered efficient, cost-effective, and straightforward as compared to most other crosslinking agents.

[0090] The “chemical crosslinking agent” in the context of the present invention can be any molecule, ion, or enzyme that induces the formation of intermolecular linkages between functional groups on polymer chains. The formation of intermolecular linkages may be through one or more of covalent, ionic, or other physicochemical interactions, preferably covalent interactions. In an embodiment, the chemical crosslinking agent induces intermolecular linkages between functional groups on the ionically crosslinkable polysaccharide and on the collagen-derivative. In an embodiment, the chemical crosslinking agent induces intermolecular linkages between functional groups on the collagen-derivative. In an embodiment, the chemical crosslinking agent induces intermolecular linkages between functional groups on the ionically crosslinkable polysaccharide.

[0091] Glutaraldehyde is known to react with primary amine groups on the collagen derivative (e.g. gelatin), primarily provided by the lysine residues, to form Schiff bases (imine bonds). In a preferred embodiment, the collagen derivative in the microcarrier is in a first step crosslinked with glutaraldehyde, thereby forming Schiff bases (imine bonds) between the collagen derivative molecules, and additionally crosslinked in a second crosslinking step (e.g. by heat treatment disclosed herein), so that the microcarrier is characterized by covalent binding between the polysaccharide and the collagen derivative as disclosed herein (e.g. by one or more of a Schiff base (C=N) bond and a pyrazine (C-N) bond and / or covalent binding is between carbonyl groups in the ionically crosslinkable polysaccharide and amino groups in the collagen derivative.

[0092] The present inventors consider that chemical crosslinking of the collagen derivative, in particular glutaraldehyde treatment thereof, affects Maillard-based crosslinking reaction of the collagen derivative with the polysaccharide. Without being bound by theory, the chemical crosslinker (e.g. glutaraldehyde) may consume amine groups, thereby reduce the number of sites available for Maillard-based crosslinking thus potentially allowing a different crosslinking reaction. In addition or alternatively, structural changes in the collagen derivative due to e.g. glutaraldehyde crosslinking can make collagen derivative less flexible and less accessible for the Maillard reaction, thus also allowing a different crosslinking reaction. In the case of alginate and gelatin, a competition between the chemical crosslinker (e.g. glutaraldehyde) and alginate-derived aldehydes for reaction with gelatin's amines could shift the balance towards glutaraldehyde crosslinking over Maillard crosslinking, thus also allowing a different crosslinking reaction.

[0093] The glutaraldehyde concentration in the crosslinking solution can be in the range of 0.01%- 50% (v / v). Preferably the glutaraldehyde concentration is 0.1-25% (v / v), more preferably 0.5- 10% (v / v), even more preferably 1-5%(v / v). The glutaraldehyde treatment can be for 1 - 120 min. Preferably, the glutaraldehyde treatment is for 5-60 min, more preferably 10-45 min, even more preferably 15-30 min. The glutaraldehyde is preferably in an aqueous buffer, more preferably water.

[0094] The microparticles are preferably washed after the chemical crosslinking step, more preferably with the washing solution disclosed herein and ending with (demineralized) water.

[0095] In an embodiment, the composite microparticles are cooled down to room temperature or lower, allowing solidification of the collagen derivative. In case of gelatin, the alginate-gelatin microparticle may be cooled down to e.g. 2-8 °C, (~ 4°C) for complete gelatin solidification.

[0096] In embodiments, the embedding method (e.g. immersion) disclosed herein allows the collagen derivative to penetrate into the polysaccharide microparticle such that the collagen derivative is distributed throughout the inside of the microcarrier and able to form crosslinks. This may particularly happen if the collagen derivative has a relatively low molecular weight (e.g. <150 kDa, preferably <100 kDa) and / or a negative charge (e.g. alkali treated gelatin and / or having an acidic isoelectric point). In case of a negatively charged polysaccharide such as alginate, the penetration of the collagen derivative into the polysaccharide microparticle and / or their crosslinking is considered to be improved if a negative or neutral charge is selected for the collagen derivative. Without being bound by theory, neutral / negatively charged molecules will interact less with the negatively charged polysaccharide (e.g. alginate) particle, and therefore may more readily infiltrate the particle.

[0097] The present inventors found that higher cell attachment and growth is observed if the collagen derivative is present in sufficient high amount on the outside of the microcarrier, e.g. forming a coating or outer shell layer. Without being bound by theory, the present inventors consider this to be the result of the adhesion sites (through RGD motifs) naturally present in collagen sequences (which e.g. polysaccharides such as alginate lack) and which promote the attachment and proliferation of anchorage-dependent cells. In addition or alternatively, the present inventors consider that collagenous materials may promote cell attachment and growth and therefore it is also advantageous to have an outer layer of gelatin (e.g. “coating or “shell”) in the microcarrier.

[0098] Hence, in the context of the present invention it is considered beneficial to preserve the outer layer (e.g. “coating” or “shell”) of collagen derivative which deposited by the embedding method (e.g. immersion) disclosed herein. To preserve the outer layer of collagen derivative, the composite microcarrier is preferably continuously kept in a solvent in which the collagen derivative has high dissolvability (referred to herein as a “good solvent”). Good solvents for collagen derivatives such as gelatin are water, aqueous solutions (with or without additives like acids or bases) or polar protic solvents like glycerol. In a preferred embodiment, the microcarriers are maintained in water or aqueous solution with high water content preferably continuously but at least during step a) and b) of the method. Aqueous solution with high water content in the context of the present invention can mean at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% water.

[0099] Ethanol (or ethanol-water mixtures with higher ethanol content) is typically used to remove excess collagen derived material that is outside the gel matrix. For instance, EP4339283A1 discloses subjecting alginate-gelatin microcarriers to ethanol to condense the gelatin (nanoparticle formation) and washing to remove gelatin from the outside. To preserve or maintain the outer layer of collagen derivative (e.g. “coating” or “shell”) in the microcarrier, the method of the invention preferably excludes a further step of removing collagen derivative (e.g. gelatin) from the outer layer of the microcarrier, e.g. after step a), such as by a method involving washing and / or condensation of gelatin. In particular, the method preferably excludes subjecting the microcarrier to ethanol or an ethanol solution.

[0100] In embodiments, the embedding method (e.g. immersion) disclosed allows the collagen derivative to be deposited substantially at the outside of the polysaccharide microparticle. This can be particularly achieved by providing a relatively high molecular weight of the collagen derivative (e.g. >80 kDa, preferably >100 kDa, more preferably >150 kDa), which may at least partially prevent the penetration of collagen derivative molecules into the polysaccharide particle. In addition or alternatively, the collagen derivative may be deposited (substantially) at the outside of the polysaccharide microparticle if a positive charge is selected for the collagen derivative (e.g. acid treated gelatin and / or having isoelectric point of 7-9). Without being bound by theory, if the polysaccharide is negatively charged such as in the case of alginate, the positively-charged collagen derivative will interact with the negatively charged microparticle at the surface, thereby reducing diffusion into the particle. The present inventors consider that crosslinked collagen-derivative substantially at the outside of the microcarrier may lead to improved solubility since the collagen-derivative- dissolving agents do not have to reach the particle core (i.e. center) by substantial intraparticle diffusion to have their mode of action.

[0101] The method of the invention may include a further step of drying, preferably after the heattreatment and crosslinking step disclosed herein. The drying may be freeze-drying. The drying is preferably spray-drying. In an embodiment, the method according to the invention is characterized in that said microparticles or microcarriers are dried, particularly by a drying method chosen from a group containing evaporation, blowing dry gas, vacuum drying, fluid bed drying, freeze drying, microwave drying, and chemical drying. Such method will remove water from the microcarriers. In, embodiment dry or dried microcarriers are formed into at least partially a free flowing powder. The drying may be aided by further addition of a drying agent. The drying agent particularly comprises at least one sugar compound, and more particularly in that said sugar compound comprises dextran, dextrin, maltodextrin, trehalose, lactose, glucose, dextrose, sucrose, fructose, maltose, isomaltose, sorbitol, mannitol, lactitol, xylitol, and / or erythritol.

[0102] Further crosslinking method

[0103] To further improve the stability and functional properties of the microcarrier, the composite microparticle disclosed herein is preferably subjected to a further crosslinking step.

[0104] The further crosslinking may allow a Maillard reaction between (functional) groups of the polysaccharide and the collagen derivative. For instance, the further crosslinking may enhance the physical and / or chemical interactions between the ionically crosslinkable polysaccharide and the collagen derivative. In a preferred embodiment, the further crosslinking disclosed herein is to cause formation of covalent bonds between the ionically crosslinkable polysaccharide and the collagen derivative. The further crosslinking may comprise one or more selected from an enzymatic, chemical, photo-crosslinking, gamma irradiation and heat-treating method. The enzymatic, chemical, photo-crosslinking or gamma irradiation method may induce the same type of bonding as described below for the heattreatment and Maillard reaction.

[0105] In embodiments, the further crosslinking may comprise carbodiimide coupling, such as using EDC (-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride) and NHS (N- Hydroxysuccinimide). The carbodiimide coupling may form amide bonds between the polysaccharide’s -COOH groups and the collagen derivative’s -NH2(lysine residues).

[0106] In embodiments, the further crosslinking is by subjecting the composite microparticle to one or more of the chemical crosslinking agents as disclosed herein.

[0107] In embodiments, the enzymatic crosslinking disclosed herein may comprise subjecting the composite microparticle to a transglutaminase, such as to catalyze amide bonds between glutamine and lysine residues in gelatin. The enzymatic crosslinking may in addition or alternatively comprise horseradish peroxide + H2O2crosslinking, such as to catalyze radical coupling of phenol / tyramine groups introduced to the polysaccharide and / or collagen derivative.

[0108] In embodiments, the photo-crosslinking method may for example comprise functionalization of functional groups such as methacrylate groups onto the polysaccharide and / or collagen derivative, and subjecting to ultraviolet (UV) or visible light to induce radical polymerization.

[0109] The gamma irradiation in the context of the present invention may be at a dose of 5 -50 kilogray (kGy), preferably 10-40 kGy, or 15-30 kGy.

[0110] Heat-treatment and Maillard reaction

[0111] To further improve the stability and functional properties of the microcarrier, the composite microparticle disclosed herein is preferably subjected to a heat-treatment step. The heattreatment step may allow a Maillard reaction between (functional) groups of the polysaccharide and the collagen derivative. The heat-treatment step disclosed herein may enhance non-covalent and / or covalent interactions in microcarrier functional groups.

[0112] For instance, the heat-treatment may enhance the physical and / or chemical interactions between the ionically crosslinkable polysaccharide and the collagen derivative, in particular when the polysaccharide is alginate and the collagen derivative is gelatin.

[0113] In addition or alternatively, the heat-treatment in step b) and / or the Maillard reaction may render proteins, cells and / or other and bioactive components to exhibit increased binding affinity with the microcarrier, due to increase presence of Maillard reaction products. For example, the Maillard reaction between reducing sugars and amino acids leads to a range of advanced glycation end-products (AGEs), which can introduce additional functional groups, such as carbonyl, carboxyl, and hydroxyl groups. These new functional groups can enhance non-covalent interactions (e.g., hydrogen bonding, electrostatic interactions, and hydrophobic interactions) that may increase the binding affinity of proteins, cells and / or other and bioactive components. The increased binding affinity can for example be beneficial in applications where prolonged or controlled release of proteins, cells and / or other and bioactive components is desired, as it may help to retain these molecules in the microcarrier matrix and / or on the microcarrier surface for a prolonged time.

[0114] Example 4 in the present disclosure describes a method to quantify the concentration of Advanced Glycation End-products (AGEs) in microcarriers, based on the presence of fluorescent AGEs. In an embodiment, the heat-treating in step b) of the method involves a time / temperature- or time / temperature / pressure combination which causes at least 10%, preferably 100%, more preferably 500% increase in the Maillard reaction, defined by a change in fluorescence signal measured at excitation wavelength 360 nm (+ / - 10 nm) and emission wavelength 440 nm (+ / - 10 nm). In an embodiment, the heat-treating in step b) of the method involves a time / temperature- or time / temperature / pressure combination which causes 2-20 fold increase, preferably 4-10 fold increase, more preferably 6-8 fold increase in the Maillard reaction, defined by a change in fluorescence signal measured at excitation wavelength 360 nm (+ / - 10 nm) and emission wavelength 440 nm (+ / - 10 nm).

[0115] Example 4 in the present disclosure describes a method to quantify the fold increase in Maillard reaction using quinine sulfate as a standard. In an embodiment, the heat-treating in step b) of the method involves a time / temperature- or time / temperature / pressure combination which yields an amount of fluorescent AGEs in the range 0.1-1000 pg / g dried microcarrier, preferably 1-100 pg / g dried microcarrier, even more preferably 2-10 pg / g dried microcarrier.

[0116] The groups of polysaccharide and the collagen derivative and / or the groups derived therefrom following the heat-treatment reaction are preferably functional groups, i.e. specific groups of the polysaccharide / collagen derivative responsible for the characteristic chemical reactions thereof.

[0117] The progression of the Maillard reaction is illustrated in the case of alginate. Alginate is a polysaccharide comprising mannuronic and guluronic acid residues, both of which have carboxyl groups (-COOH). In the Maillard reaction, carboxyl groups in alginate can form carbonyl groups (C=O), typically under conditions that promote oxidation or the breakdown of the polymer structure. In addition or alternatively, the degradation of alginate, leads to smaller sugar molecules (e.g. monosaccharides, oligosaccharides) having free aldehyde groups (comprising carbonyl group) which act as reducing sugars, and which can react with the amino groups in the collagen derivative. Carbonyl groups can interact with the amino groups (-NH2) in the collagen derivative, such as in highly present in lysine. The amino groups act as nucleophiles that react with the carbonyl groups provided by alginate. The carboxyl group in the polysaccharide may be transferred to carbonyl so that the interaction between the divalent or multivalent cation and the cation-crosslinked polysaccharide is broken, causing a release of the divalent or multivalent cation.

[0118] The Maillard reaction between the polysaccharide and the collagen derivative under heating disclosed herein typically will include the steps of condensation and Amadori Rearrangement. In the condensation step, the amino group from the collagen derivative reacts with the carbonyl group from alginate, in particular a reducing sugar, to form a Schiff base (an imine). In the Amadori Rearrangement step, the Schiff base undergoes a rearrangement, leading to the formation of more stable compounds, which can then further react and polymerize to form complex Maillard reaction products.

