Process for culturing stem cells

Abstract

The present invention relates to a support matrix and its utility in the culturing of stem cells. In particular, the present invention relates to a support matrix comprising cellulose nanofibers upon which stem cells readily proliferate for industrial scale production.

Description

[0001] Process for culturing stem cells

[0002] Technical field of the invention

[0003] The present invention relates to a support matrix and its utility in the culturing of stem cells. In particular, the present invention relates to a support matrix comprising cellulose nanofibers upon which stem cells readily proliferate for industrial scale production.

[0004] Background of the invention

[0005] Stem cells, with their remarkable ability to self-renew and differentiate into various cell types, hold immense promise for biomedical research and regenerative medicine. In biomedical research, stem cells provide valuable insights into cell biology, developmental processes, and gene expression regulation. In regenerative medicine, cultured stem cells may be used for tissue engineering to replace damaged or dysfunctional tissues or as cellbased therapies to treat conditions such as spinal cord injuries, heart diseases, and neurodegenerative disorders.

[0006] One type of stem cells of great interest for therapeutic applications are human mesenchymal stem cells (hMSCs). These stem cells are derived from sources such as bone marrow, adipose tissue and umbilical cords and are capable of differentiating into adipocytes, chondrocytes, osteoblasts, cartilage, bone, skeletal muscle among other cell types. They hold promise for treating diseases due to their therapeutic properties, including immunomodulation and angiogenesis, and clinical investigations are currently underway. hMSCs secrete factors, such as immunosuppressive, angiogenic, anti-apoptotic and anti-oxidative agents, which play important roles in tissue repair and suppression of graft versus host response. Furthermore, emerging evidence suggests that extracellular vehicles (EVs) produced by hMSCs also exhibit therapeutic effects. Therefore, hMSC culture systems offer a dual therapeutic approach, utilizing both the cells themselves and the EV-containing culture supernatants.

[0007] Unfortunately, challenges to unlock the full potential of stem cells, such as hMSCs, persist and include identifying suitable donors, optimizing culture methodologies to uphold cell quality and minimizing production costs.

[0008] Conventional monolayer-cultured hMSCs that undergo progressive passage diminish growth kinetics, downregulate expression of sternness-associated genes and decrease secretion of factors. To overcome the limitations of monolayer culturing, use of support matrix systems may be utilised for cell attachment and growth in an in v / o-like environment. As such, researchers have explored synthetic polymers, hydrogels, and natural biomaterials as support materials for stem cell culturing, but a support matrix that delivers the right balance between scalability, cost-effectiveness and maintaining cell quality and sternness has to this point remained largely elusive.

[0009] Thus, there is an unmet need for provision of a support matrix for stem cell culturing which enables scalable production of high quality stem cells in a cost-effective manner.

[0010] Hence, it would be advantageous to provide a support matrix with a high capacity for hosting a stem cell culture in an environment that retain the native properties of the cells.

[0011] Specifically, it would be advantageous to provide a support matrix that facilitates strong cell attachment and reduces the cell doubling time.

[0012] Summary of the invention

[0013] Herein are presented a method for scaling up hMSC production using a cellulose-based support matrix that provides a three-dimensional (3D) surface for cell attachment and growth. The support matrix offers an environment for long term cell culture mimicking in vivo conditions, maintaining cell quality and sternness.

[0014] The cellulose-based support matrix is a durable material that can withstand the shear forces that it is subjected to during operation of industrial bioreactors, such as a packed bed bioreactors. It also offers unique properties such as surface functionalization and versatility in size and porosity, making it a suitable choice for scaling production from labscale to industrial scale.

[0015] Thus, an object of the present invention relates to the provision of a method for culturing stem cells that preserve the native properties of the stem cells.

[0016] Another object of the present invention relates to provision of a method for producing stem cells, such as hMSCs, in a cost-effective and scalable manner.

[0017] Thus, an aspect of the present invention relates to a method for culturing of stem cells, said method comprising the steps of: providing a container comprising a support matrix;

[0018] - seeding stem cells on said support matrix; and incubating said stem cells; wherein said support matrix comprises cellulose nanofibers. Another aspect of the present invention relates to use of a nanofibrous cellulose scaffold as described herein as a support matrix for culturing of stem cells.

[0019] Yet another aspect of the present invention relates to a support matrix comprising a nanofibrous cellulose scaffold as described herein.

[0020] A further aspect of the present invention relates to a nanofibrous cellulose scaffold comprising cellulose nanofibers with a mean length of less than about 250 pm, and wherein the cellulose nanofibers are crosslinked with a crosslinking agent.

[0021] A still further aspect of the present invention relates to a method of preparing a nanofibrous cellulose scaffold, said method comprising the steps of:

[0022] (i) providing an initial cellulose nanofiber material,

[0023] (ii) dividing said initial cellulose nanofiber material into a processed cellulose nanofiber material,

[0024] (iii) crosslinking the processed cellulose nanofiber material, and

[0025] (iv) drying the processed cellulose nanofiber material, thereby providing the nanofibrous cellulose scaffold.

[0026] An even further aspect of the present invention relates to a nanofibrous cellulose scaffold obtainable from a method as described herein.

[0027] Brief description of the figures

[0028] Figure 1 shows (A) Scanning electron microscopy (SEM) image of the nanofibrous cellulose scaffold. (B) FTIR spectrum of crosslinked cellulose. (C) Comparison of FTIR spectra of (top to bottom) cellulose acetate, regenerated cellulose, QA functionalized and crosslinked cellulose.

[0029] Figure 2 shows (A) Comparison of a traditional, cut, nanofibrous sheet (left) and a moulded nanofibrous cellulose scaffold (right). (B) Nanofibrous cellulose scaffold moulded to fit labscale bioreactors (left) and pilot-scale bioreactors (right). (C-D) Force (N) versus deformation (%) behaviour of the nanofibrous cellulose scaffold (cured and dry QA 2%).

[0030] Figure 3 shows (A) bright field microscope images capturing hMSC adherence to the scaffold within 5 hours post-inoculum in well plates, as demonstrated by the absence of cells in the surrounding medium around the scaffold. (B) Cell growth of hMSCs on QA functionalized and non-functionalized scaffolds that have been coated with vitronectin, presented as fold change over cell growth on uncoated scaffolds (without vitronectin) across a time-course experiment. Data are presented as means ± SEM, using technical replicates. (C) Cell growth on scaffolds with different crosslinker concentrations shown as fold change over medium crosslinker (0.2 pl / mg corresponding to a molar ratio of crosslinking agent to cellulose of 19.0*10-3mol / mol) analysed at day 6 post-inoculum.

[0031] Figure 4 shows cell growth of hMSCs on nanofibrous cellulose scaffolds. (A) Growth curve of live cells presented as fold change over seeded cells across two independent timecourse experiments. Data are presented as means ± SEM, using biological replicates. (B) Viability of hMSCs throughout the culture period. Data are presented as means ± SEM, using biological replicates. (C) Counts of live cells per scaffold coated with either high (5- 10 pg / ml) or low (2.5 pg / ml) concentrations of vitronectin. Data are presented as mean ± SEM, using technical replicates. (D) Microscopic visualization of Giemsa-stained hMSCs on scaffolds. Scale bars 100 pm.