[0119] The present inventors found that improved cell performance on the microcarriers was positively related to the formation of Maillard reaction products and degree of crosslinking. It was found that the crosslinking method disclosed herein leads to overall improved performance of cells when cultured on the microcarrier. Without being bound by theory, the present inventors consider that the specific crosslinking between polysaccharide, in particular alginate, and the collagen derivative and / or formation of specific Maillard reaction products may underlie this observation. In particular, the formation of Maillard reaction products appears associated with an anti-inflammatory and / or anti-oxidant effect of the microcarriers, such as measurable with the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, as the skilled person is familiar with.

[0120] The enhanced properties of the microcarriers due to Maillard browning may be (further) due to one or more of enhanced mechanical stability and structural integrity, enhanced surface chemistry for cell adhesion, improved bioactivity and cellular response, improved protein and / or nutrient adsorption and release.

[0121] The present inventors consider that the heat-treatment step may promote cell attachment and growth. Without being bound by theory, the heat-treatment step may fragment the (poly)peptide chains of the collagen derivative into shorter ones, decreasing the molecular weight. The reduced molecular weight may allow for improved cell adhesion or growth, depending on the cell type or application. In addition or alternatively, the reduced molecular weight may allow for improved functionality and / or interaction with cells, such as by the binding through integrins, cadherins, selectins, immunoglobulins and the like.

[0122] In addition or alternatively, heat-mediated fragmentation may otherwise change the surface structure (e.g. roughness) of the microcarrier. For example, it is assumed that an improved micro-roughness of the microcarrier may allow for improved cell adhesion and growth, depending on the cell type or application. By fragmenting the collagen derivative during and / or after the incorporation with the microcarrier, it is circumvented that excessive amount of collagen derivative infiltrated into the microparticle during step a) of method. This may be desirable if a core-shell structure is to be obtained.

[0123] The term “roughness” (Ra) as used herein relates to the surface roughness of the microcarrier or microcarrier-embedded granular substrate. The “roughness” (Ra) preferably relates to the mean roughness (e.g. based on sufficient measurements performed on a sample to reduce the standard deviation to no more than 20%). The roughness of the microcarrier as disclosed herein is preferably measured by atomic force microscopy (AFM). In a preferred protocol, the roughness of a surface is measured using an atomic force microscopy (AFM, Easy Scan, or similar) recording in tapping mode at room temperature in air.

[0124] In an embodiment, the heat-treatment (in step b) as disclosed causes an increase in the roughness (Ra) of the microcarrier. In an embodiment, the microcarrier (after the heattreatment) has an average surface roughness (Ra) of 0.1-100 nm (nanometer), preferably 0.5 - 50 nm, more preferably 1-30 nm, most preferably 5-15 nm. In an embodiment, the microcarrier (after the heat-treatment) has an average surface roughness (Ra) of 1-30 nm, preferably 5-15 nm.

[0125] Hence, the heat-treatment in step b) of the method preferably establishes a Maillard reaction between groups of the ionically crosslinkable polysaccharide and the collagen derivative. As a result, a crosslinked microcarrier is obtained characterized by covalent binding between the ionically crosslinkable polysaccharide and the collagen derivative.

[0126] As part of the Maillard reaction, covalent bonds may be formed between the ionically crosslinkable polysaccharide and the collagen derivative. Hence, in an embodiment, the Maillard reaction causes covalent binding between the ionically crosslinkable polysaccharide and the collagen derivative so that step c) of the method obtains a crosslinked microcarrier characterized by covalent binding between carbonyl groups in the polysaccharide and amino groups in the collagen derivative.

[0127] In an embodiment, a covalent binding between the polysaccharide (e.g. alginate) and the collagen derivative is between carbonyl groups in the alginate and amino groups (e.g. in lysine) in the collagen derivative. In an embodiment, the carbonyl groups are formed from carboxyl groups in the polysaccharide, e.g. in mannuronic and guluronic acid residues in case of alginate. In an embodiment, reducing sugar having free aldehyde group provide for covalent binding between the alginate and the collagen derivative. In an embodiment, as part of the Maillard reaction, the amino group from the collagen derivative reacts with the carbonyl group from the polysaccharide to form a Schiff base (an imine). In an embodiment, the Schiff base undergoes a rearrangement as disclosed herein, and may lead to the formation of more which can then further react and polymerize to form complex Maillard reaction products.

[0128] Typically, in the Maillard reaction, functional groups including amino groups (especially from lysine) are reduced while the reaction progresses, while the amount of those associated to Maillard products such as Amadori compounds (C=O), Schiff base (C=N), and pyrazines (C- N) increase through Maillard reaction. In an embodiment, the covalent binding in the microcarrier disclosed herein is by one or more of a Schiff base (C=N) bond and a pyrazine (C-N) bond.

[0129] To characterize the Maillard reaction between the polysaccharide and the collagen derivative (before and after the Maillard reaction), several analytical methods can be used to detect the formation of Maillard reaction products (MRPs), by analyzing the changes in chemical composition. A preferred method in the context of the present invention is use of UV-Vis spectroscopy to measure changes in absorbance at characteristic wavelengths, such as 320 nm (for early Maillard reaction products) and 420 nm (for advanced browning products). UV- Vis absorbance at 420 nm can be particularly used as a measure of browning, which indicates Maillard reaction progression. The L\*, a\*, b\* color values (measured with a colorimeter) can quantify changes in color, especially if browning is visible to the naked eye. Another preferred method in the context of the present invention is use Fourier Transform Infrared (FTIR) spectroscopy, which can identify functional groups involved in the reaction, such as amide (from collagen derivative, e.g. gelatin) and carboxyl (from polysaccharide, e.g. alginate) changes. New peaks or shifts in FTIR, such as changes in carbonyl (C=O) stretching around 1650 cm-1or 1740 cm-1, can indicate the formation of Amadori products or other MRPs

[0130] The browning effect can be based on the colour (change) in one or more of the L*a*b* values as defined by the International Commission on Illumination (CIE). The one or more of the L*a*b values as disclosed herein can be measured by any suitable method known to the skilled person. Widely accepted methods are colorimetry or spectrophotometry. In an embodiment, the L*a*b is measured by colorimetry, using a standard white calibration (e.g. L*=97.10, a*=0.19, b*=1.95), CIE standard illuminant D65, and 10° standard observer angle setting.

[0131] The delta E (dE) is defined as the difference between two colors in an L*a*b* colorspace. The dE can be calculated using the formula, wherein L2, a2, and b2 are the values corresponding to samples before browning and / or the heat-treatment as disclosed herein, and L1, a1 and b1 are the values corresponding to samples after browning and / or the heat-treatment as disclosed herein: al)2+ (62 - bl)2

[0132] Without being to be bound to a definition, the dE value and the color difference between the input and output material as perceived by the human eye would typically be correlated as follows:

[0133] • dE <= 1.0: color difference not perceptible by the human eye;

[0134] • dE 1-2: color difference perceptible through close observation;

[0135] • dE 2-10: color difference perceptible at a glance;

[0136] • dE11-49: colors are more similar than the opposite;

[0137] • dE 100: colors are opposite.

[0138] In embodiments, the dE value for the microcarriers (as a result of the heat-treatment) is in the range 2-100, preferably 5-50, more preferably 10-25. In an embodiment, the dE value for the microcarriers (as a result of the heat-treatment) is 1-2. In an embodiment, the dE value for the microcarriers (as a result of the heat-treatment) is 2-10. In an embodiment, the dE value for the microcarriers (as a result of the heat-treatment) is 11-49. In an embodiment, the dE value for the microcarriers (as a result of the heat-treatment) is 50-100.

[0139] In an embodiment, the microcarrier as disclosed herein has a colour profile defined by the following L*a*b* values:

[0140] - L* in the range of +50 to +100, preferably +60 to +90; and / or

[0141] - a* in the range of -30 to +30, preferably -5 to +10; and / or

[0142] - b* in the range of +10 to +80, preferably +30 to +60

[0143] In an embodiment, the microcarrier as disclosed herein has a colour profile defined by the following L*a*b* values [e.g. (light) yellow)]:

[0144] - L* in the range of +80 to +100; and / or

[0145] - a* in the range of -5 to +10; and / or

[0146] - b* in the range of +50 to +80. In an embodiment, the microcarrier as disclosed herein has a colour profile defined by the following L*a*b* values (e.g. yellow-brown):

[0147] - L* in the range of +50 to +80; and / or

[0148] - a* in the range of +5 to +20; and / or

[0149] - b* in the range of +30 to +60.

[0150] In an embodiment, the microcarrier as disclosed herein has a colour profile defined by the following L*a*b* values (e.g. green-yellow):

[0151] - L* in the range of +60 to +90; and / or

[0152] - a* in the range of -30 to -5; and / or

[0153] - b* in the range of +30 to +60.

[0154] In an embodiment, the microcarrier as disclosed herein has a colour profile defined by the following L*a*b* values [e.g. (tan-like) brown]:

[0155] - L* in the range of +60 to +90, preferably and / or

[0156] - a* in the range of +5 to +20; and / or

[0157] - b* in the range of +10 to +30.

[0158] Another preferred method to measure the colour of samples is using a Helliges comparator of color, normalized using a standard series of color solutions at 10% dry material.

[0159] The heat-treatment is preferably a wet (i.e. moist) heat-treatment method. In addition or alternatively, the microparticle is preferably provided in a liquid suspension (i.e. as a slurry) during the heat-treating. The term “slurry” in the context of the present invention means a mixture of solid particles suspended in a liquid phase, e.g. creating a fluid-like mixture.

[0160] The present inventors consider that with wet heating and / or by providing the microcarriers in a slurry during the heat-treatment, the crosslinking reaction between ionically crosslinkable polysaccharide and collagen derivative may already be efficiently induced at relatively lower temperatures (e.g. starting already from 60-70 °C or higher) and / or at atmospheric pressure.

[0161] “Wet heating” or “wet heat-treatment” in the context of the present invention means a use of water or steam to transfer heat to a material. In contrast, the term “dry heating” or “dry heattreatment” in the context of the present invention means heating methods that do not involve water or moisture.

[0162] The heat-treatment disclosed herein preferably excludes thermal treatment wherein the microcarrier is in dry form or has too low water content (e.g. below 10 wt.%) and / or preferably excludes drying and / or method such as dry heat sterilization. More preferably, the water content in the slurry is preferably at least 10 wt.%, more preferably at least 25 wt.%, even more preferably at least 50 wt.%, such as in the range 70-99 wt.%, or 80-95 wt.%, or 85-90 wt.%. or The water content in the slurry is most preferably 70-95 wt.% or 80-90 wt.%.

[0163] In a preferred embodiment, the microparticle does not achieve a water content of less than 10 wt.%, more preferably les than 5 wt.%, even more preferably less than 2% during step a) and / or step b) in the method disclosed herein.

[0164] The concentration of particles in the slurry during heat-treatment is preferably 2-50% by weight or volume, more preferably 5-25% by weight or volume, even more preferably 10-20% by weight or volume.

[0165] The skilled person is able to select appropriate temperature / time- or temperature / time / pressure combination to achieve a desirable Maillard reaction, e.g. depending on the chosen polymers and the design of the microcarrier and / or whether or not to achieve chemical covalent bonding between the polysaccharide and the collagen derivative. For instance, the skilled person can select the appropriate heating conditions to obtain a desirable degree of crosslinking as disclosed herein. In addition and / or alternatively, the skilled person can select the appropriate heating conditions to obtain a desirable type of chemical bond between the polysaccharide and the collagen derivative.

[0166] The heat-treatment, in particular the temperature / time- or temperature / time / pressure combination thereof, can be chosen appropriately such as to provide the desired crosslinking (%). The degree of crosslinking may be at least 1%, 5%, 10%, 20%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% or 90% and / or no more than 95%, 90%, 85%, 80%, 75%, 70%, 65% 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10% or 5%. In an embodiment, the degree of crosslinking is 5-50%, preferably 10-40% more preferably 20- 30%. In an embodiment, the degree of crosslinking is 30-80%, preferably 40-70% more preferably 50-60%.

[0167] The degree of crosslinking is determined by measuring the amount of unreacted groups in the collagen derivative. A preferred method of determining the degree of crosslinking using a UV assay of uncrosslinked e-amino groups before and after crosslinking according Ofner et al (1996. Pharm Res 13:1821-1827). A preferred protocol is as follows:

[0168] 11 mg of the sample is mixed with 1 mL of 4% NaHCCh and 1 mL of 0.5% 2,4,6- trinitrobenzenesulfonic acid TNBS, and heated at 40 °C for 4 h

[0169] 3 mL of 6 N HCI is added and the mixture was maintained at 60 °C for 2 h. • the reaction mixture is extracted with ethyl ether.

[0170] • a 5 mL aliquot of the aqueous phase is removed from the sample and heated for 15 min in a hot water bath, cooled to room temperature, and diluted again with 15 mL of water.

[0171] The Maillard reaction gives rise to characteristic XRD or FTIR peaks, which can be used to determine the amount of crosslinking and / or covalent binding. Hence, the XRD or FTIR results can be used to determine the appropriate heat-treatment to obtain the microcarrier disclosed herein, in particular the time / temperature combination needed.

[0172] In an embodiment, the crosslinked microcarrier is characterized by an XRD pattern showing a peak at 20 of 14.4 - 17.0°.

[0173] In an embodiment, the crosslinked microcarrier is characterized by an XRD pattern showing a peak at 20 of 25.0 - 35.0°.

[0174] In an embodiment, the crosslinked microcarrier is characterized by an XRD pattern showing a peak at 20 of 39.0 - 50°.

[0175] The peak at 20 in the context of the present invention is when measured with a diffractometer from Bruker equipped with a Cu-Ka radiation (k = 1.5418 A° ) operating at 40 kV and 40 mA and the 20 diffraction diagram determined between 5 and 90 at a scanning rate of 0.02 / min.

[0176] In an embodiment, the microcarrier comprises one or more melanoids in a total amount of 0.1-10 wt%, preferably 0.2-5 wt.%, more preferably 0.4-1 wt.%, even more preferably 0.5-0.7 wt.%, calculated on total dry weight of the microcarrier.

[0177] In an embodiment, the microcarrier is characterized by covalent crosslinking between dicarbonyls and amino groups in the collagen derivative and / or between dicarbonyl groups and hydroxyl groups in the polysaccharide, in particular alginate.

[0178] Some preferred embodiments for heat-treatment are provided below and which appear particularly suitable.