[0032] Figure 5 shows (A) cell growth of hMSCs on nanofibrous cellulose scaffolds prepared from cellulose suspensions of varying concentration (0.2%, 0.5%, 1.0% and 2.0%) presented as fold change over cell growth on scaffold of 0.2% cellulose and analyzed at day 8 postinoculum. Data are presented as means± SEM, using biological replicates. (B) Viability of hMSCs throughout the culture period. Data are presented as means ± SEM, using biological replicates.

[0033] Figure 6 shows pictures of Giemsa-stained nanofibrous cellulose scaffolds (A) containing hMSCs and (B) after hMSCs harvest. (C) Cell growth rate under 2D culture conditions for hMSCs passaged from either conventional 2D culture or post cell culturing on a nanofibrous cellulose scaffold. Data are presented as means. (D) Viability of hMSCs throughout the culture period. Data are presented as means ± SEM, using biological replicates. (E) Brightfield microscope pictures of hMSCs passaged into 2D culture following culturing on a nanofibrous cellulose scaffold and observed at 4 days in 2D culture. Scale bar 100 pm. (F) Flow cytometry shows the presence of CD90, CD73, CD105 and CD44 markers along with the negative expression of CD19, CD34, CDllb, HLA-DR and CD45, confirming that that the cells growing on the nanofibrous cellulose scaffolds have retained characteristics typical of mesenchymal stem cell as compared to the 2D cultures from the same passage. Data are presented as means ± SEM, using technical replicates from different macrocarrier productions.

[0034] Figure 7 shows examples of fibers cut by laser. (A) The cellulose material cut by laser results in pieces of cellulose that trap air bubbles and float. (B) Electrospun cellulose material cut with a laser. The laser burns the cellulose sheets. (C) SEM images of cellulose nanofibers cut by laser. The cellulose nanofibers are melted and fused together.

[0035] Figure 8 shows determination of cellulose nanofiber diameters. (A-B) SEM micrographs of cellulose nanofibers after 1 hour dispersing at 18000 rpm. (C) Histogram depicting the cellulose nanofiber diameter distribution of five separate samples. Approximately 2500 individual cellulose nanofibers were measured using ImageJ software.

[0036] Figure 9 shows scanning electron microscopy (SEM) images of cellulose nanofibers blended (left) or dispersed (right). The cellulose nanofibers were mixed for different periods of time; 1 min (A-B), 5 min (C-D), 15 min (E-F) or 60 min (G-H).

[0037] Figure 10 shows histograms of size (length) distributions of cellulose nanofibers that have been divided by (A) blending or (B) dispersing. The histograms are for samples divided for 1, 5, 15, or 60 min (left to right). Nanofiber lengths are displayed as relative frequency of mean nanofiber lengths in bins of 200 pm.

[0038] Figure 11 shows scanning electron microscopy (SEM) images of electrospun nanofibers; (A) PCL, (B) PLA, (C) PLA / PCL, and (D) cellulose. All images are recorded at 600x magnification, scale bar is 50 pm.

[0039] Figure 12 shows representative photographs (left) and SEM images (right) of different electrospun nanofibers subsequent to 5 minutes of mixing in a blender. (A-B) PCL, (C-D) PLA, (E-F) PLA / PCL. Scale bar of SEM images is 200 pm.

[0040] Figure 13 shows representative photographs (left) and SEM images (right) of different electrospun nanofibers subsequent to 5 minutes of mixing in a disperser. (A-B) PCL, (C- D) PLA, (E-F) PLA / PCL. Scale bar of SEM images is 50 pm.

[0041] Figure 14 shows nanofibrous cellulose scaffolds prepared at different cellulose concentrations; (A) 2%, (B) 1%, (C) 0.5%, (D) 0.25%, (E) 0.125%, (F) 0.06%, (G) 0.03%, (H) 0.015%.

[0042] Figure 15 shows nanofibrous cellulose scaffolds made with varying cellulose concentrations; (I) 0.03%, (II) 0.06%, (III) 0.125%, (IV) 0.25%, and different volumes in the moulds; (A) 25 ml, (B) 17 ml, (C) 10 ml, (D) 3 ml. Figure 16 shows (A) the setup for mechanical testing; (left) scaffold mounted between two support disks on stirring rod, (middle) stirring rod immersed in 500 ml beaker, (right) visible breakage of sample IIB (0.06% cellulose and 17 ml volume). (B) Recovery of scaffolds after stirring in mock-up reactor; samples were as follows according to the reference numbers (A) IA, (B) IIA, (C) IIB, (D) IIIA, (E) IIIB, (F) IIIC, (G) IVA, (H) IVB, (I) IVC.

[0043] Detailed description of the invention

[0044] Definitions

[0045] Prior to outlining the present invention in more details, a set of terms and conventions is first defined:

[0046] Nanofiber

[0047] In the present context, the term "nanofiber" refers to fibers with diameters in the range of 10-2000 nm. The fibers may be generated from different types of polymers, such as cellulose.

[0048] Cellulose nanofiber material

[0049] In the present context, the term "cellulose nanofiber material" refers to an initial material prepared from cellulose. The cellulose nanofiber material may be prepared by any method suitable for preparing sheets of cellulose that can subsequently be processed as described herein to provide the nanofibrous cellulose scaffold. These methods include, but are not limited to, electrospinning, meltblowing, and drawing of cellulose nanofibers.

[0050] Preferably, the cellulose nanofiber material is prepared by electrospinning of cellulose nanofibers. Electrospinning may be performed from a solution of cellulose acetate.

[0051] Container

[0052] In the present context, the term "container" refers to any delimited vessel suitable for culturing of cells. The container is preferably a conventional culturing vessel, including, but not limited to, a bioreactor, a cell culturing plate, a cell culture bottle, a spinner flask, a shaker flask, and a roller bottle, a petri dish, and a tube.

[0053] The nanofibrous cellulose scaffold as described herein is readily scalable and the container may therefore be of any volume suitable for culturing cells.

[0054] Dispersing In the present context, the term "dispersing" refers to the process of dividing the cellulose nanofiber material by means of a disperser. Dispersing is preferably performed in a liquid.

[0055] In the present context, a disperser is a high-speed mixing device that can comminute a solid, such as a cellulose sheet, into smaller fragments. The disperser comprises one or more heads which constitute the means of dividing the cellulose nanofiber material. The head may be in the form of a disk blade. The head of the disperser may comprise a blade with a propeller design. The disperser creates a turbulent flow and a vortex which ensures homogenous dividing of the cellulose nanofiber material into smaller fragments, i.e. cellulose nanofibers of reduced length compared to the initial cellulose nanofiber material.