[0179] The temperature used in heat-treatment step may be 50-165 °C, preferably 60 - 140 °C, more preferably 80 - 120 °C , even more preferably 90-110 °C. In a preferred embodiment, the heating, preferably wet heating, is performed at relatively low temperature such as 50-100 °C, preferably 60-90°C, more preferably 70-80 °C. Wet heating can be efficiently performed under atmospheric pressure conditions. Preferably, the time of the heat-treatment or wet heat-treatment is at least 1 min, 5 min, 10 min, 15 min, 30 min, 60 min, 90 min, 120 min, 3 h, 4 h and / or no more than 8 h, 6 h, 4 h, 120 min, 90 min, 60 min, 30 min, 15 min, 10 min or 5 min. In a preferred embodiment, heattreatment or wet heat-treatment is for 15 min - 120 min, preferably 30-90 min, more preferably 45-60 min.

[0180] In a preferred embodiment, the heat-treating in step b) is

[0181] - at 50-140 °C and / or

[0182] - for 5-60 min,

[0183] In a preferred embodiment, the heat-treating in step b) is performed under pressurized steam conditions, for example 5-100 psi, preferably 10-50 psi (pounds per square inch).

[0184] The heat-treating under step b) can be performed under pressurized steam conditions of at least 10 psi, 20 psi, 30 psi, 40 psi, 50 psi, 100 psi, 200 psi and / or no more than 1000 psi, 900 psi, 800 psi, 700 psi, 600 psi, 500 psi, 400 psi, 300 psi, 200 psi, 100 psi or 50 psi.

[0185] In addition or alternative to psi, the pressure can be expressed in pascal (Pa) or kilopascal (kPa) in the context of the present invention. The skilled person is aware how to convert psi to Pa, or vice versa. The pressure value in Pa is preferably derived by multiplying the pressure value in psi by 6894.76.

[0186] In embodiments, the heat-treating under step b) can be performed under pressurized steam conditions of at least at least 50kPa, 100 kPa, 200 kPa, 250 kPa, 300 kPa, 350 kPa, 400 kPa, 450 kPa or 500 kPa and / or no more than 1000 kPa, 750 kPa, 500 kPa, 400 kPa, 350 kPa, 300 kPa, 250 kPa or 200 kPa.

[0187] Pressurized steam heating (e.g. autoclaving) microcarriers was found to achieve desirable chemical crosslinking, and improved microcarriers. In an embodiment, the heat-treating in step b) is a pressurized steam heating method. Autoclaving was surprisingly found to reduce LPS content / activity in the microcarriers which is advantageous for the reasons disclosed herein. As a further advantage of autoclaving, a sterile product is immediately also achieved, hence removing the need of additional sterilization step.

[0188] The term “pressurized steam heating” in the context of the present invention means any method that utilizes heated steam under pressure. The pressurized steam heating disclosed herein can encompass common autoclaving or steam sterilization methods.

[0189] A preferred embodiment of an autoclaving and / or steam sterilization method is: -at 100-140°C, preferably 110-130 °C; and / or

[0190] - at 5-60 min, preferably 10-30 min; and / or

[0191] - at 5-45 psi, preferably 10-20 psi.

[0192] Another preferred embodiment of an autoclaving and / or steam sterilization method is

[0193] - at 110-150°C, preferably 120-140 °C; and / or

[0194] - at 1-15 min, preferably 2-10 min; and / or

[0195] - at 10-50 psi, preferably 20-40 psi.

[0196] The present inventors consider that alkaline pH typically leads to most efficient heat-induced crosslinking disclosed herein. Preferably, the pH during heat-treating is 8-13, preferably 9-12, more preferably 10-11. In addition or alternatively, the pH is preferably at least 10, 11 , 12 or 14 and / or no more than 14, 13, 12 or 11. It is considered by the present inventors that the degree of crosslinking increases with pH and hence stability of the final microcarrier. The pH can for instance be adjusted by adding aqueous NaOH. However, the present inventors also consider that more acidic pH can be more beneficial depending on desired speed of the reaction and the types of MRPs sought. Hence, in certain embodiments, the pH is 4-8, preferably 5-7.

[0197] Embodiments of the method of the invention include a further step of introducing one or more agents in the solution which can enhance the Maillard reaction between the ionically crosslinkable polysaccharide and the collagen derivative and / or allow better control over the electrostatic interactions. The (Maillard-) enhancing agent is preferably one or more selected from the group consisting of a reducing agent, preferably a reducing sugar, a polyelectrolyte, a salt, a pH modifier, a metal catalyst and an ionic influencer. In an embodiment, alginate, pectin and / or one or more collagen derivatives as disclosed herein are further added in the microcarrier suspension since they can contribute in enhancing the Maillard reaction.

[0198] Further purification step(s)

[0199] Advantageously, the microcarrier of the invention is found to be surprisingly stable when subjected to further purification and / or washing treatments. For example, the microcarrier of the invention may be simply subjected to further purification with chelating agents and washing after its preparation. Such an approach may circumvent the need to first purify the polysaccharide or collagen derivative prior raw materials. For example, purification of the microcarrier after its preparation may circumvent the need for (more complex and timeconsuming) ultrafiltration and / or dialysis treatments of the raw materials. The method of the invention may comprise one or more further steps of purification and / or washing of the microcarrier. The purification and / or washing step may preferably be to reduce the content of one or more contaminants. The contaminant may be one or more selected from the group consisting of unbound salt, unbound ions, metal contaminants including heavy metals, a residual reagent, endotoxin and microbial burden. In embodiments, the one or more purification and / or washing steps may reduce the content of one or more elemental impurities, more preferably one or more heavy metals selected from the group consisting of lead, cadmium, mercury and arsenic.

[0200] In embodiments, the purification and / or washing step includes a chelation treatment. The chelation treatment may involve use of one or more chelators selected from the group consisting of EDTA, EGTA (Ethylene glycol-bis(P-aminoethyl ether)-N,N,N',N'-tetraacetic acid), citrate (sodium citrate I citric acid), DTPA (Diethylenetriaminepentaacetic acid) and NTA (Nitrilotriacetic acid), preferably EDTA. The chelation treatment may in particular be beneficial for reduction of one or more heavy metals as disclosed herein.

[0201] The chelation treatment may use a concentration of chelator, preferably EDTA, in a concentration of 0.1 - 10 mM, preferably 0.5-5 mM, more preferably 1-2.5 mM, even more preferably 1.5-2.0 mM. In embodiments, the chelation treatment is preferably at a temperature of 2-8 °C, more preferably 3-6 °C. In embodiments, the chelation treatment is preferably at a temperature of 30-40 °C, more preferably 34-38 °C. The chelation treatment may also be performed at other temperatures, such as around room temperature. In embodiments, the chelation treatment is for 1-30 min, preferably 5-20 min, more preferably 10-15 min. In embodiments, the chelation treatment is for 0.5-5 hours, or 1-4 hours, or 2-3 hours.

[0202] After and / or in between the one or more purification and / or washing steps, it may be beneficial to subject the microcarriers to a re-crosslinking step. The re-crosslinking may be achieved by reintroducing ions (e.g. Ca2+) such as to allow re-establishing microcarrier crosslinking and integrity. For example, the ionically crosslinkable polysaccharide may be recrosslinked with the divalent or higher cation as disclosed herein. In addition or alternatively, a crosslinker for re-crosslinking may be one or more of CaCh, BaCh, SrCh and MgCh. The concentration of the crosslinker may be the same as disclosed above for the initial crosslinking.

[0203] In embodiments, purification and / or washing as disclosed herein may result in a microcarrier with reduced or no measurable elemental impurities and / or total heavy metals. For example, the amount of elemental impurities and / or total heavy metals may be less than 500 parts per million (ppm), preferably less than 50 ppm, more preferably less than 5 ppm, even more preferably less 0.5 ppm, most preferably less than 0.05 ppm. In the context of the present invention, the amount of elemental impurities and total heavy metals is preferably calculated on the dry weight of the crosslinkable polysaccharide and / or the dry weight of the microcarrier. In embodiments, the microcarrier comprises lead in an amount of less than 5 ppm, preferably less than 0.5 ppm, more preferably less 0.05 ppm, even most preferably less than 0.005 ppm. In embodiments, the microcarrier comprises cadmium in an amount of less than 5 ppm, preferably less than 0.5 ppm, more preferably less 0.05 ppm, even most preferably less than 0.005 ppm. In embodiments, the microcarrier comprises mercury in an amount of less than 5 ppm, preferably less than 0.5 ppm, more preferably less 0.05 ppm, even most preferably less than 0.005 ppm. In embodiments, the microcarrier comprises arsenic in an amount of less than 5 ppm, preferably less than 0.5 ppm, more preferably less 0.05 ppm, even most preferably less than 0.005 ppm. The amount of heavy metal is preferably calculated on the dry weight of the crosslinkable polysaccharide and / or the dry weight of the microcarrier. In an embodiment, the microcarrier is essentially free of elemental impurities and / or heavy metals, meaning that it is not present or at least not detectable by conventional analytical techniques.

[0204] Further microcarrier properties, core-shell structure, plurality of microcarriers, cell-laden microcarrier

[0205] Further microcarrier properties

[0206] The term “microcarrier” as used herein means particle bodies with physical dimensions typically in the order of micrometers. The term “microcarrier” can pertain to discrete particles or to microcarrier bodies (e.g. as gel particles, particularly gel microparticles) which form the grains of granular compositions or granular substrates. The term “microcarrier” therefore encompass gel particles that are deposited onto or embedded in substrate, such as to form a micro-rough surface. In a preferred embodiment, the microcarrier disclosed herein is a discrete particle.

[0207] The microcarrier may be in any suitable shape, such as a sphere, hemisphere, ellipsoid, rod, disk and the like, but is most preferably spherical or substantially spherical. The microcarrier may be particularly suitable for use in culturing cells by providing a substrate or scaffold onto which cells may attach and subsequently grow, however the use is not limited thereto. For example, in addition or alternatively, the microcarrier may be used for immobilization of cells. The term ‘immobilization of cells” encompasses both cell adhesion (e.g. mediated by normal cell surface-receptors and techniques that (further) promote cell attachment, such as promote attachment, e.g. by providing one or more cell-adhesive molecules, reactive groups on the microcarrier allowing cell binding through cell surface receptors and entrapment of cells in the microcarrier or a coating on the microcarrier. In addition or alternatively, the microcarrier may be used as drug delivery system and / or adjuvant systems such as vaccine adjuvants. In addition or alternatively, the microcarrier may be used to deliver active ingredients, e.g. in cosmetic or therapeutic applications. In addition or alternatively, the microcarrier may be used to immobilize active ingredients, such as bacteria (e.g. suitable for wastewater treatment or bacteria cultivation), or yeast (e.g. suitable for yeast cultivation), or enzymes (e.g. suitable for use in food processing, pharmaceuticals, biofuel production). In addition or alternatively, the microcarrier may be used as biosensor. In addition or alternatively, the microcarrier may be used as for the cryopreservation (i.e. freeze storage) of cells. In addition or alternatively, the microcarrier may be used to encapsulate active ingredients (e.g. pesticides or herbicides in agricultural applications).

[0208] In an aspect, the present invention pertains to a use of the microcarrier and / or plurality of microcarriers disclosed herein. In a preferred embodiment, the use is selected from the group consisting of pharmaceutical production, vaccine production, gene therapy, regenerative medicine, cell therapy, biologies manufacturing such as antibody or recombinant protein production, viral vector production, cosmetic testing, toxicology testing, 3D cell culture, extracellular vesicle and / or exosome production, bioreactor culture and cultivated meat or fish production. In an embodiment, the microcarrier and / or plurality of microcarriers is provided in an injectable or used in an in-body therapeutic application, such as in embolization, wound healing, or an advanced therapy medicinal product (ATMP) where the microcarrier is part of the implant / cell therapy.

[0209] The microcarrier of the present invention is preferably for one or more of culturing adherent cells, immobilization of cells and / or active ingredients, drug delivery, encapsulation of cells / and / or active ingredients, preferably culturing of adherent cells.

[0210] The microcarrier of the present disclosure is preferably a composite microcarrier, meaning a microcarrier that includes two or more polymers.

[0211] The microparticle or microcarrier may be a discrete body or be deposited or embedded on a substrate so that it is provided as part of a granular substrate. The microparticle or microcarrier thus formed preferably is spherical or substantially spherical. Alternatively, the microparticle or microcarrier thus formed may be ellipsoidal, rod-shaped, sheet-shaped or polygonal shape. In addition or alternatively, the microparticle or microcarrier may have an (average) diameter in particular ranging from 1-1000 pm (micrometers), preferably 10 - 500 pm, more preferably 100-300 pm, even more preferably 150-250 pm. The diameter may be at least 1 , 5, 10, 20, 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 500, 600 or 700 pm and in addition or alternatively, at most 1000, 900, 800, 700, 600, 500, 400, 350, 300, 250200, 150 or 100 pm including ranges between any of the foregoing values. The size may refer to size (average particle size for measurement) of the dry particle or the size of swelling in water or aqueous medium at 20° C for 30 min. Particle size can be preferably determined using for example digital image analysis of microscopic photographs or dynamic light scattering (DLS).ln addition or alternatively, the particle size can be preferably determined by any method described in the Examples. In an embodiment, the particle size or average diametric dimension as disclosed herein is measured with a dynamic image particle size analyzer. A preferred method of measuring particle size or average diametric dimension is by static and / or dynamic digital image-based size analysis (e.g., using QicPic size analyzer).

[0212] For certain applications, such as when used as granule in packed bed reaction, the microparticle or microcarrier disclosed herein may have dimensions up to the mm range, e.g. up to 10 mm in diameter. In embodiments, the microparticle or microcarrier has a diameter of 0.1 - 10 mm, preferably 0.5 - 5 mm, more preferably 1 - 2.5 mm.

[0213] In an embodiment, the amount of ionically crosslinkable polysaccharide and / or collagen derivative in the microcarrier in dry state is 0.1- 90 wt.%, preferably, 0.2 - 75 wt.%, more preferably 0.5-30 wt.%, even preferably 2-25 wt.%, most preferably 5-20 wt.% such as 10-15 wt.%. For the microcarrier in dry state, the amount of ionically crosslinkable polysaccharide and / or collagen derivative can be at least 0.1 wt.%, 0.5 wt.%, 1 wt.%, 5 wt.%, 10 wt.%, 20 wt.%, 30wt.%, 40 wt.%, 50 wt.%, 60 wt.%, 70 wt.%, 80 wt.%, 90 wt.%, 95 wt.%, 97.5 wt.% or 99 wt.% and / or no more than 99 wt.%, 97.5 wt.%, 95 wt.%, 90 wt.%, 80 wt.%, 70 wt.%, 60 wt.%, 50 wt.%, 40 wt.%, 30 wt.%, 20 wt.% or 10 wt.%, calculated on the total dry weight of the microcarrier.