[0056] Any type of disperser may be used, including, but not limited to, laboratory high-speed disperser, pilot-scale high-speed dispersers, and production-scale high-speed dispersers. The disperser can be chosen according to the batch size to be divided, and the diameter of the disperser blades are adjusted accordingly. The disperser blade may be raised and lowered during dispersing to eliminate stratification, such mechanism may be automatic. For large batches the disperser may be a floor-mounted or tank-mounted model and / or be a multishaft model.

[0057] Crosslinking agent

[0058] In the present context, the term "crosslinking agent" refers to any chemical or biological molecule capable of connecting multiple cellulose nanofibers to each other. Preferably, the crosslinking agent can connect cellulose nanofibers via reaction with hydroxyl groups of the cellulose backbone to create covalently crosslinked cellulose nanofibers.

[0059] Thus, a crosslinking agent may be a molecule comprising at least two functional groups capable of reacting with hydroxyl groups.

[0060] Functional moiety

[0061] In the present context, the term "functional moiety" refers to a chemical or biological group or molecule positioned on the nanofibrous cellulose scaffold, and which interacts with the cells associated with scaffold. A functional moiety may interact with the cells via interactions including, but not limited to, electrostatic interactions, affinity, and hydrophobicity / hydrophilicity. The functional moiety may promote attachment / adhesion of cells to the nanofibrous cellulose scaffold, induce cell differentiation and proliferation, and / or assist maintenance of in vivo cellular functions. Chemical moieties may have one or more positive or negative charges to induce electrostatic interaction with the charged cell membranes. Examples hereof include, but are not limited to, quaternary ammonium (QA), carboxymethyl (CM), and diethylaminoethyl (DEAE). For many cells, the cell membrane would be negatively charged, and electrostatic interactions would be induced for nanofibrous cellulose scaffolds functionalized with positively charged functional moieties, such as QA or DEAE.

[0062] Biological moieties may be any type of biological molecule that can secure cell attachment to the nanofibrous cellulose scaffold and / or promote cell proliferation, including but not limited to, proteins, lipoproteins, lipid anchors, cell-adhesion molecules (CAMs), antigens, receptors, glycoproteins and antibodies. CAMs include integrins, immunoglobulins, cadherins, and selectins. Biological moieties may also assist cellular differentiation through receptor-ligand interactions, antigen-antibody interactions, protein interactions, and / or immune stimulatory signalling. The biological moiety may be a glycoprotein found in the extracellular matrix, such as vitronectin.

[0063] Adherent cell

[0064] In the present context, the term "adherent cells" refers to any cell that requires a surface or artificial substrate, such as a support matrix, to form an adherent cell culture. Preferably, the adherent cell is derived from a solid tissue.

[0065] Adherent culture is to be distinguished from suspension culture in which cells are grown freely floating in suspension.

[0066] Mean diameter (of cellulose nano fiber)

[0067] In the present context, the term "mean diameter" refers to the average diameter of cellulose nanofibers in the nanofibrous cellulose scaffold. The mean diameter may be determined from SEM images of the nanofibrous cellulose scaffold. Preferably, the mean diameter is determined from measurement of at least 100 individual nanofibers within the sample, e.g. by use of image analysis software, such as Image!

[0068] The mean diameter of the cellulose nanofibers can be adjusted in the process of preparing the initial cellulose nanofiber material. This may be achieved by varying the parameters of e.g. the electrospinning process.

[0069] Preferably, the mean diameter of the cellulose nanofibers in the nanofibrous cellulose scaffold is from about 250 nm to about 750 nm, such as about 400 nm to about 600 nm. Mean length (of cellulose nanofiber)

[0070] In the present context, the term "mean length" refers to the average length of cellulose nanofibers in the nanofibrous cellulose scaffold. The mean length is determined as the volume weighted mean value (D[4,3]) measured by light scattering, e.g. on a Malvern Mastersizer S. D[4,3] is also known as the De Brouckere mean value.

[0071] The mean fiber length in a sample may be determined using the following settings on a Malvern Mastersizer S:

[0072] Range lens: 300RF mm

[0073] Presentation: 3OHD

[0074] Analysis model: Polydisperse

[0075] Particle refractive index: (1.5295, 0.1000)

[0076] Dispersant refractive index: (1.33000)

[0077] Density: 1.5000 g / cm3

[0078] Surface area

[0079] In the present context, the term "surface area" refers to the Brunauer-Emmett-Teller (BET) surface area. The BET surface area may be determined by measurement of the physiosorption of a gas, usually nitrogen, to give a value of the sample. The BET method can accurately determine the surface area of the nanofibrous cellulose scaffold since the gas molecules can travel within the nanofibrous cellulose scaffold to probe also interior surfaces.

[0080] The surface area is given as area per unit mass (e.g. cm2 / g) and can be measured according to ISO 9277:2022 - Determination of the specific surface area of solids by gas adsorption — BET method.

[0081] Degree of substitution (DS)

[0082] In the present context, the term "degree of substitution (DS)" refers to the average number of functional moieties attached per base unit of the condensation polymer cellulose. The base unit of cellulose is 0(1— >4) linked D-glucose, which comprise three hydroxyl groups that may be subjected to substitution. Accordingly, the theoretical maximum value of DS is 3.

[0083] Degree of substitution (DS) may be determined using the following formula:

[0084] DS = (162N / (1400-CAxN)) where 162 is the molecular weight of the anhydrous glucose unit (AGU); N is the percentage of nitrogen; CA is the molecular weight of the cationic reagent. Substitution of the cellulose nanofibers can also be quantified as equivalent of charge per base unit mass of cellulose, and is given in units of meq / g. This value can be determined by zeta potential measurements, pH titration or electrokinetic chromatography.

[0085] Mercerization

[0086] In the present context, the term "mercerization" refers to a process comprising swelling of the cellulose nanofiber material in an aqueous or ethanolic NaOH solution to break internal hydrogen bonds of cellulose and increase the number of available hydroxyl groups (■OH).

[0087] Support matrix

[0088] In the present context, the term "support matrix" refers to any material upon which adherent cells may grow in adherent culture. The support matrix described herein is based on a nanofibrous cellulose scaffold.

[0089] The support matrix may also be referred to as "microcarrier" or "macrocarrier". Both microcarriers and macrocarriers can be microporous materials. A macrocarrier has a macroscopic structure as opposed to a microcarrier, which is defined only by its microstructure. Thus, the macrocarriers described herein comprise cross-linked nanofibers and may be moulded for specific containers or applications.

[0090] It is to be understood that the support matrix prepared herein is not a hydrogel, which amongst others are characterized by its ability to absorb and retain large amounts of water creating a highly viscous environment and are generally used for 3D cell culture in small scale or static conditions. In particular, hydrogels are not suited as support matrices for large scale cell production, such as in a bioreactor. While hydrogels can be alternatively used as shear-thinning, the shear stress in stirred-tank or perfusion bioreactors may disrupt their structure. Instead, the support matrix herein is robust and can be efficiently agitated in bioreactors, providing a surface for anchorage-dependent cells to attach and grow.

[0091] Packed bed bioreactor

[0092] In the present context, the term "packed bed bioreactor" refers to a bioreactor wherein the support matrix is immobilised in a bed within the bioreactor. Fresh medium is continuously circulating within the system. The means for providing circulation (or agitation) within the bioreactor include, but is not limited to, a pump, a stirrer, such as an impeller, or a pneumatic means. Cells are seeded on the support matrix in the bed. Packed bed bioreactors fall in two overall categories; fixed bed bioreactors where the packed bed is stationary, and dynamic bed bioreactors wherein the packed bed is in motion.

[0093] Dry continuous material

[0094] In the present context, the term "dry continuous material" refers to a dry material that can be produced as a single entity that extends significantly in three dimensions. Extending significantly includes, but is not limited to, extending at least about 0.1 mm in all three dimensions.

[0095] A dry continuous material is to be distinguished from materials that only gain their volumetric form from adding individual entities together. Examples of non-continuous materials include, but are not limited to, a high density population of solid spherical particles, or a collection of stacked disks.

[0096] Resilient

[0097] In the present context, the term "resilient" refers to the ability of a material to return to its original shape and size after being subjected to external forces or stresses. Thus, a resilient material is able to absorb energy when it is deformed, and then release that energy when the deforming force is removed, causing the material to return to its original shape. This property is often referred to as "elasticity" or "elastic deformation". A resilient material is both compressible and has a sufficient elasticity allowing the material to decompress and expand back to the initial shape when the force on the material is removed.

[0098] Accordingly, a resilient material can be forced into a container or void of a smaller volume, and adopt the shape of that container or void. This change of shape is, in principle, reversible in nature, as release from the smaller volume or void will return the material to its original shape and size.

[0099] Compressive strength

[0100] In the present context, the term "compressive strength" refers to the ability of a material to withstand a compressive load. The compressive strength may be given by the force required to cause a specific deformation. The value can be given in units of mm / N.

[0101] As a benchmark, the compressive strength of a material can be presented as the force necessary to obtain 60% deformation of the material. The compressive strength can be measured according to ISO 604:2002 - Plastics - Determination of compressive properties.

[0102] Molar ratio

[0103] In the present context, the term "molar ratio" refers to the number of moles of a first moiety to the number of moles of a second moiety, such as moles of crosslinking agent per moles of cellulose nanofibers. The moles of cellulose nanofibers are given per glucose unit, i.e. anhydrous glucose unit (AGU, molecular weight of 162 g / mol).

[0104] The molar ratio may be denoted "mol / mol" or alternatively "mol% / mol%".

[0105] About

[0106] Wherever the term "about" is employed herein in the context of amounts, for example absolute amounts, such as numbers, purities, weights, concentrations, sizes, etc., or relative amounts (e.g. percentages, equivalents or ratios), timeframes, and parameters such as temperatures, pressure, etc., it will be appreciated that such variables are approximate and as such may vary by ±10%, for example ± 5% and preferably ± 2% (e.g. ± 1%) from the actual numbers specified. This is the case even if such numbers are presented as percentages in the first place (for example 'about 10%' may mean ±10% about the number 10, which is anything between 9% and 11%).

[0107] Process for culturing stem cells

[0108] Herein are described a method for culturing of stem cells based on utilization of a support matrix comprising cellulose nanofibers with excellent surface-to-volume ratio and physical properties that mimic the collagen and elastin fiber structures that make up the human extracellular matrix. Stem cells grown on the support matrix retain their sternness and can be efficiently produced in a simple and scalable manner.

[0109] Thus, an aspect of the present invention relates to a method for culturing of stem cells, said method comprising the steps of: providing a container comprising a support matrix;

[0110] - seeding stem cells on said support matrix; and incubating said stem cells; wherein said support matrix comprises cellulose nanofibers. It is to be understood that the term "seeding" can be used interchangeably with the term "inoculating", and that the term "incubating" in the present context is equivalent to "expansion / expanding" which is a commonly used term to describe growth of cells.

[0111] By processing the cellulose nanofibers into shorter lengths, a material with greatly enhanced surface-to-volume ratio is obtained. Without being bound by theory, it is contemplated that the shorter nanofiber strands may contribute to the mechanical strength of the support matrix, as shorter fiber strands in general will appear stiffer than their longer counterparts. However, it is also contemplated that too short cellulose (e.g. below 10 pm) nanofibers will not be suitable for building a support matrix with a macroscopic structure comprising the required porosity. The mechanical strength of the support matrix can be further adjusted by crosslinking of the processed cellulose nanofibers. Crosslinking of the individual cellulose nanofibers is preferably achieved by the addition of a crosslinking agent.

[0112] Therefore, an embodiment of the present invention relates to the method as described herein, wherein the cellulose nanofibers have a mean length of less than about 250 pm, and wherein the cellulose nanofibers are crosslinked with a crosslinking agent.

[0113] Herein has been identified cellulose nanofiber dimensions that produce a highly porous support matrix with great surface-to-volume ratio, while at the same time retaining sufficient mechanical stability when moulded to yield a macroscopic structure. This makes the support matrix ideal for interacting with cells seeded thereupon and capable to withstand shear stress experienced when in operation, such as in an agitated bioreactor.

[0114] Thus, an embodiment of the present invention relates to the method as described herein, wherein the cellulose nanofibers have a mean length in the range of about 30 pm to about 250 pm, such as about 40 pm to about 200 pm, such as about 50 pm to about 175 pm, such as about 60 pm to about 150 pm, preferably about 70 pm to about 120 pm.

[0115] Another embodiment of the present invention relates to the method as described herein, wherein the cellulose nanofibers have a mean diameter in the range of about 10 nm to about 2000 nm, such as about 50 nm to about 1500 nm, such as about 100 nm to about 1000 nm, preferably about 250 nm to about 750 nm.

[0116] Electrospinning is a versatile method for producing nanofibers with tuneable properties, and provide the desired dimensions of cellulose nanofibers. Electrospinning relies on an electrostatic field to create fibers. By adjusting process parameters such as voltage, flow rate, and distance between the spinneret and collector, it is possible to control the fiber dimensions and morphologies. For example, increasing the voltage produce thinner nanofibers, nanofiber diameter can be adjusted by selecting the flow rate, and the distance between the spinneret and collector determines the stretching and alignment of the fibers. The properties of the polymer solution (such as viscosity, concentration, and surface tension) impact the dimensions and morphologies of the final electrospun cellulose nanofiber. Electrospinning, in contrast to other cellulose nanofiber fabrication methods, such as defibrillation from a raw pulp material, provides precise control over nanofiber properties. The cellulose nanofibers are consequently preferably obtained by electrospinning.

[0117] Therefore, an embodiment of the present invention relates to the method as described herein, wherein the cellulose nanofibers are electrospun cellulose nanofibers.

[0118] Crosslinking of the individual cellulose nanofibers can be achieved by the addition of a crosslinking agent. The crosslinking agent is not limited to any specific type of crosslinking agent. The cellulose backbone comprises hydroxyl groups that may be used to bridge the nanofibers via the crosslinking agent. Thus, the crosslinking agent preferable has functional groups that may react with the hydroxyl groups to create covalent bonds.