[0214] In addition or alternatively, in an embodiment, the amount of collagen derivative in the microcarrier in dry state is 0.2-40 wt.%, preferably 1-30 wt.%, more preferably 2-20 wt.%, even more preferably 5-10 wt.%, calculated on the total dry weight of the microcarrier.

[0215] In a wet state (i.e. swollen), the microcarrier may comprise 0.1-15 wt.%, preferably 1-10 wt.%, more preferably 2-8 wt.% of the ionically crosslinkable polysaccharide, calculated on total weight including liquid (e.g. water) contained by the gel network. In a wet state (i.e. swollen), the microcarrier may comprise 0.01-25 wt.%, preferably 0.1-15 wt.%, more preferably 1- 10 wt.% of the collagen derivative, calculated on total weight including liquid (e.g. water) contained by the gel network. For the microcarrier in wet state, the amount of ionically crosslinkable polysaccharide and / or collagen derivative can be at least 0.1 wt.%, 0.5 wt.%, 1 wt.%, 5 wt.%, 10 wt.%, 20 wt.%, 30wt.%, 40 wt.%, 50 wt.%, 60 wt.%, 70 wt.%, 80 wt.%, 90 wt.%, 95 wt.%, 97.5 wt.% or 99 wt.% and / or no more than 99 wt.%, 97.5 wt.%, 95 wt.%, 90 wt.%, 80 wt.%, 70 wt.%, 60 wt.%, 50 wt.%, 40 wt.%, 30 wt.%, 20 wt.% or 10 wt.%, calculated on total weight including liquid (e.g. water) contained by the gel network.

[0216] In various embodiments, the microcarrier has a relative density of from about 0.9 g / cm3- 1.3 g / cm3, preferably 1.0 - 1.2 g / cm3.

[0217] In an embodiment, the microcarrier as disclosed herein is edible, which is to be interpreted broadly to refer to suitable for animal or human consumption. In an embodiment, the microcarrier as disclosed herein is “food grade”, which means that it is approved for human consumption by a relevant authority in a jurisdiction, e.g., The European Food Safety Authority (EFSA), the Food and Drug Administration (FDA) or national food agencies.

[0218] The present inventors surprisingly found that the efficiency of cell culture and cell recovery was particularly high when providing and / or obtaining a low lipopolysaccharide (i.e. LPS) content in the microcarrier. Therefore, preferably, the LPS is no more than 3000 Endotoxin Units (EU) / g or more preferably no more than 1000 EU / g. In an embodiment, the LPS content is preferably no more than 100 EU / g, or more preferably no more than 10 EU / g. The LPS content is calculated on the dry weight of the microcarrier, ionically crosslinkable polysaccharide and / or collagen derivative. Preferably the LPS content refers to the amount calculated in the dry weight amount of the collagen derivative (e.g. gelatin). The terms lipopolysaccharide and endotoxin can be used interchangeably and synonymously in the content of the present invention.

[0219] Widely-used and preferred methods for measuring LPS level in the context of the current invention are the Limulus Amebocyte Lysate (LAL) test or the recombinant Factor C (rFC) test, which the skilled person is familiar with. The term means 'endotoxin units'.

[0220] Considering that a positively charged particle may be desirable for the reasons given herein, in an embodiment, the microcarrier has a positive zeta potential, preferably in the range +10 mV to +50 mV or +20 mV to +40 mV or +25 mV to +35 mV. Methods to measure the zeta potential of microparticles are common in the art. A preferred way is by using a zeta potential analyzer. An alternative method is by using dynamic light scattering (DLS) instrument.

[0221] In an embodiment, the net charge and / or surface charge of the microcarrier is modified by endowing the substrate according to the invention with charged molecules, particularly by the addition of one or more poly-electrolytes or polysaccharides selected from the group consisting of agar, alginate, chitosan, dextran, poly(ethylene glycol), collagen, gelatin, hyaluronic acid, carrageenan, fibroin, fibronectin, poly-L-Lysine (PLL), cellulose, graphene, polyethylenimine (PEI), poly(amidoamine) (PAA), dextran sulfate, silk, silk fibroin, Pectin, K- carrageenan, lota carrageenan, Gellan gum, Guar gum, Tragacanth gum, Xanthan gum, Acacia gum, Karaya gum and sodium carboxymethyl cellulose (S-CMC). The polyelectrolytes or polysaccharides may be naturally derived materials and / or synthetically derived materials including recombinant proteins and / or derivatives of these materials.

[0222] The microcarriers obtainable by the method were found to be highly stable yet easily digestible, e.g. with a chelating agent commonly used in cell culture. For instance, the microcarrier can be digested with a chelating agent selected from the group consisting of ethylenediaminetetraacetic acid (EDTA), glycol ether diamine tetraacetic acid (EGTA), 1 ,2- bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetate (BAPTA), N'-(2 hydroxyethyl)ethylenediamine-N,N,N'-triacetic acid (HEDTA) and nitrilotriacetic acid (NTA), sodium citrate, diethylenetriaminepentaacetic acid (DTPA) most preferably EDTA.

[0223] In embodiments of the invention, the microcarrier of the invention is digestible or digested by a precipitation reaction. For example a phosphate or carbonate buffer can dissolve the microcarrier disclosed herein (e.g. alginate-gelatin microcarrier) by forming a calciumphosphate or calcium-carbonate precipitate.

[0224] In an embodiment, the microcarrier is preferably dissolved or digested with a reagent comprising or consisting of a protease and / or a chelator, preferably a mixture of a protease and a chelator, wherein the protease is preferably trypsin (or TrypLE and the like) and the chelator EDTA.

[0225] In an embodiment, the microcarrier is preferably digested with a digestion reagent comprising at least a protease and / or a salt that precipitates with calcium, preferably a mixture of a protease and phosphate-based buffer salt or a carbonate-based buffer salt, more preferably a mixture of a protease and a phosphate-buffered saline (PBS) solution or sodium bicarbonate solution, wherein the protease is preferably trypsin, TrypLE or the like. The term “protease” in the context of the present invention, preferably refers to a protease capable of cleaving peptide bonds between specific amino acids in a collagen-derived material, more preferably, targeting the carboxyl side of lysine and arginine residues within the amino acid chains of a collagen-derived material. The protease disclosed herein may be preferably selected from the group consisting of trypsin, TrypLE, Accutase, collagenase, dispase and papain. The protease disclosed herein may be a recombinant protease and / or a protease blend. For example “TrypLE” is a recombinant protease blend similar to trypsin in functionality but nonmammalian in origin. “Accutase” is a blend of proteases similar to trypsin in functionality but non-mammalian in origin.

[0226] The concentration of the chelating agent used for digestion (e.g. EDTA) is preferably 0.01-10 mM (i.e., milli molar), more preferably 0.05-5 mM, even more preferably 0.1-1 mM.

[0227] In addition or alternatively the microcarrier can be digested with a collagenase, e.g. at a concentration of preferably 0.1-2 mg / mL, preferably 0.2-1 mg / ml, more preferably 0.3-0.5 mg / ml. In addition or alternatively the microcarrier can be digested with a protease such as trypsin (or TrypLE and the like), e.g. at a concentration of 0.1 -5 mg / mL, preferably 0.5-2.5 mg / ml, more preferably 1-1.5 mg / ml.

[0228] In an embodiment, the microcarrier is free or substantially free of one or more of a polygalacturonic acid compound, a pectic acid, a partially esterified pectic acid and / or a partially amidated pectic acid and salts thereof. The cell release from commercially available microcarriers based on polygalacturonic acid compounds requires the use of additional (less biocompatible and / or non-mammalian) non-proteolytic enzymes such as pectinase. The present inventors considers the use of said non-proteolytic enzymes impractical and less compatible with the intended use of the microcarriers, hence use of polygalacturonic acid compounds is preferably avoided.

[0229] In an embodiment, the microcarrier is free or substantially free of dextran and / or agar. In an embodiment, the microcarrier is free or substantially free of agar.

[0230] In embodiments of the invention, the microcarrier comprises one or more bio-adhesive moieties, which allow binding of cellular (transmembrane) receptors hence improving cell adhesion and / or growth. The term “bio-adhesive moiety” in the context of the present invention means a molecular group or functional element attached to the surface of the microcarrier that may enhance cell attachment, interaction and / or growth, preferably by mimicking the natural extracellular matrix components or cell surface signals. The bio-adhesive moieties is preferably covalently linked, tethered and / or bound to the polysaccharide or collagen derivative by a linker molecule. For example the linker may comprise an amine functional group. In an embodiment, the linkage (e.g. covalent linkage) between a polysaccharide and the linker molecule is characterized by having one or more bonds between a carbonyl group and an amino group. In an embodiment, the bio-adhesive moieties are able to bind to one or more from the group of integrin, cadherin, selectin, and immunoglobulin.

[0231] In an embodiment, the microcarrier can bind and / or activate biological entities, in particular bind to and / or activate cells selected from the group of induced pluripotent stem cells, T cells such as Chimeric Antigen Receptor T cells, adult stem cells, hematopoietic stem cells. In a further embodiment, the microcarrier comprises a bio-adhesive moieties that is able to bind to one or more from the group of integrin, cadherin, selectin, and immunoglobulin, more specifically one ore more selected from the group of a4pi (VLA-4), al_p2 (LFA-1), a5pi, a6p4, avp3, avp6, E-cadherin, N-cadherin, VE-cadherin, P-cadherin, E-selectin, P-selectin, L- selectin, ICAM-1 (CD54), VCAM-1 (CD106), PECAM-1 (CD31), NCAM (CD56), CD3 and CD28. These integrins, cadherins, selectins, and immunoglobulins are central to cellular adhesion, migration, and signaling processes that are crucial for cell and gene therapy applications, particularly in the context of immune modulation, stem cell targeting and tissue repair.

[0232] In an embodiment, a product, powder, or slurry comprising microcarriers or microparticles according to the invention contains no or substantially no unbound biological entity-binding moieties, or contains less than 1 mM, preferably less than 100 pM, even more preferably less than 10 pM, and most preferably less than 1 pM of unbound biological entities, for example in the range of 1 pM to 1 pM. In another embodiment, a product, powder, or slurry comprising microcarriers or microparticles according to the invention has all or substantially all biological entity-binding molecules bound, attached, or tethered to the microcarriers or microparticles.

[0233] In an embodiment, the microcarrier or microparticle of the invention includes a bio-active moiety, peptide sequence, or peptide epitope capable of interacting with a cell surface receptor, cytokine, or other bio-active moiety. More particularly, the bio-active moiety, peptide sequence, or peptide epitope may support binding or adhesion of cells, growth factors, extracellular matrix components, or ions. Even more specifically, the bio-active moiety, peptide sequence, or peptide epitope may be derived from one or more compounds selected from the group comprising Fibronectin, Laminin, Collagen, Nidogen, CAM, VCAM, Netrin, Thrombospondin, Cadherin, BMP-2, TGF-B1 , BMP-7, VEGF, Osteonectin, Osteopontin, Angiopoietin, FGF-2, Phage-display, BDNF, NGF, NOAM, Myxinidin, Melittin, Ovalbumin, T- cells, Enolase, H1 N1, Heparin, Polysaccharide, Decorin, Amelogenin, Dentin phosphoprotein, Statherin, Perlecan, Elastin, Agrin, Fibulin, EMILIN, Periostin, Transglutaminase, Versican, Tenascin, Fibrillin, Fibrinogen, Bone sialoprotein, Factor X, and iC3b, or one or more peptides may offer synergistic or additive effects, such as enhanced cell adhesion.

[0234] In an embodiment, the microcarrier or microparticle according to the invention includes one or more peptide sequences selected from the group consisting of RGD, IKVAV, YIGSR, DGEA, REDV, LRE, QAGTFALRGDNPQG, HAVDI, LDV, with RGD being preferred.

[0235] In an embodiment, the microcarrier or microparticle of the invention includes one or more moieties capable of binding a cluster of differentiation (CD) marker, particularly selected from CD19, CD20, CD22, CD30, CD33, CD123, CD133, CD276 (B7-H3), CD70, CD3, CD4, CD25, CD47, CD7, and CD28. More specifically, the CD-binding moiety may be an antibody, preferably an immunoglobulin, or a VHH (Variable Heavy domain of Heavy chain, also known as sdAb, llama-body, or nanobody).

[0236] In an embodiment, the substrate and / or microcarrier of the invention includes antibodies capable of binding T-cell surface receptors, specifically targeting CD3 and / or CD28, enabling convenient in vitro activation of human T cells without requiring antigen-presenting cells. In one specific embodiment, human T cells are stimulated with anti-CD3 and anti-CD28 antibodies coated on microbeads.

[0237] In an embodiment, the substrate and / or microcarrier of the invention exhibits magnetic properties. More specifically, the microparticle or microcarrier may contain magnetic or paramagnetic features, preferably derived from magnetic or magnetizable particles. In one embodiment, the substrate and / or microcarrier includes magnetic compounds selected from the group consisting of Magnetite (Fe3O4), Maghemite (y-Fe2O3), Cobalt Ferrite (CoFe2O4), Nickel Ferrite (NiFe2O4), Superparamagnetic Iron Oxide Nanoparticles (SPIONs), General Ferrites (MFe2O4, where M = Mn or Zn), with magnetite being preferred.

[0238] In an embodiment, the microparticle of the invention is suitable for the cultivation of induced pluripotent stem cells (iPSCs), wherein the microparticle comprises one or more proteins or derivatives thereof selected from the group consisting of fibronectin, collagen IV, laminin, and vitronectin. Core-shell structure

[0239] The present inventors found that in certain embodiments (e.g. for cell attachment purposes), it may be sufficient to provide the collagen derivative and / or other biologic entity-binding moieties (mostly) on the outside of the microcarrier. When the collagen derivative is mostly or only on the outside, it maximizes the available attachment area, enabling cells to more easily access and anchor to the microcarrier surface. Moreover, the amount of required biologic entity-binding and / or collagen derivative can be reduced, without affecting the functionality of the microcarrier. In addition or alternatively, the microcarrier may be more easily dissolvable when needed by providing the collagen derivative mostly or only as a shell layer.