[0119] Therefore, an embodiment of the present invention relates to the method as described herein, wherein crosslinking agent is selected from the group consisting of epichlorohydrin, formaldehyde dimethyl acetate (FDA), maleic anhydride (MAL), 3- glycidoxypropyltrimethoxysilane (GPS), citric acid, and 1,2,3,4-butanetetracarboxylic acid.

[0120] Another embodiment of the present invention relates to the method as described herein, wherein the crosslinking agent comprises at least two functional groups that can react with hydroxyl groups to form covalent bonds.

[0121] A further embodiment of the present invention relates to the method as described herein, wherein the crosslinking agent comprises at least two functional groups selected from the group consisting of carboxylic acids, aldehydes, epoxides, halides, anhydrides, silanes, and azetidinium.

[0122] A still further embodiment relates to the method as described herein, wherein the crosslinking agent comprises at least two azetidinium groups. Some crosslinking agents have been identified as particular useful for crosslinking of cellulose nanofibers. In particular, it is demonstrated herein that crosslinking with polyamide epichlorohydrin results in a support matrix with large surface-to-volume ratio and great mechanical properties.

[0123] Accordingly, an embodiment of the present invention relates to the method as described herein, wherein the crosslinking agent is a polyamide epichlorohydrin resin.

[0124] Another embodiment of the present invention relates to the method as described herein, wherein the crosslinking agent is a hexanedioic acid, polymer with Nl-(2-aminoethyl)-l,2- ethanediamine and 2-(chloromethyl)oxirane.

[0125] Hexanedioic acid is also known as adipic acid and has the chemical formula (CH2)4(COOH)2. Epichlorohydrin is also known as 2-(chloromethyl)oxirane and is an organochlorine compound and an epoxide. Nl-(2-aminoethyl)-l,2-ethanediamine is also known as diethylenetriamine and is an organic compound with the formula HN(CH2CH2NH2)2. It is N- alkylated upon reaction with epoxide groups forming crosslinks.

[0126] An embodiment of the present invention relates to the method as described herein, wherein the crosslinking agent is adipic acid diethylenetriamine epichlorohydrin copolymer.

[0127] It is to be understood that copolymers may also be referred to as resins.

[0128] The amount of crosslinking agent may be varied to modify the properties of the support matrix. For example, increasing the amount of crosslinking agent will create more dense and mechanically strong material. It has been found that certain ratios of cellulose nanofibers to crosslinking agent promotes properties such as high available surface area and sufficient mechanical strength.

[0129] The amount of crosslinking agent may be presented as the moles of crosslinking agent per moles of cellulose nanofibers. The molar ratio is relevant because it sets out how many crosslinking units that connects with glucose units within the support matrix. By altering the number of crosslinkers per glucose unit, the properties of the support matrix will change. Thus, a higher molar ratio may increase mechanical strength and reduce porosity. Herein, the moles for the cellulose nanofibers is given per glucose unit. Favourable molar ratios of crosslinking agent to cellulose have been identified that provide a support matrix which increase cell proliferation. The selected molar ratios of crosslinking agent to cellulose nanofibers are advantageous in that they result in a support matrix that allow cells to freely migrate within the porous structure but at the same time provide sufficient mechanical strength for the support matrix to be suitable for used in bioreactors which cause shear stress during operation. Importantly, rapid and robust proliferation of cells translates to a decreased cell population doubling time, which is a significant parameter for designing a cost-effective process for production of stem cells.

[0130] Thus, an embodiment of the present invention relates to the method as described herein, wherein the molar ratio of crosslinking agent to cellulose nanofibers is in the range of about 2*10-3mol / mol to about 175*10-3mol / mol, such as about 5*10-3mol / mol to about 100*10-3mol / mol, such as about 10*10-3mol / mol to about 80*10-3mol / mol, such as about 12*io-3mol / mol to about 50*10-3mol / mol, such as about 15*10-3mol / mol to about 25*10-3mol / mol.

[0131] Another embodiment of the present invention relates to the method as described herein, wherein the molar ratio of crosslinking agent to cellulose nanofibers is in the range of about 5*10-3mol / mol to about 50*10-3mol / mol, such as about 10*10-3mol / mol to about 4O*io-3mol / mol, such as about 12*10-3mol / mol to about 30*10-3mol / mol, such as about 16*10-3mol / mol to about 24*10-3mol / mol.

[0132] A preferred embodiment of the present invention relates to the method as described herein, wherein the molar ratio of crosslinking agent to cellulose nanofibers is in the range of about 19*10-3mol / mol.

[0133] It has been found that for some varieties of the support matrix it can be advantageous to decrease the molar ratio of crosslinking agent to cellulose nanofibers. Without being bound by theory, it is contemplated herein that the lower crosslinking degree may assist the migration of cells within the support matrix, resulting in better cell growth.

[0134] Therefore, an embodiment of the present invention relates to the method as described herein, wherein the molar ratio of crosslinking agent to cellulose nanofibers is in the range of about 90*10-3mol / mol, such as less than about 75*10-3mol / mol, such as less than about 50*10-3mol / mol, such as less than about 40*10-3mol / mol, such as less than about 3O*io-3mol / mol, such as less than about 25*10-3mol / mol, such as less than about 20*10"3mol / mol. A preferred embodiment of the present invention relates to the method as described herein, wherein the concentration of cellulose nanofibers is about 0.2 wt% to about 2 wt%, with respect to the total weight of the support matrix, and the molar ratio of crosslinking agent to cellulose nanofibers is in the range of about 15*10-3mol / mol to about 25*10-3mol / mol.

[0135] It is to be understood that the moles of the cellulose material refers to the moles per glucose unit in the processed cellulose nanofiber material.

[0136] The cellulose nanofibers of the support matrix may be functionalized with one or more functional moieties. Functionalization may be achieved by chemical coupling or coating of the cellulose nanofibers. The functional moieties attached to the cellulose nanofibers may be of either chemical or biological origin. In particular, positively charged groups may be advantageous as they induce electrostatic interactions between the support matrix and cells with a negatively charged membrane, thereby increasing attachment of cells to the support matrix. Biological moieties include proteins and peptides that are normally an integral part of the interaction between the cell and the extracellular environment. This interaction may further promote attachment of cells to the nanofibrous cellulose scaffold.

[0137] Thus, an embodiment of the present invention relates to the method as described herein, wherein the cellulose nanofibers comprise one or more functional moieties selected from chemical moieties and / or biological molecules.

[0138] Another embodiment of the present invention relates to the method as described herein, wherein the chemical moieties are selected from the group consisting of negatively charged groups, positively charged groups, zwitterionic groups, hydrophobic groups, hydrophilic groups, amphiphilic groups, ligands, and combinations thereof.

[0139] Still another embodiment of the present invention relates to the method as described herein, wherein the chemical moieties are positively charged groups.