[0240] The present inventors found that the Maillard reaction may provide for a rigid and stable shell structure of collagen derivative around the core. Without Maillard reaction, surfaces coated with the collagen derivative may be less stable and suboptimal for supporting long-term cell adhesion, since cells may render the shell structure to deteriorate over time. In particular, it is considered by the present inventors that the collagen derivative shell layer is easily detached or released in absence of Maillard reaction disclosed herein. In particular, the Maillard reaction may provide for covalent crosslinking between the shell (collagen derivative) and core (polysaccharide) components.

[0241] Since the present inventors consider it advantageous to have an outer layer of collagen derivative (e.g. as “coating” or “shell”), in certain embodiments, the microcarrier has a coreshell structure defined by a core comprising ionically crosslinkable polysaccharide, the core optionally comprising collagen derivative, with a collagen derivative layer disposed around the core.

[0242] The ratio’s of two or more components herein refer to the molar ratio, unless indicated otherwise.

[0243] In the core-shell structure, the core may contain a polymer matrix comprising crosslinked ionically crosslinkable polysaccharide and collagen derivate. The core may have a ratio of ionically crosslinkable polysaccharide (e.g. alginate) and collagen derivative (e.g. gelatin) in the range of 10000:1 to 1 :1000, preferably 1000:1 to 1 :100, more preferably 100:1 to 1:10, even more preferably 10:1 to 1 :1. In an embodiment, the core preferably has a ratio of ionically crosslinkable polysaccharide and collagen derivative (e.g. gelatin) of 10000:1 to 1 :1, preferably 1000:1 to 10:1 , more preferably 100:1 to 50:1. In embodiments, the core may contain the ionically crosslinkable polysaccharide or collagen derivative in an amount of more than 50% by weight or volume, more than 60% by weight or volume, more than 70% by weight or volume, more than 80% by weight or volume, more than 90% by weight or volume or more than 95% by weight or volume, more than 97% by weight or volume or more than 99% by weight or volume, with respect to the total dry weight or volume of the polymer matrix contained in the core and / or less than 99% by weight or volume, less than 97% by weight or volume, less than 95% by weight or volume, less than 90% less than by weight or volume, less than 80% by weight or volume, less than 70% by weight or volume, less than 60% by weight or volume, with respect to the total dry weight or volume of the polymer matrix contained in the core.

[0244] In the core-shell structure, the shell may have a ratio of collagen derivative (e.g. gelatin) to ionically crosslinkable polysaccharide (e.g. alginate) in the range of 10000:1 to 1 :1000, preferably 1000:1 to 1 :100, more preferably 100:1 to 1:10, even more preferably 10:1 to 1 :1. In an embodiment, the shell may have a ratio of collagen derivative (e.g. gelatin) to ionically crosslinkable polysaccharide (e.g. alginate) in the range of 10000:1 to 1:1 , preferably 1000:1 to 10:1, more preferably 100:1 to 50:1.

[0245] In embodiments, the shell may comprise the collagen derivative in an amount of more than 50% by weight or volume, more than 60% by weight or volume, more than 70% by weight or volume, more than 80% by weight or volume, more than 90% by weight or volume, more than 95% by weight or volume, more than 97% by weight or volume or more than 99% by weight or volume, with respect to the total dry weight or volume of the polymer matrix contained in the shell. The collagen derivative may also be contained in an amount of 100% by weight or volume or less, 99% by weight or volume or less, 97% by weight or volume or less, 95% by weight or volume or less, 90% by weight or volume or less, 80% by weight or volume or less or 70% by weight or volume or less, with respect to the total dry weight or volume of the polymer matrix contained in the shell.

[0246] In an embodiment, the collagen derivative is comprised in a shell structure and the core is (substantially) free of collagen derivative or comprises collagen derivative in a low amount. In embodiments, the collagen derivative is comprised in a shell structure as disclosed herein and the core comprises less than 10% or less than 5 % collagen derivative by weight or volume of the core. In embodiments, the collagen derivative is comprised in a shell structure as disclosed herein and the core comprises less than 2% or less than 1% collagen derivative by weight or volume of the core. In an embodiment, the shell layer may consist of collagen derivative and / or be free or substantially free of ionically crosslinkable polysaccharide . In an embodiment, the microcarrier has a core-shell structure defined by:

[0247] -a core comprising ionically crosslinkable polysaccharide in an amount of at least 80%, preferably at least 90%, more preferably at least 95% by weight or volume of the core, and -a shell layer comprising at least 80%, preferably at least 90%, more preferably at least 95% collagen derivative by weight or volume of the shell, more preferably wherein the shell layer is an outer layer of the microcarrier consisting of collagen derivative.

[0248] In embodiments, the microcarrier has a core-shell structure defined by a core comprising ionically crosslinkable polysaccharide in an amount of at least 50%, by weight or volume of the core. In embodiments, the microcarrier has a core-shell structure defined by a core comprising ionically crosslinkable polysaccharide in an amount of at least 60%. In embodiments, the microcarrier has a core-shell structure defined by a core comprising ionically crosslinkable polysaccharide in an amount of at least 70%. In embodiments, the microcarrier has a core-shell structure defined by a core comprising ionically crosslinkable polysaccharide in an amount of at least 80%. In embodiments, the microcarrier has a coreshell structure defined by a core comprising ionically crosslinkable polysaccharide in an amount of at least 90%. In embodiments, the microcarrier has a core-shell structure defined by a core comprising ionically crosslinkable polysaccharide in an amount of at least 95%. In embodiments, the microcarrier has a core-shell structure defined by a core comprising ionically crosslinkable polysaccharide in an amount of at least 99%. In embodiments, the microcarrier has a core-shell structure defined by a core consisting of, or consisting essentially of, ionically crosslinkable polysaccharide.

[0249] In addition or alternatively, in embodiments, the microcarrier has a core-shell structure defined by a shell layer comprising at least 50% collagen derivative by weight or volume of the shell. In embodiments, the microcarrier has a core-shell structure defined by a shell layer comprising at least 60% collagen derivative. In embodiments, the microcarrier has a coreshell structure defined by a shell layer comprising at least 70% collagen derivative. In embodiments, the microcarrier has a core-shell structure defined by a shell layer comprising at least 80% collagen derivative. In embodiments, the microcarrier has a core-shell structure defined by a shell layer comprising at least 90% collagen derivative. In embodiments, the microcarrier has a core-shell structure defined by a shell layer comprising at least 95% collagen derivative. In embodiments, the microcarrier has a core-shell structure defined by a shell layer comprising at least 99% collagen derivative. In embodiments, the microcarrier has a core-shell structure defined by a shell consisting of, or consisting essentially of, collagen derivative. In an embodiment, the shell is defined as the outer 5 pm layer of the microcarrier. In an embodiment, the shell is defined as the outer 10 pm layer of the microcarrier. In an embodiment, the shell is defined as the outer 25 pm layer of the microcarrier. In an embodiment, the shell is defined as the outer 50 pm layer of the microcarrier. In an embodiment, the shell is defined as the outer 100 pm layer of the microcarrier.

[0250] In an embodiment, the shell is defined as the outer 1 nm layer of the microcarrier. In an embodiment, the shell is defined as the outer 5 nm layer of the microcarrier. In an embodiment, the shell is defined as the outer 10 nm layer of the microcarrier. In an embodiment, the shell is defined as the outer 25 nm layer of the microcarrier. In an embodiment, the shell is defined as the outer 50 nm layer of the microcarrier. In an embodiment, the shell is defined as the outer 100 nm layer of the microcarrier. In an embodiment, the shell is defined as the outer 500 nm layer of the microcarrier. In embodiments, the shell is defined as the outer layer of the microcarrier which encompasses at least 0.001% by volume or of the weight of the microcarrier, calculated on the total dry weight. In embodiments, the shell is defined as the outer layer of the microcarrier which encompasses at least 0.005% by volume or of the weight of the microcarrier, calculated on the total dry weight. In embodiments, the shell is defined as the outer layer of the microcarrier which encompasses at least 0.01 % by volume or of the weight of the microcarrier, calculated on the total dry weight. In embodiments, the shell is defined as the outer layer of the microcarrier which encompasses at least 0.05% by volume or of the weight of the microcarrier, calculated on the total dry weight. In embodiments, the shell is defined as the outer layer of the microcarrier which encompasses at least 0.1% by volume or of the weight of the microcarrier, calculated on the total dry weight.

[0251] In addition or alternatively, embodiments, the shell is defined as the outer layer of the microcarrier which encompasses no more than 1 % by volume or of the weight of the microcarrier, calculated on the total dry weight. In embodiments, the shell is defined as the outer layer of the microcarrier which encompasses no more than 0.5% by volume or of the weight of the microcarrier, calculated on the total dry weight. In embodiments, the shell is defined as the outer layer of the microcarrier which encompasses no more than 0.1% by volume or of the weight of the microcarrier, calculated on the total dry weight. In embodiments, the shell is defined as the outer layer of the microcarrier which encompasses no more than 0.05% by volume or of the weight of the microcarrier, calculated on the total dry weight. In embodiments, the shell is defined as the outer layer of the microcarrier which encompasses no more than 0.01 % by volume or of the weight of the microcarrier, calculated on the total dry weight. In an embodiment, the shell is defined as the outer layer of the microcarrier which encompasses 1% by volume or of the weight of the microcarrier, calculated on the total dry weight.

[0252] In an embodiment, the shell is defined as the outer layer of the microcarrier which encompasses 2% by volume or of the weight of the microcarrier, calculated on the total dry weight.

[0253] In an embodiment, the shell is defined as the outer layer of the microcarrier which encompasses 5% by volume or of the weight of the microcarrier, calculated on the total dry weight.

[0254] In an embodiment, the shell is defined as the outer layer of the microcarrier which encompasses 10% by volume or of the weight of the microcarrier, calculated on the total dry weight.

[0255] In an embodiment, the shell is defined as the outer layer of the microcarrier which encompasses by volume or 20% of the weight of the microcarrier, calculated on the total dry weight.

[0256] The core-shell structure can for instance be demonstrated using photoelectron spectroscopy (XPS), elemental analysis and / or determining the charge (change) of the particle.

[0257] Moreover, it can be confirmed by taking an infrared (IR) photograph of the characteristic peak of the ionically crosslinkable polysaccharide relative to the collagen derivative (e.g. gelatin) characteristic peak of the produced microcarriers. In addition or alternatively, the distribution of the collagen derivative may be determined by using a collagen derivative which is labelled (e.g. with fluorescent dye, biotin or radioisotope) before forming the microcarrier. The distribution of collagen derivative can be visualized (e.g. by microscopy such as confocal microscopy). The distribution of collagen derivative can be quantified by image analysis and / or extraction and a fluorometric assay. A preferred way is to use a collagen derivative which is fluorescently labelled, and visualizing and / or quantifying as aforementioned.

[0258] Plurality of microcarriers

[0259] Furthermore provided as part of the present disclosure is a plurality of microcarriers disclosed herein.

[0260] In an embodiment, the plurality of microcarriers is provided as a dry powder. In an embodiment, the plurality of microcarriers is provided as a liquid suspension such as a wet slurry. The plurality of microcarriers is preferably provided as a slurry because this avoids the need for an additional rehydration step. Moreover, if the heat-treatment is by autoclaving or the like, also the need for a further sterilization step can be circumvented.

[0261] The present inventors consider it advantageous to have a collection of microcarriers which are as uniform as possible with respect to their shape and size, which for example ensures that cells reach confluence at approximately the same time. In various embodiments, the present invention provides for a plurality of microcarriers as disclosed herein, have a coefficient of variation of the diameter of less than 20%, less than 15%, less than 10%, less than 9%, less than 8%, less than 7%, less 6%, less than 5%, less than 4%, less than 3% or less than 2% and / or more than 0.2%, more than 0.5%, more than 1%, more than 2%, more than 3%, more than 4% or more than 5%. In an embodiment, the monodispersity of the microcarriers disclosed herein is characterized by a coefficient of variance of the particle size distribution of less than 15%, preferably less than 10%, more preferably 0.5-5%.

[0262] Cell laden microcarrier

[0263] The present invention pertains also to a cell-laden microcarrier, comprising the microcarrier disclosed herein and one or more adherent cells on the surface of the microcarrier.

[0264] The microcarrier of the present invention is suitable for culture of any type of adherent cell. The term “adherent cell” in the context of the present invention means a type of cell that requires attachment to a solid or semi-solid surface to grow. The adherent cell in the context of the present invention is not limited to a particular type, although several examples can be provided. For example, the adherent cell may be a cell used in the manufacturing of meat or fish alternatives and / or a cell present in the final edible product, such as a myoblast (i.e. muscle cell), adipocyte (i.e. fat cell), smooth muscle cell, induced pluripotent stem cells or fibroblast. In addition or alternatively, the adherent cell may be an undifferentiated (stem) cell including but not limited to embryonic stem cell, endothelial progenitor cells, induced pluripotent stem cell, neural stem cell, cardiac progenitor cell, mesenchymal stem cells, adipose-derived stem cell and vascular endothelial progenitor cell.

[0265] In addition or alternatively, the adherent cell may be a cell or cell line used in biologies manufacturing, such as cells 3T3 cells, HEK 293 cells, Vero cells, MRC5 cells, CHO cells, BHK-21 cells, Hela cells or hybridomas. Several other examples of adherent cell types, e.g. suitable for tissue repair are chondrocytes, keratinocytes or osteoblasts. In a preferred embodiment, the adherent cell is an induced pluripotent stem cells (iPSCs).

[0266] The terms “adherent cell” and “anchorage-dependent cell” can be used interchangeably and synonymously in the context of the present invention. The terms “mesenchymal stem cell”, “multipotent stromal cell” and “multipotent mesenchymal stromal cell” can be used interchangeably in the context of the present invention to refer to the MSC cell type meeting the minimal criteria as defined by the Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy (Dominici et al. Cytotherapy . 2006;8(4):315-7. doi: 10.1080 / 14653240600855905) .

[0267] The cell-laden microcarrier can be provided in an aggregate of cell-laden cell microcarriers which optionally further comprises a carrier material (e.g. scaffold material) known in the art.