[0140] Yet another embodiment of the present invention relates to the method as described herein, wherein the chemical moieties are selected from the group consisting of quaternary ammonium (QA), carboxymethyl (CM), diethylaminoethyl (DEAE), 4-(l- bromoethyl)benzoic acid, 4-(dimethylamino)pyridine, epoxide, and combinations thereof, preferably quaternary ammonium (QA). A further embodiment of the present invention relates to the method as described herein, wherein the chemical moiety is quaternary ammonium (QA).

[0141] It is to be understood that the chemical moieties may be attached to the cellulose backbone using conventional chemistry. Thus, reagents such as, but not limited to, 3- chloro-2-hydroxypropyl trimethyl ammonium chloride (CHPTAC), 2-chloro-N,N diethylethylamine hydrochloride (DAECH), and monochloro acetic acid (MCAA) may be used for attachment of QA, DEAE, and CM, respectively.

[0142] The cellulose nanofibers can also be functionalized and / or coated with a variety of biological molecules. Decorating the support matrix with biological molecules can assist in creating a three-dimensional milieu that mimics the extracellular matrix that the stem cells normally experience in their native environment. As such, preferred biological molecules include proteins normally found in the extracellular matrix (ECM), i.e. ECM proteins. Imitating the native environment of the stem cells is beneficial because it facilitates proliferation of the cells and promotes their ability to retain sternness identity during culturing.

[0143] Thus, an embodiment of the present invention relates to the method as described herein, wherein the biological molecules are selected from the group consisting of proteins, peptides, antibodies, amino acids, polypeptides, glycoproteins, lipoproteins, and antigens, and combinations thereof.

[0144] Another embodiment of the present invention relates to the method as described herein, wherein the biological molecule is a glycoprotein of the extracellular matrix selected from the group consisting of vitronectin, fibronectin, collagen, proteoglycans, laminin, tenascins, and integrins, preferably vitronectin.

[0145] It has been found that coating of the cellulose nanofibers with vitronectin promotes proliferation of stem cells seeded onto the support matrix. In particular, beneficial molar ratios of vitronectin to cellulose nanofibers have been identified. Without being bound by theory, it is contemplated that the specific molar ratios closely mimic the native environment of the extracellular matrix and therefore promote cell proliferation and retention of sternness identity.

[0146] Thus, a preferred embodiment of the present invention relates to the method as described herein, wherein the biological molecule is vitronectin. Another preferred embodiment of the present invention relates to the method as described herein, wherein the molar ratio of vitronectin to cellulose nanofibers is in the range of about 5*10-7mol / mol to about 75*10-6mol / mol, such as about IO-6mol / mol to about 5O*io-6mol / mol, such as about 2*10-6mol / mol to about 25*10-6mol / mol, such as about 4*10-6mol / mol to about 12*10-6mol / mol.

[0147] Yet another embodiment of the present invention relates to the method as described herein, wherein the molar ratio of vitronectin to cellulose nanofibers is about 8*10-6mol / mol.

[0148] A further preferred embodiment of the present invention relates to the method as described herein, wherein the one or more functional moieties comprises quaternary ammonium (QA) and vitronectin.

[0149] The support matrix may be used for seeding of stem cells of various types. A preferred type is mesenchymal stem cells (MSC), which may differentiate into multiple cell lineages useful in regenerative medicine and for therapeutic applications.

[0150] Therefore, an embodiment of the present invention relates to the method as described herein, wherein the stem cells are selected from the group consisting of mesenchymal stem cells (MSCs), induced pluripotent stem cells (iPSCs), embryonic stem cells (ESCs), hematopoietic stem cells (HSCs), neural stem cells (NSCs), cardiac stem cells, amniotic fluid stem cells, epidermal stem cells, adipose derived stem cells (ASC) and endothelial progenitor cells (EPCs).

[0151] A preferred embodiment of the present invention relates to the method as described herein, wherein the stem cells are mesenchymal stem cells (MSCs).

[0152] Another embodiment of the present invention relates to the method as described herein, wherein the stem cells are of human origin.

[0153] A further embodiment of the present invention relates to the method as described herein, wherein the human stem cells are derived from bone marrow.

[0154] The stem cells are incubated in a liquid environment, such a liquid cell culturing medium. Any conventional cell culturing medium suitable for proliferation of stem cells may be used. Advantageously, the cell culturing medium may be supplemented with an expansion medium once the initial lag phase of culturing the stem cells has passed. It is to be understood that the expansion medium is a booster medium different from the cell culturing medium.

[0155] Thus, an embodiment of the present invention relates to the method as described herein, wherein the container comprises a solvent.

[0156] Another embodiment of the present invention relates to the method as described herein, wherein the solvent comprises a cell culturing medium.

[0157] A further embodiment of the present invention relates to the method as described herein, wherein an expansion medium is added to the container at a time between about 24 hours and about 72 hours after seeding of said stem cells, such as between about 36 hours and 60 hours after seeding of said stem cells, preferably about 48 hours after seeding of said stem cells.

[0158] It is to be understood that cell culture medium and stem cells may be added in two separate steps or simultaneously. Preferably, the cell culture medium is added to the container first followed by seeding of the stem cells on the support matrix. The support matrix can be added to the container before or after addition of the solvent, e.g. cell culturing medium.

[0159] Following incubation, the proliferated stem cell population can be extracted from the container and used for any downstream application, or the proliferated stem cell population can be directly used for downstream applications without extraction from the container (such as collecting culture supernatants).

[0160] Therefore, an embodiment of the present invention relates to the method as described herein, wherein the step of incubating the stem cells is followed by extracting the proliferated stem cell population from the container.

[0161] The container is not limited to any particular container as long as it is a suitable vessel for culturing of stem cells.

[0162] Therefore, an embodiment of the present invention relates to the method as described herein, wherein the container is selected from the group consisting of a bioreactor, a cell culturing plate, a well plate, a cell culture bottle, a spinner flask, a shaker flask, and a roller bottle, a petri dish, and a tube. Bioreactors are of particular importance to large scale industrial production. They may hold larger volumes than laboratory- or pilot study equipment and are used in upstream processes to expand and scale cell culture for production. A typical process involves initial expansion of cells in a smaller vessel followed by successive expansion into larger culture vessels. When the culture volume and density is optimal, the cells are transferred to a production bioreactor, which offers a controlled microenvironment and nutrient delivery to regulate cell growth and differentiation, improving standardization and reproducibility. The support matrix described herein is suitable for use at any point in this process and is particularly advantageous at the production scale with packed bed bioreactors where other support materials or microcarriers may not possess the characteristics for scalability of high quality cells to be feasible for commercial use.

[0163] Accordingly, a preferred embodiment of the present invention relates to the method as described herein, wherein said container is a bioreactor.

[0164] In packed bed bioreactors, the support material is positioned in a bed (or compartment) within the bioreactor. By proliferating the stem cells in a bed, they experience less stress than when freely floating in a bioreactor. However, the support matrix in the bed is still subject to shear stress caused by agitation, e.g. perfusion of solvent, in the bioreactor.