[0268] Method of culturing cells

[0269] In a further aspect, the present invention pertains to a method of culturing adherent cells, comprising contacting one or more adherent cells with the microcarrier disclosed herein in an aqueous medium, e.g. cell culture medium, salt solution or the like. Contacting the cells with the microcarriers allows for cell adhesion and proliferation / culture on the microcarrier. Preferably the microcarriers are mixed and / or agitated during the adhesion and / or culture such as by means of a stirring method such as provided by a spinner device and / or magnetic stirrer. Other mixing methods include shakers and / or rockers and the like. In embodiments, as cells proliferate on microcarriers, part of the microcarriers can be replaced or new microcarriers can be added to further expand the culture. Cells can colonize new microcarriers by transferring to new beads in suspension or by including a static culture period to enforce close proximity of all microcarriers and cells.

[0270] In an embodiment, the method of culturing adherent cells comprises a step of de-attaching at least part of the adherent cells by digesting the microcarrier by a method disclosed herein, preferably with a digestion reagent comprising a protease and / or a chelator, preferably a mixture of a protease and a chelator, wherein the protease is preferably trypsin, TrypLE or the like, and the chelator is preferably ethylenediaminetetraacetic acid (EDTA). The digestion reagent is preferably free or substantially free of an enzyme, preferably non-proteolytic enzyme, more preferably a pectinase. In an embodiment, digesting the microcarrier is done such that all of the microcarrier is solubilized. In embodiment, digesting the microcarrier is done such that least 50%, 60%, 70%, 80%, 90%, 95% or 99% of the microcarrier is solubilized.

[0271] In processes where high cell densities are required, such as in the production of biologies, tissue engineering, or regenerative medicine, the microcarrier or plurality of microcarriers disclosed herein may be provided in a packed bed configuration. A packed bed reactor design comprises one or more columns filled with solid support materials (known also as packing) where cells can adhere and grow. In particular, a packed bed cell culture reactor may include a chamber containing the (plurality of) dissolvable microcarriers, which serve as a scaffold for cell attachment and growth. The microcarriers can be packed densely within the reactor chamber, forming a structured bed that provides a high surface area-to-volume ratio, optimized for efficient cell adhesion and proliferation. The packed bed reactor may provide for a dissolution mechanism which enables easy harvesting of the cultured cells without the need for mechanical disruption. In such a case, microcarrier dissolution may be achieved by perfusing the packed bed with the harvesting solution. In an embodiment the dissolution rate of the microcarriers is controlled by the polymer composition, crosslinking degree, or pH of the culture conditions and / or reactor environment. In the context of a packed bed cell culture reactor, the “pellet size” means the size of the aggregated cell masses or cell-laden particles that form within the packed bed. In the case of a packed bed cell culture reactor, the pellet size may be typically 1-10 mm.

[0272] General definitions

[0273] -The terms ‘comprising’ or ‘to comprise’ and their conjugations are used in the context of the current invention in their non-limiting sense to indicate that items following the word are included, but items not specifically mentioned are not excluded.

[0274] -Reference to an element by the indefinite article ’a’ or ‘an’ does not exclude the possibility that more than one of the elements 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’.

[0275] -In the context of the current invention, a level is considered “increased” or “decreased” when it is at least 1% (such as at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%) higher or lower, respectively, than the corresponding level in a control or reference. In addition or alternatively, a level is considered increased or decreased when it is statistically significantly higher or lower, respectively, compared to a level in a control or reference (including compared to an earlier time point), irrespective of the size of the change. The term “to reduce” may in the context of the current invention be used interchangeably with the term “to decrease”.

[0276] FIGURE LEGENDS

[0277] Figure 1. A. Total cell yield after 24 h of static well-plate culture on the lamF A (according to invention) and Cytodex-1 (comparative) microcarriers. B. Harvest efficiency of bone marrow derived human mesenchymal stromal cells cultured for 24 h in a static well-plate format on the lamF A (according to invention) and Cytodex-1 (comparative) microcarriers. C. Population doubling time of cells cultured in a spinner flask on the lamF A (according to invention) and Cytodex-1 (comparative) microcarriers. Significant differences determined using ANOVA with Tukey’s post-hoc test (n=3), **** P < 0.0001 ; ‘ns’ indicates not significant.

[0278] Figure 2. Visual inspection of spinner culture of bone marrow-derived human mesenchymal stromal cells on lamF A (according to the invention) and Cytodex-1 (comparative) microcarriers after 9 days of culture in a spinner flask.

[0279] Figure 3. Visualization of the dissolution of the lamF A microcarriers and release of bone marrow-derived human mesenchymal stromal cells during 15 minute incubation with TrypLE and EDTA.

[0280] Figure 4. Cell attachment comparison between microcarriers coated at pH 6 (Alg / Gel+, according to the invention) and pH 11 (Alg / Gel-, comparative example). Scalebar represents 500 pm.

[0281] Figure 5. Dissolvability comparison between microcarriers coated at pH 6 (Alg / Gel+, according to the invention) and pH 11 (Alg / Gel-, comparative example), using either EDTA alone or Tryple + EDTA. Scalebar represents 500 pm.

[0282] EXAMPLES

[0283] Example 1

[0284] Example 1 describes a method of preparing of alginate-gelatin microcarriers and the technical specifications thereof.

[0285] Method

[0286] Solution preparation

[0287] A liquid preparation of 1 wt.% alginate is prepared in demineralized water. The alginate solution is placed on a stirring plate and stirred (magnetic stirred, 600 RPM) overnight. A crosslinker solution (0.1M CaCh 10 wt% EtOH) is prepared.

[0288] A gelatin solution (2 wt.%) is prepared in demineralized water and placed in a 60 °C water bath and stirred (magnetic stirred, 250 RPM) for 1.5 h. The gelatin used is high molecular weight gelatin (300 bloom, type A).

[0289] A CaCh washing solution (0.2M) is prepared by dissolving CaCh(dihydrate) in demineralized water.

[0290] Alginate microparticle production

[0291] Microparticles are produced by reacting liquid droplets of the alginate solution with a crosslinking solution of 0.1M CaCh + 10% EtOH. The microparticles are collected in a beaker pre-filled with dH2O. In the present example, the microparticles are produced using an in-air microfluidics method, although any other method allowing formation of liquid droplets with appropriate dimensions are suitable.

[0292] The alginate microparticles are transferred in liquid to an autoclavable container and washed once in dH2O by letting the sample sediment and aspirating the supernatant or sieving the particles. dH2O is added once again to regain the original volume. Sedimentation should take around 10 minutes. Samples are stored in the fridge (2-8 °C) until further use.

[0293] Particle coating with gelatin

[0294] The 2 wt% gelatin solution is heated to 60°C in a water bath for 30-90 minutes. All other liquids are warmed up to room temperature (RT).

[0295] The 2 wt% gelatin solution is added to the microparticle solution in 1 :1 ratio to obtain a final gelatin concentration of 1 wt% to the bottle with particles.

[0296] The bottle with gelatin is placed in a magnetic stirring water bath at 60 °C and the gelatin is allowed to coat the particles for 2 h under stirring (magnetic stirrer, 300 RPM).

[0297] After coating, the particles are washed by letting the particles sediment and aspirating the supernatant. The particles are washed fully 2 times with 0.2M CaCI2at RT and 1 time with dH2O at RT, ending with a wash in distilled water (dH2O).

[0298] Glutaraldehyde is added to reach a 2% v / v concentration and the gelatin is allowed to crosslink for 30 min (stir plate, 300 RPM).

[0299] The particles are washed by letting the particles sediment and aspirating the supernatant. Particles are washed fully 2 times with 0.2M CaCI2at RT and 1 time with dH2O at RT, ending with a wash in dH2O. The samples are stored in the fridge (2-8 °C) until further use.

[0300] Autoclaving procedure

[0301] Samples are autoclaved according to a standard autoclaving protocol (121 °C, 15 psi, 15 min, no drying).

[0302] Samples are stored in the fridge (2-8 °C) until further use. Alternatively, the samples are stored at room temperature.

[0303] Particle size determination

[0304] The particle size is determined using static and / or dynamic digital image-based size analysis (e.g., using QicPic size analyzer)

[0305] Freeze drying

[0306] Maltodextrin (DE 13-17) powder is dissolved in Milli-Q water (demi water can also be used) up to 60% w / v. Indicatively, particles can be preserved with 7-10% w / v Maltodextrin cryoprotectant (CPA). Preferably, before CPA infusion the particles must be in Milli-Q water. CaCh in the solution could affect the process. The particles are strained with a cell strainer. Ideally the strainer has a mesh size not smaller than half of the particle diameter, that way the particles will strain faster. Infusion time is proportionate to particle size. For hydrogel particles with a diameter of 150pm, >2 h is recommended. Overnight is suggested.

[0307] Optionally particles are strained to remove excess cryoprotectant, e.g., using aspiration, a cell strainer or sieve, and the sample is frozen, e.g., by placing it on the metal surface of a -80 freezer, so that the sample is in direct contact with the metal plate. Alternatively, the sample could be frozen by covering it with liquid nitrogen. Optionally, particles are rinsed with water prior to freezing, to remove excess CPA from the particle surface. The frozen samples are transferred to the freeze drier, while preventing thawing and following standard freeze drying protocol.

[0308] Results

[0309] Diameter: > 90% of microcarriers have a diameter between 170 - 250 pm (average -220 pm), measured after swelling in Dulbecco’s Modified Eagle’s Medium (DMEM).

[0310] Coefficient of variation: < 10%

[0311] Circularity: > 80%

[0312] Surface area: - 2200 cm2 / g dry weight

[0313] Relative density: 1.014 ± 0.001 g / cm3(determined in DMEM medium) Dissolvability: the microcarriers are dissolvable using TrypLE + EDTA or Trypsin + EDTA (see Example 2 for protocol)

[0314] Example 2

[0315] Example 2 describes an example cell culture and harvesting protocol on the microcarrier as obtained in Example 1. The cell culture protocol is however also suitable for embodiments of the microcarrier, other than the specific embodiment in Example 1.

[0316] Microcarrier preparation

[0317] 1. Disperse the microcarriers in dhW and let hydrate for at least 30 minutes at room temperature. Use 50 mL dhW per gram of microcarriers.

[0318] 2. Let the microcarriers settle, remove the supernatant and replace it with 50 mL fresh dhW per gram of microcarriers. Let the carriers rest in the fresh dhW for 3 minutes, repeat this step two times. Finish in fresh dhW (50 mL per gram of microcarrier).

[0319] 3. Optionally sterilize the microcarriers in an autoclave at 121 °C, 15 psi for 15 minutes. 4. Prior to use, suspend the microcarriers well by gently shaking the container and transfer the desired amount of stock microcarrier solution to a new sterile container. Unused resuspended microcarriers should be stored at 4 - 8°C. It is recommended to use them within 1-3 months after preparation.

[0320] 5. Remove the supernatant and wash the microcarriers once in warm cell culture medium for 3 minutes (use 50 mL medium per gram of microcarrier).

[0321] 6. Add fresh warm cell culture medium. The microcarriers are now ready for use and can be transferred to the culture vessel.

[0322] Example culture protocols

[0323] To find the right culture conditions, it is recommended to first use the microcarriers in a static experiment in a well plate, before initiating dynamic (spinner flask) culturing. The optimal cell seeding density (cells / cm2) can vary per cell type and / or passage number. It is recommended to start with the standard cell seeding density as used in 2D culture and to optimize from there. An example of a first procedure is described below both for static and dynamic experiments.

[0324] Static culture

[0325] 1 . Prepare a working solution of 5 cm2 / mL microcarriers in cell culture medium by following the steps as previously described under Microcarrier preparation.

[0326] 2. Add 4 mL of the microcarrier working solution per well of a 6-well ultra-low attachment plate to obtain a total surface area of 20 cm2per well.

[0327] 3. Inoculate the culture with 1x105cells, resulting in a density of 5x103cells / cm2, or use the recommended 2D seeding density of the cell type in use.

[0328] 4. Incubate the cells with the microcarriers for at least 2 days under standard cell culture conditions (typically 37°C and 5% CO2) and check the plate regularly under the microscope to observe cell attachment and growth.

[0329] Dynamic culture

[0330] 1. Prepare a working solution of 20 cm2 / mL microcarriers in cell culture medium by following the steps outlined above.

[0331] 2. Add the suspension to the spinner flask and equilibrate the environment for at least 20 minutes at 35 RPM. 3. Seed cells at a concentration of 800 cells / cm2.

[0332] 4. Incubate the spinner flask cells with microcarriers under standard cell culture conditions (typically 37°C and 5% CO2).

[0333] 5. Start the culture with a 2-hour static incubation, followed by a continuous loop of 6 hours mixing and 2 hours static

[0334] Visualization

[0335] To visualize cells on microcarriers cultured in a spinner flask, use pipet to take a sample of the suspended culture and transfer to a well plate. Phase-contrast microscopy generally allows for easier visualization than brightfield microscopy. Alternatively, fluorescent staining of the cells and visualization using a fluorescent microscope may aid in visualization.

[0336] Harvesting

[0337] Cell harvesting from microcarriers can be performed at high efficiency with either TrypLE-EDTA (1x TrypLE supplemented with 2.5mM EDTA) or Trypsin-EDTA (0.25%) or. An example of a harvesting protocol using these harvesting solutions is described below.

[0338] TrypLE-EDTA (recommended):

[0339] 1. Prepare the harvesting solution to obtain a 1X TrypLE solution containing 2.5mM EDTA in PBS. Prepare 2 mL of harvesting solution per 3 mL of culture volume. The recommended harvesting solution (final volume 5 mL) has the following composition: TrypLE 10x (0.5 mL), EDTA 0.5 M (0.25 mL), PBS 1X (4.25 mL).

[0340] 2. Transfer the microcarrier suspension to a 15 mL or 50 mL centrifuge tube and let the microcarriers settle down for approximately 1 minute.

[0341] 3. Remove 2 / 3 of the solution without disturbing the microcarrier pellet.

[0342] 4. Add the same amount of 1x harvesting solution (TrypLE-EDTA) as the volume that was removed.

[0343] 5. Incubate the tube containing the cell-laden microcarriers with harvesting solution for at least 5-15 minutes at 37°C. Invert the tube occasionally to mix the solution. Check whether the microcarriers are completely dissolved by transferring a small sample to a wells plate and visualizing using a microscope. If there are still microcarriers visible, keep incubating until all microcarriers are completely dissolved. 6. Wash 2x by first forming a pellet through centrifugation (300 RCF, 3-5 minutes) and then gently replacing 90-95% of supernatant with fresh cell culture medium using a micropipette and without disrupting the pellet.

[0344] 7. Once microcarriers are completely dissolved, the cell suspension is ready for further use.

[0345] Trypsin-EDTA:

[0346] 1. Transfer the microcarrier suspension to a 15 mL or 50 mL centrifuge tube and let the microcarriers settle down for approximately 1 minute .

[0347] 2. Remove the supernatant and wash the microcarrier suspension at least once with 1X PBS (wash with the same volume as the amount of supernatant removed).

[0348] 3. Replace the supernatant with harvesting solution (T rypsin-EDTA, 0.25%), gently mix by swirling the tube, and incubate for at least 5 - 15 minutes at 37 °C. Invert the tube occasionally to mix the solution. Check whether the microcarriers are completely dissolved by transferring a small sample to a wells plate and visualizing using a microscope. If there are still microcarriers visible, keep incubating until all microcarriers are completely dissolved.

[0349] 4. Wash 2x by first forming a pellet through centrifugation (300 RCF, 3-5 minutes) and then gently replacing 90-95% of supernatant with fresh cell culture medium using a micropipette and without disrupting the pellet.

[0350] 5. The cell suspension is now ready for further use.

[0351] Example 3 describes the efficiency of cell attachment and growth on alginate-gelatin microcarriers in comparison to commercially available microcarriers.

[0352] Method

[0353] Microcarriers

[0354] The microcarriers of the invention are denoted in this Example as “lamF A”. The The lamF A microcarriers are produced according to Example 1. As a comparative example, commercially available Cytodex-1 microcarriers are included. Cytodex-1 are microcarriers (typically ~ 100-200 micrometers) based on a core of crosslinked dextran. The microcarriers of the invention are compared to Cytodex-1 as Cytodex-1 is the market standard microcarrier used in industry.

[0355] Preparation of microcarriers

[0356] The microcarriers are diluted at 250 cm2 / mL in cell culture medium.

[0357] The Cytodex-1 microcarriers are prepared according to the procedure below (according to manufacturer’s instructions):

[0358] - a stock solution of Cytodex-1 microcarriers is prepared at a microcarrier concentration of 7 g / L (weight: 1.89 g, volume 0,27 L)

[0359] - Hydrate the microcarriers in Ca2+ and Mg2+ free PBS for at least 3 hours at room temperature.

[0360] - Decant the supernatant and wash the microcarriers in fresh Ca2+ and Mg2+ free PBS for 2 to 3 minutes.

[0361] - Discard the PBS and replace with fresh Ca2+ and Mg2+ free PBS

[0362] - Sterilize the microcarriers in an autoclave at 115°C, 15 psi for 15 minutes.

[0363] - Prior to use, allow the microcarriers to settle and remove the supernatant.

[0364] - Rinse the microcarriers briefly in warm culture medium.

[0365] Cell seeding

[0366] Bone marrow-derived human mesenchymal stromal cells (BM-MSC) are used. After thawing, cells are seeded 900 cells / cm2and incubated for 6 days in BM-MSC cell culture medium. Medium is refreshed every 3-4 days until use.

[0367] For lamF A microcarriers, 4 mL of 5 cm2 / mL microcarriers is added to ultra-low attachment plate (3471 ; Corning). For Cytodex-1 , 952 pL of the microcarrier solution is added to the wells and medium added up to 4 mL.

[0368] The BM-MSC are seeded at a density of 5000 cells / cm2 (1 E05 cell / well) (added cells were suspended in ~150uL). The 6-well plates are incubated static at 37°C and 5% CO2. A monolayer control of BM-MSC is seeded with 5000 cells / cm2onto T25 flasks.

[0369] Cell expansion in spinner flasks • 5 mL BM-MSC cell suspension is added to a T-25 flask

[0370] • 50 mL containing microcarriers with a total surface area of 1050 cm2is added to the spinner flask

[0371] • Begin equilibrating the environment within the spinner flask for at least 20 minutes at 35 rpm using the stirring regime, 2 hour static followed by 6 hour mixing, in a continuous loop.

[0372] • Harvest BM-MSC from pre-culture and determine cell numbers.

[0373] • Seed 800 cells / cm2onto T-25 and in spinner flasks.

[0374] • T25 = 2e4 cells

[0375] • spinner flasks = 8.4e5 cells

[0376] • Start the culture with 2 hour static to facilitate the cells to attach to the microcarriers

[0377] • The microcarrier surface is expanded when the cell density is between 3000 - 5000 cells / cm2. This is done by expanding the surface 3 times by diluting the volume (Remove homogenous MC-suspension until 16.7 mL is left and add 33.3 mL of microcarrier solution).

[0378] Harvesting

[0379] The following harvesting solutions were used:

[0380] For IAMF A: TrypLE (1x) with 2.5mM EDTA (0.5 mL of 0.5M EDTA stock was added to 100 mL of TrypLE (1x))

[0381] For Cytodex-1 : TrypLE (1x) (according to manufacturer’s instructions).

[0382] Harvesting was performed by transferring the complete cell-microcarrier suspension to a 50 mL tube and letting the microcarriers settle down for 5 minutes.

[0383] After washing the cell-microcarriers 3x with PBS, harvest solution is added (40 mL harvest solution added to -5-10 mL sample).

[0384] The samples are incubated for at least 15 minutes at 37°C in a water bath and homogenize every few minutes by inverting or pipetting in case of clumps.

[0385] Analyses

[0386] The following analyses were performed (day 1 and / or 2 and 4,7,8 up to day 9) -qualitative imaging was performed e.g. to evaluate confluency and morphology -cells were counted with the NC250 NucleoCounter

[0387] -harvesting efficiency was determined by a) separating bound and unbound cells using a 40pm strainer to retrieve the occupied microcarrier and b) count & viability determination using NucleoCounter. Harvesting efficiency is quantified based on original seeding density & attached cells as quantified through attachment efficiency - the population doubling time was quantified from cell growth data

[0388] Results

[0389] After 24 hours, cells seeded on lamF A (1.36e05 ± 9.13e03 cells) yielded more cells than on Cytodex-1 (2.14e04 ± 6.71 e03 cells)(P < 0.0001) (Figure 1A).

[0390] It was found that attachment efficiency was 92.1 ± 2.2% on lamF A microcarriers and 92.4 ±11.3% on Cytodex-1 microcarriers.

[0391] The harvest efficiencies of lamF A and Cytodex-1 were 138.3 ± 9.1% and 22.5 ± 5.8%, hence the harvesting efficiency for IAMF A was significantly higher (P<0.0001)(Figure 1B).

[0392] The lamF A microcarriers yielded 2.42E07 ± 9.62e05 cells after 9 days of spinner culture, whereas the Cytodex-1 microcarriers gave the lower cell yield of 1.14e06 ± 5.78e05 cells, which was a significant difference at day 9 (P<0.0001). Viability was over the whole culture highest on the lamF A microcarriers (>95%).

[0393] An improved growth on lamF A microcarriers was confirmed in view of the reduced doubling time. For cells grown on lamF A, the cell doubling time was 35 ± 1 h, whereas this was 54 ± 14 h for cells grown on Cytodex-1 (Figure 1C).

[0394] Figure 2 shows the visual inspection of spinner culture of BM-MSCs on lamF A and Cytodex- 1 after 9 days expansion. Improved cell growth and overall coverage of microcarriers can be seen on lamF A microcarriers compared to Cytodex-1 microcarriers.

[0395] During harvesting, the microcarriers were visually inspected for dissolution. The images are shown for lamF A in Figure 3. After T=3 minutes, rounded cells are seen, which means that the cells are detaching. At T = 5 min, the density of the microcarriers decreases, and at T = 10 min, all the cells have detached, but some microcarriers are still visible. These are dissolved at T = 15 min.

[0396] Similar good results were obtained using other cell types. For example, Vero cells were subjected to similar microcarrier culture conditions as described under present Example 3, which yielded also good results. Vero cells are a type of cell line widely used in for instance scientific research, virology, vaccine production, and drug testing, Based on the findings, it is considered that the microcarriers are suitable for many / most adherent cell types. Example 4

[0397] Example 4 describes the measurement of a Maillard reaction in Microcarriers prepared according to Example 1.

[0398] Method

[0399] Digestion buffer preparation (100 mM Tris pH 7.4, 5 mM CaC , 0.02% NaN3)

[0400] -3.94 g Tris is added to a 250 mL volumetric flask

[0401] -0.1838 g CaCh is added

[0402] -0.05 g of NalXh is added

[0403] - the 250 mL of volumetric flask is filled with de-ionized water and make up the volume to 250 mL

[0404] - the buffer is filtered with 0.45 pm filters.

[0405] Sample preparation

[0406] - samples are equilibrated in a climatic chamber to ensure equal moisture content

[0407] - 0.050 g of sample (+ / - 0.0005 g) is weighed in a 15 mL plastic tube

[0408] - 0.995 g of digestion buffer is added

[0409] - samples are let to well for 30 min on an orbital shaker (200 rpm)

[0410] - samples are placed in an oven at 37°C on an orbital shaker until most particles are dissolved

[0411] - 10 pL of freshly prepared trypsin solution (stock 50 mg / mL in digestion buffer) is added

[0412] - samples are incubated (ca. 16h) in an oven at 37°C on an orbital shaker (200 rpm)

[0413] - fluorescence is measured at emission 360 nm (+ / - 10 nm) and emission 440 nm (+ / - 10 nm) in a black 96-well plate (100 pL / well)

[0414] Evaluation of the Maillard reaction

[0415] As the Maillard reaction progresses, it creates intermediate products that can eventually lead to Advanced glycation end-products (AGEs). AGEs are commonly associated with Maillard browning.

[0416] The fold-increase in Maillard reaction (e.g. due to heat-treatment) is measured with the equation: (fluorescence signal after treatment) / (fluorescence signal before treatment). Quinine sulfate can be used as a standard to compare fluorescence intensities because it has a similar fluorescence profile to many AGEs, which typically show excitation around 340-370 nm and emission around 420-460 nm. For quantitative analysis, a calibration curve is prepared using known concentrations of quinine sulfate to convert fluorescence intensity to concentrations. Results

[0417] The Maillard reaction for the alginate-gelatin microcarrier of Example 1 is confirmed following autoclaving (121°C, 15 psi, 15 min, no drying), both in terms of the fold-increase in Maillard reaction and increase in fluorescent AGEs.

[0418] Similar results are found for wet heating of alginate-gelatin microcarriers under different conditions 70-90°C for 30-240 min.

[0419] Hot air drying at 70-90°C leads to less favorable reaction and less favorable alginate-gelatin microcarriers.

[0420] Example 5

[0421] Example 5 provides an overview of the attachment, proliferation, (differentiation) potency and functionality of different cell types on alginate-gelatin microcarriers in different reactor types (shown in Table 1). The microcarrier are as obtained in Example 1. The cell culture and harvesting protocol is generally as described in Example 1 , but can be varied and optimized depending on the cell type and reactor.

[0422] The well plate described in Table 1 is either of the static and / or dynamic type (e.g. orbital shaker). The spinner flask described in Table 1, is preferably of a rotating impeller type.

[0423] As can be seen, excellent attachment, proliferation, (differentiation) potency and functionality is achieved for different cell types under different culture conditions and reactor types.

[0424] Table 1.

[0425] Example 6 Example 6 compares two types of alginate-gelatin (Alg / Gel) microcarriers and establishes how coating conditions - either favoring or disfavoring electrostatic interactions between the alginate and gelatin - affect cell-related performance, integrity and dissolvability of the microcarriers.

[0426] Method Alginate microparticle production

[0427] The method described in Example 1 is used to produce the alginate microparticles.

[0428] Particle coating with gelatin

[0429] The method for producing alginate-gelatin microcarriers is based on the method described in Example 1 (see Example 1, “solution preparation” and “Particle coating with gelatin”). The gelatin used is also high molecular weight gelatin (300 bloom, type A), having an isoelectric point of ~7-9. Different from Example 1 , the alginate microparticles are coated with gelatin in a final 4 wt.% gelatin solution (i.e. 8 wt.% gelatin solution which is diluted 1 :1 in the alginate solution).

[0430] Alg / Gel+ microparticles:

[0431] In the example according to the invention (“Alg / Gel+”), the pH of the alginate-gelatin solution (4 wt.% gelatin) is adjusted to pH 6.0. At pH 6.0, the gelatin has a net positive charge.

[0432] Alg / Gel- microparticles:

[0433] In the comparative example (“Alg / Gel-”), the pH of the alginate-gelatin solution (4 wt.% gelatin) is adjusted to pH 11.0 by adding 1 M NaOH. At pH 11.0, the gelatin has a net negative charge.

[0434] Alginate has carboxyl groups (-COOH) on each monomer that can deprotonate. Since alginate does not contain amino groups, it does not become positively charged under normal pH. At either pH 6.0 or 11.0, the alginate has a net negative charge.

[0435] Autoclaving procedure

[0436] Samples are autoclaved and stored the same way described in Example 1.

[0437] Cell attachment, proliferation and dissolvability during standard cell culture conditions The Alg / Gel microcarriers were tested for cell attachment and dissolvability.

[0438] For cell attachment study, VERO cells (ATCC; CCL-81) were used. A vapor phase nitrogen frozen stock was thawed and passaged once before testing began to allow the cells to mitigate any proliferation issues due to recent thawing from cryogenic conditions. For dissolvability, an extra condition was added where only EDTA (2.5 mM) was added as dissolution agent instead of EDTA + TrypLE (2.5 mM 12.5X). Cell cultures were incubated at 37 °C supplemented with a 5% CO2 gas concentration.

[0439] Particle integrity during constant low shear application The Alg / Gel microcarriers were tested for particle integrity over time when subjected to constant shear applied using a magnetic stir bar. Of the original microcarriers suspension stock, 1.5 mL homogeneous suspension was transferred aseptically to a 50 mL tube. Sterile complete cell culture medium (DMEM (Gibco) + high glucose + pyruvate -glutamine; 10% fetal bovine serum (FBS); 1% (penicillin / streptomycin) P / S; 1% GlutaMAX) was added to the tube to provide a final volume of 15 mL. Sterile magnetic cylindrical stir bars (10 mm) were added to the tubes to provide constant shear.

[0440] Particles were incubated in a magnetic stirring water bath at 37 ± 0.2 °C with the stirring rate set at 400 RPM. Medium changes were performed on day 3 and 5. Images were taken preincubation on day 0, day 3, day 5 and day 7. 1 mL of homogenous microcarrier suspension was removed for imaging purposes and kept separately after imaging. Imaged particles and supernatant from media refreshes were collected and stored in a 37 °C incubator for future references.