[0165] Therefore, an embodiment of the present invention relates to the method as described herein, wherein said bioreactor comprises a compartment containing the support matrix. Packed bed bioreactors come in two main categories. One wherein the bed is stationary within the reactor, this is typically referred to as a fixed bed bioreactor. Another wherein the bed is moving within the reactor, this is also known as a dynamic bed bioreactor or moving bed bioreactor. In the latter type of bioreactor, the bed may be connected to one or more shafts that moves within the bioreactor, such as by rotation. The support matrix may be used with any type of packed bed bioreactor.

[0166] Accordingly, an embodiment of the present invention relates to the method as described herein, wherein said container is a packed bed bioreactor, such as a fixed bed bioreactor or a dynamic bed bioreactor.

[0167] Another embodiment of the present invention relates to the method as described herein, wherein the bioreactor is a fixed bed bioreactor.

[0168] A further embodiment of the present invention relates to the method as described herein, wherein the bioreactor is a dynamic bed bioreactor. The method for culturing stem cells is preferably performed with agitation of the cell culture within the container to promote better transport of nutrients and oxygen to the stem cells. The method for stem cell culturing may be performed in a packed bed bioreactor. In this type of bioreactor typical means of agitation includes, but is not limited to, stirring by an impeller and / or perfusion by a circulation pump.

[0169] Thus, an embodiment of the present invention relates to the method as described herein, wherein incubating said stem cells is performed under agitation.

[0170] Another embodiment of the present invention relates to the method as described herein, wherein agitation is effected by mechanical agitation, circulation pump, and / or pneumatic means.

[0171] In packed bed bioreactor particularly the agitation creates a flow of solvent within the bioreactor that continuously supplies the cells attached to the support matrix within the bed with fresh nutrients and oxygen. The direction of the flow may vary depending on the type of bioreactor.

[0172] Therefore, an embodiment of the present invention relates to the method as described herein, wherein said agitation comprises perfusion of said solvent through the container.

[0173] A further embodiment of the present invention relates to the method as described herein, wherein said perfusion of solvent is longitudinal and / or radial with respect to the container.

[0174] The support matrix is suitable for use all the way from pilot scale / product development (1 L to 100 L) to production scale (100 L to 1000 L). If required, the material may be also used as support matrix in even larger bioreactors.

[0175] Thus, an embodiment of the present invention relates to the method as described herein, wherein the volume of the container is in the range of about 1 L to about 1000 L, such as about 100 L to about 1000 L.

[0176] Another embodiment of the present invention relates to the method as described herein, wherein the volume of the container is at least 10 L, such as at least 50 L, such as at least 100 L, such as at least 200 L, such as at least 500 L, such as at least 1000 L. The container is not limited to any particular material but may be made from e.g. stainless steel or of a disposable material, the latter of which provides flexibility and reduces downtime caused by the need for cleaning and sterilization of the container.

[0177] The cellulose nanofibers of the support matrix may be provided as a nanofibrous cellulose scaffold as described herein. Methods for producing the nanofibrous cellulose scaffold is provided herein and yield durable scaffolds with large surface areas that advantageous for use as a support matrix for culturing demanding cells, such as stem cells. The nanofibrous cellulose scaffold described herein offers all the traits (high surface-to-volume ratio, adequate mechanical strength, customizable) that are necessary for use as an efficient support matrix for packing within a packed bed bioreactor. Importantly, the nanofibrous cellulose scaffold can easily be scaled for industrial use without cost and applicability of the support matrix when used at large volumes being a hindrance.

[0178] Thus, an aspect of the present invention relates to use of a nanofibrous cellulose scaffold as described herein as a support matrix for culturing of stem cells.

[0179] Another aspect of the present invention relates to a support matrix comprising a nanofibrous cellulose scaffold as described herein.

[0180] The nanofibrous cellulose scaffold is founded on the cellulose nanofibers, crosslinking agent, and functional moieties as described above and those features may readily be utilized in the nanofibrous cellulose scaffold.

[0181] Accordingly, an aspect of the present invention relates to a nanofibrous cellulose scaffold comprising cellulose nanofibers with a mean length of less than about 250 pm, and wherein the cellulose nanofibers are crosslinked with a crosslinking agent.

[0182] The nanofibrous cellulose scaffold comprises an intricate network of cellulose nanofibers connected by a crosslinking agent to present stem cells with a three-dimensional platform upon which they can proliferate under native-like conditions. The interplay between the short cellulose nanofibers and the crosslinking agent guides the formation of pores into which the stem cells can migrate and proliferate. If the cellulose nanofibers are too long, they will filter and bundle, ultimately causing the scaffold to lose its macrostructure. If the cellulose nanofibers are too short (only few pm or below e.g. 10 pm), the three- dimensional network will become too dense and not accessible to the cells and / or the macrostructure cannot be formed. Thus, an embodiment of the present invention relates to the nanofibrous cellulose scaffold as described herein, wherein the cellulose nanofibers are connected by the crosslinking agent in a three-dimensional network.

[0183] Another embodiment of the present invention relates to the nanofibrous cellulose scaffold as described herein, wherein the crosslinking agent is selected from the group consisting of epichlorohydrin, formaldehyde dimethyl acetate (FDA), maleic anhydride (MAL), 3- glycidoxypropyltrimethoxysilane (GPS), citric acid, and 1,2,3,4-butanetetracarboxylic acid.

[0184] However, the crosslinking agent are not particularly limited, and it is to be understood that crosslinking agents described elsewhere herein are suitable for use with the nanofibrous cellulose scaffold.

[0185] A further embodiment of the present invention relates to the nanofibrous cellulose scaffold as described herein, wherein the cellulose nanofibers have a mean length in the range of about 30 pm to about 250 pm, such as about 40 pm to about 200 pm, such as about 50 pm to about 150 pm, preferably about 60 pm to about 100 pm.

[0186] A still further embodiment of the present invention relates to the nanofibrous cellulose scaffold as described herein, wherein the cellulose nanofibers have a mean diameter in the range of about 10 nm to about 2000 nm, such as about 50 nm to about 1500 nm, such as about 100 nm to about 1000 nm, preferably about 250 nm to about 750 nm.

[0187] The dimensions of the cellulose nanofibers (length and diameter) within the nanofibrous cellulose scaffold may be as described elsewhere herein.

[0188] The nanofibrous cellulose scaffold may preferably comprise one or more functional moieties to promote stem cell proliferation and retention of sternness identity. One preferred coating is by vitronectin, potentially in combination with quaternary ammonium (QA). However, functional moieties described elsewhere herein are equally suitable for the nanofibrous cellulose scaffold.