[0441] Comparison of the effect of PBS washing

[0442] Particles were tested for particle integrity when washed with PBS (Gibco) to simulate media refreshes during regular cell culture. Particles were first reconstituted in DMEM. 1 mL of particle suspension was transferred to an appropriate container in which particles were washed twice with DMEM, removing 80% and 70% of the supernatant respectively during the washing steps whilst particles were allowed to sediment prior to supernatant removal. Particles were washed three times with 1x PBS by letting the particles sediment and removing >90% of the supernatant to be replaced with an equal volume of PBS. The final wash was performed in 10X PBS to speed up the process. Particles were imaged after each washing step to observe any changes.

[0443] Results

[0444] Cell attachment and proliferation

[0445] Both types of microcarriers (Alg / Gel+ and Alg / Gel-) showed similar cell attachment and growth. In both cases, most carriers were fully covered by cells after 7 days of culture. The results of 7 days in culture are shown in Figure 4.

[0446] Dissolvability

[0447] There was a clear difference between Alg / Gel+ and Alg / Gel- microcarriers in terms of dissolvability when only EDTA was added as dissolution agent. The Alg / Gel- microcarriers (coated at pH 11) dissolved within 5 minutes of incubation in the EDTA solution, whereas the Alg / Gel+ microcarriers (coated at pH 6) were stable for up to 15 minutes. When TrypLE and EDTA were both added, both microcarrier formulations dissolved within 15 minutes. The results are visually represented in Figure 5.

[0448] Particle integrity during constant low shear application

[0449] Over time, a clear difference in terms of particle integrity was observed where Alg / Gel+ or Alg / Gel- microcarriers were fragmented as a result of the constant subjection to shear. However, a clear difference between the amount of fragments that formed over time between both particles is clearly visible. This was already visible after three days of applying shear where significant fragments were visible in the Alg / Gel- (pH 11) condition where almost no fragments were visible in the Alg / Gel+ (pH 6) condition. This phenomenon only became more apparent over time where a significant number of particles were destroyed in the Alg / Gel- (pH 11) condition resulting in the debris found in between the particles as clearly visible after 5 or 7 days. This is in clear contrast with the Alg / Gel+ (pH 6) particles where the constant application of particles did result in some debris, however, significantly less in comparison to the Alg / Gel- (pH 11) condition. A semi-quantitative analysis was performed of particle intactness, which is shown in Table 2. No visual effect could be observed as a result from the media refreshes as particles did not seem to swell with any significance between the different timepoints.

[0450] Table 2. Semi-quantitative analysis of particle intactness after shear exposure (+ fully intact, ± semi-intact or intact, - severely fragmented). T=time in days

[0451] Comparison of the effect of PBS washing

[0452] Both Alg / Gel+ and Alg / Gel- particles did not show large changes in particle integrity when observing the particles up until washing step 3 as a result of the PBS washing. However, a clear difference can be seen after the 4th washing step with 10x PBS where a clear distinction between the two particle formulations can be observed. The Alg / Gel+ particles (pH 6) are still visible after this washing step, whereas the Alg / Gel- particles (pH 11) have completely disappeared and thus have completely dissolved. The analysis of particle intactness is shown in Table 3. It should be noted that Alg / Gel- particles do appear to lose their contrast to some extent with the surrounding liquid which could indicate near dissolution.

[0453] Table 3. Particle intactness after multiple washes with PBS. (+ particles intact, - particles dissolved).

[0454] Conclusions

[0455] Assessment of the two kinds of microcarriers has shown distinct differences when assessing their performance when subjected to several parameters which are relevant to real-world scenarios encountered during cell culture applications. The individual tests have shown clear benefits of coating the alginate particles using Alg / Gel+ microcarriers where a pH provided below the isoelectric point of gelatin such that the gelatin has a net positive charge when contacted with the alginate (having a net negative charge).

[0456] The Alg / Gel+ microcarriers resulting microparticles were superior in all tests, showing improved particle integrity in adverse conditions such as when subjected to constant shear or strong salt solutions. Moreover, no adverse difference could be observed when culturing microcarriers with VERO cells in static conditions when comparing the Alg-Gel+ and ALg-Gel- microcarriers. Individual conclusions per experiment are elucidated below.

[0457] Cell attachment, proliferation and dissolvability during standard cell culture conditions

[0458] No difference in cell attachment and proliferation could be observed during the 7-day cell culture period. This indicates that there are no benefits or drawbacks to controlling the pH during coating which might affect the main purpose of the microcarrier.

[0459] When assessing the dissolvability of the two microcarriers, a clear distinction in performance was observed. Using only EDTA to dissociate the carriers resulted Alg / Gel- microcarrier (pH 11) dissolving, therefore indicating poor stability when a chelator is introduced, in contrast to the Alg / Gel+ microcarrier (pH 6) which remained stable. Utilizing standard cell dissociation media consisting of TrypLE and EDTA, the moment during cell culture where dissolution of microcarriers is desired, both microcarriers dissolved.

[0460] Particle integrity during constant low shear application

[0461] Under constant shear, much fragmentation and formation of debris was observed for the Alg / Gel- microcarrier (pH 11). The Alg / Gel+ microcarriers (pH 6) showed only limited fragmentation, to a much lesser extent. This indicates Alg / Gel+ microcarriers (pH 6) retain their integrity under constant shear for at least 5 days and most particles retain their integrity for 7 days or longer.

[0462] Comparison of the effect of PBS washing

[0463] Washing the particles with 1x PBS showed no clear difference in particle integrity when washed three times. However, washing with a much more potent PBS washing solution, the Alg / Gel- microcarriers (pH 11) dissolved completely in contrary to Alg / Gel+ microcarriers (pH 6) which remained mostly intact.

Claims

- 68 -CLAIMS1. Method for producing a microcarrier, comprising a) providing a microparticle comprising ionically crosslinkable polysaccharide having a negative charge in a solvent containing a collagen derivative having a positive charge to obtain a composite microparticle, subjecting the composite microparticle to a chemical crosslinking agent to chemically crosslink the collagen derivative, and b) providing further crosslinking to obtain a crosslinked microcarrier characterized by covalent binding between the polysaccharide and the collagen derivative.

2. Method according to claim 1 , wherein in step b) the further crosslinking is by one or more of an enzymatic, chemical, photo-crosslinking, gamma irradiation or heat- treating method.

3. Method according to claim 1 or 2, wherein in step b) the further crosslinking is by heat-treating the composite microparticle with a time / temperature combination such as to provide a Maillard reaction between groups of the polysaccharide and the collagen derivative4. Method according to claim 3, wherein the heat-treating in step b) is by wet heating.

5. Method according to claim 3 or 4, wherein during the heat-treating in step b) the composite microcarrier is provided in liquid suspension.

6. Method according to any one of claims 3-5, wherein the heat-treating in step b) is performed- at 50-140 °C, and / or- for 5-60 min7. Method according to any one the previous claims, wherein step b) is performed until formation of one or more of a Schiff base (C=N) bond and a pyrazine (C-N) bond between the polysaccharide and the collagen derivative.

8. Method according to any one of the previous claims, wherein the ionically crosslinkable polysaccharide is one or more selected from the group consisting of alginate, pectin, galacturonic acid and carrageenan.- 69 -9. Method according to claim 8, wherein the ionically crosslinkable polysaccharide is alginate.

10. Method according to any one of the previous claims, wherein the collagen derivative is hydrolysed or partially hydrolysed collagen, preferably gelatin or hydrolysed gelatin.

11. Method according to claim 10, wherein the collagen derivative is gelatin.

12. Method according to any one of the previous claims, wherein the collagen derivative in step a) has a weight-average molecular weight of more than 100 kilodalton (kDa), preferably more than 150 kDa and / or is type A collagen derivative, preferably type A gelatin.

13. Method according to any one of the previous claims, wherein the collagen derivative is chemically crosslinked before the heat-treating, by subjecting to one or more crosslinking agents selected from the group consisting of glutaraldehyde, genipin, a carbodiimide, an epoxy compound and a transglutaminase, more preferably glutaraldehyde.

14. Method according to any one of the previous claims, wherein the alginate provided in step a) is ionically crosslinked with a divalent or higher cation, preferably with one or more of Ca2+, Mg2+, Sr2+and Ba2+, most preferably Ca2+.

15. Method according to any one of the previous claims, wherein the method comprises one or more further steps of purification and / or washing of the microcarrier obtained in step b), wherein the purification and / or washing step preferably is to reduce the content of one or more contaminants selected from the group consisting of unbound salt, unbound ions, metal contaminants including heavy metals, a residual reagent, endotoxin and microbial burden.

16. Method according to claim 15, wherein the one or more further steps of purification and / or washing includes a chelation treatment, preferably using ethylenediaminetetraacetic acid (EDTA) and / or preferably to reduce the content of one or more elemental impurities, more preferably one or more heavy metals selected from the group consisting of lead, cadmium, mercury and arsenic.- 70 -17. Microcarrier, optionally obtainable by the method according to any one of claims 1- 16, wherein the microcarrier comprises an ionically crosslinkable polysaccharide crosslinked with a collagen derivative, wherein the collagen derivative is chemically crosslinked with one or more chemical crosslinking agents, wherein the microcarrier is characterized by covalent binding of the polysaccharide and the collagen derivative and a core-shell structure comprising:-a core comprising ionically crosslinkable polysaccharide in an amount of at least 80% by dry weight or volume of the core layer, and-a shell layer comprising more than 50% collagen derivative by dry weight or volume of the shell layer.

18. Microcarrier according to claim 17 , wherein the microcarrier has a core-shell structure defined by:-a core comprising ionically crosslinkable polysaccharide in an amount of at least 80%, preferably at least 90%, more preferably at least 95%, calculated on dry weight or volume of the core, and-a shell layer comprising at least 80%, preferably at least 90%, more preferably at least 95% collagen derivative by dry weight or volume of the shell, more preferably wherein the shell layer is an outer layer of the microcarrier consisting of collagen derivative.

19. Microcarrier according to claim 17 or 18, wherein the microcarrier is spherical and has an average diametric dimension of 10 - 500 pm, preferably 100-300 pm, more preferably 150-250 pm, as measured with a dynamic image particle size analyzer.

20. Microcarrier according to any one of claims 17-19, wherein the ionically crosslinkable polysaccharide is one or more selected from the group consisting of alginate, pectin, galacturonic acid and carrageenan.

21. Microcarrier according to claim 20, wherein the ionically crosslinkable polysaccharide is alginate.

22. Microcarrier according to claim any one of claims 17-21 , wherein the collagen derivative is hydrolysed or partially hydrolysed collagen, preferably gelatin or- 71 - hydrolysed gelatin.

23. Microcarrier according to claim 22, wherein the collagen derivative is gelatin.

24. Microcarrier according to any one of claims 17-23, wherein the covalent binding is by one or more of a Schiff base (C=N) bond and a pyrazine (C-N) bond and / or the covalent binding is between carbonyl groups in the ionically crosslinkable polysaccharide and amino groups in the collagen derivative.

25. Microcarrier according to any one of claims 17-24, comprising covalent crosslinking between dicarbonyls and amino groups in the collagen derivative and / or between dicarbonyl groups and hydroxyl groups in the ionically crosslinkable polysaccharide.

26. Microcarrier according to any one of claims 17-25, wherein the collagen derivative is chemically crosslinked with one or more chemical crosslinking agents selected from the group consisting of glutaraldehyde, genipin, a carbodiimide, an epoxy compound and a transglutaminase, preferably glutaraldehyde.

27. Microcarrier according to any one of claims 17-26, wherein the ionically crosslinkable polysaccharide is ionically crosslinked with a divalent or higher cation, preferably with one or more of Ca2+, Mg2+, Sr2+and Ba2+, most preferably Ca2+.

28. Microcarrier according to any one of claims 17-27, wherein the collagen derivative has an isoelectric point of 7-9.

29. Microcarrier according to any one of claims 17-28, wherein the collagen derivative is a type A collagen derivative, preferably type A gelatin.

30. Microcarrier according to any one of claims 17-29, wherein the microcarrier has a positive zeta potential, preferably +10 mV to +50 mV or +20 mV to +40 mV.31 . Microcarrier according to any one of claims 17-30, wherein the lipopolysaccharide content is no more than 3000 Endotoxin Units (EU) / g, preferably no more than 1000 EU / g, more preferably no more than 100 EU / g, even more preferably no- 72 - more than 10 Ell / g, calculated on the dry weight of the microcarrier.

32. Microcarrier according to any one of claims 17-31 , wherein the microcarrier comprises total elemental impurities and / or total heavy metals in an amount of less than 500 parts per million (ppm), preferably less than 50 ppm, more preferably less than 5 ppm, even more preferably less 0.5 ppm, most preferably less than 0.05 ppm, calculated on the dry weight of the crosslinkable polysaccharide and / or the dry weight of the microcarrier.

33. Microcarrier according to any one of claims 17-32, wherein the microcarrier comprises one or more of lead, cadmium, mercury and arsenic in an amount of less than 5 ppm, preferably less than 0.5 ppm, more preferably less 0.05 ppm, even most preferably less than 0.005 ppm, calculated on the dry weight of the crosslinkable polysaccharide and / or the dry weight of the microcarrier.

34. Microcarrier according to any one of claims 17-33, wherein the microcarrier is for one or more of culturing adherent cells, immobilization of cells and / or active ingredients, drug delivery, encapsulation of cells / and / or active ingredients, preferably culturing of adherent cells.

35. Plurality of microcarriers as defined in any one of claims 17-34, provided as a dry powder or in a liquid suspension and / or a slurry.

36. Plurality of microcarriers according to claim 35, wherein the monodispersity of the microcarriers is characterized by a coefficient of variance of the particle size distribution of less than 15%, preferably less than 10%, more preferably 0.5-5%.

37. Use of the microcarrier according to any one of claims 17-34 or the plurality of microcarriers according to claim 35 or 36 in an application selected from the group consisting of pharmaceutical production, vaccine production, gene therapy, regenerative medicine, cell therapy, vaccine production, biologies manufacturing, a vaccine, antibody or recombinant protein production, viral vector production, cosmetic testing, extracellular vesicle and / or exosome production, toxicology testing, 3D cell culture, bioreactor culture and cultivated meat or fish production.

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