[0189] Accordingly, an embodiment of the present invention relates to the nanofibrous cellulose scaffold as described herein, wherein the cellulose nanofibers are functionalized with one or more functional moieties. The nanofibrous cellulose scaffold may be provided in the form of a dry continuous material, the form of which may be adapted to suit existing and future cell culturing devices, such as packed bed bioreactors. An advantage of the nanofibrous cellulose scaffold is that any shape and dimension can be produced. This means that thicker discs, cylinders, or sheets of the material can be formed, thereby obviating need of stacking thin discs or sheets directly on top of each other to achieve the desired volume of the microcarrier or support matrix. Stacking of support matrix entities on top of each other may cause inconsistent culturing since the homogeneity of the support matrix is lost in the interface between. Overall, the nanofibrous cellulose scaffold may act as a homogeneous support matrix solution which provides a higher surface-to-volume ratio than existing solutions.

[0190] Thus, an embodiment of the present invention relates to the nanofibrous cellulose scaffold as described herein, wherein the nanofibrous cellulose scaffold is provided as a dry continuous material.

[0191] Another embodiment of the present invention relates to the nanofibrous cellulose scaffold as described herein, wherein the physical form of the nanofibrous cellulose scaffold is a geometry selected from the group consisting of a disk, cylinder, cube, sheet, and sphere.

[0192] In a preferred variant of the nanofibrous cellulose scaffold, it is in the form of a small disk of a diameter suitable for packing in the bed of a bioreactor. Such packing can be in random-oriented fashion within the bed.

[0193] Therefore, an embodiment of the present invention relates to the nanofibrous cellulose scaffold as described herein, wherein nanofibrous cellulose scaffold is a disk with a diameter in the range of about 8 mm to about 16 mm, such as about 10 mm to about 14 mm, preferably about 12 mm.

[0194] For some applications it may be preferred to provide the nanofibrous cellulose scaffold in larger dimensions. For example, a thicker disk may be advantageous because it can better withstand shear forces in a bioreactor than a smaller disk. Furthermore, thick disks provide a voluminous continuous scaffold for the stem cells to proliferate within without artifacts from spaces at the interface of stacked disks. Artifacts at the interphase between separate scaffolds (e.g. disks) include, but is not limited to, irregular flow dynamics and increased shear stress, which may disturb native cell proliferation. The nanofibrous cellulose scaffold can be moulded in any shape and it has been demonstrated to have excellent compressive strength and resiliency at larger dimensions. Therefore, an embodiment of the present invention relates to the nanofibrous cellulose scaffold as described herein, wherein the nanofibrous cellulose scaffold has a thickness in all three dimensions of at least about 0.1 mm, such as at least about 0.2 mm, such as at least about 0.3 mm, such as at least about 0.4 mm, such as at least about 0.5 mm.

[0195] Another embodiment of the present invention relates to the nanofibrous cellulose scaffold as described herein, wherein the nanofibrous cellulose scaffold has a thickness in all three dimensions of at least about 0.5 mm, such as at least about 1 mm, such as at least about 1.5 mm, such as at least about 2 mm, such as at least about 3 mm, such as at least about 4 mm, such as at least about 5 mm, such as at least about 6 mm, such as at least about 7 mm, such as at least about 8 mm, such as at least about 9 mm, such as at least about 10 mm.

[0196] Yet embodiment of the present invention relates to the nanofibrous cellulose scaffold as described herein, wherein the nanofibrous cellulose scaffold is a disk or cylinder with a thickness in the range of about 0.2 mm to about 20 mm, such as about 0.5 mm to about 15 mm, such as about 1 mm to about 10 mm, such as about 1.5 mm to about 8 mm, such as about 2 mm to about 5 mm.

[0197] Another embodiment of the present invention relates to the nanofibrous cellulose scaffold as described herein, wherein the nanofibrous cellulose scaffold is a disk or cylinder with a thickness in the range of about 0.5 mm to about 3 mm, such as about 1 mm to about 2.5 mm, preferably about 2 mm.

[0198] A further embodiment of the present invention relates to the nanofibrous cellulose scaffold as described herein, wherein nanofibrous cellulose scaffold is a disk with a diameter in the range of about 8 mm to about 70 mm, such as about 10 mm to about 60 mm, such as about 10 mm to about 40 mm, such as about 10 mm to about 30 mm, such as about 10 mm to about 20 mm.

[0199] A still further embodiment of the present invention relates to the nanofibrous cellulose scaffold as described herein, wherein nanofibrous cellulose scaffold is a disk with a diameter in the range of about 40 mm to about 70 mm, such as about 50 mm to about 60 mm, preferably about 57 mm.

[0200] The nanofibrous cellulose scaffold can conveniently be provided and stored as a lyophilised material that can easily be kept until use. Upon use it is simply added to the cell culturing container, e.g. a bioreactor. Here it may for instance be packed in a bed. The flexibility of production method means that any shape of nanofibrous cellulose scaffold can be produced to fit existing or future cell culturing devices. The material is both microporous to allow stem cell migration and proliferation within the support matrix and resilient enough to withstand the shear forces in a packed bed bioreactor.

[0201] Therefore, an embodiment of the present invention relates to the nanofibrous cellulose scaffold as described herein, wherein the nanofibrous scaffold is provided as a lyophilized material.

[0202] Another embodiment of the present invention relates to the nanofibrous cellulose scaffold as described herein, wherein the nanofibrous cellulose scaffold is microporous.

[0203] A further embodiment of the present invention relates to the nanofibrous cellulose scaffold as described herein, wherein the nanofibrous scaffold is resilient.

[0204] The compressive strength of the nanofibrous scaffold may be modified by adjusting the amount of cellulose nanofibers and crosslinking agent.

[0205] An embodiment of the present invention relates to the nanofibrous cellulose scaffold as described herein, wherein the nanofibrous scaffold has a compressive strength in the range of about 0.1 mm / N to about 30 mm / N.

[0206] Yet another embodiment of the present invention relates to the nanofibrous cellulose scaffold as described herein, wherein the compressive strength is measured in accordance with ISO 604:2002 - Plastics - Determination of compressive properties.

[0207] The nanofibrous cellulose scaffold has a low density. Without being bound by theory, it is contemplated that the low density is caused by the homogenous distribution of the short cellulose nanofibers in the material, which ensures that large entanglements and clusters of nanofibers are avoided. Thus, the nanofibrous cellulose scaffold has a significantly reduced density compared to that of pure cellulose in the form of sheets, which is 1.5 g / cm3. Despite the low density, the nanofibrous cellulose scaffold provides a superior available surface area that is advantageous for culturing of adherent cells, such as stem cells.

[0208] Accordingly, an embodiment of the present invention relates to the nanofibrous cellulose scaffold as described herein, wherein the nanofibrous cellulose scaffold has a density in the range of about 0.0005 g / cm3to about 0.5 g / cm3, such as about 0.001 g / cm3to about 0.1 g / cm3, such as about 0.001 g / cm3to about 0.040 g / cm3.

[0209] Another embodiment of the present invention relates to the nanofibrous cellulose scaffold as described herein, wherein the nanofibrous cellulose scaffold has a BET surface area of at least about 40000 cm2 / g, such as at least about 50000 cm2 / g, such as at least about 55000 cm2 / g, such as at least about 60000 cm2 / g, such as at least about 70000 cm2 / g, such as at least about 80000 cm2 / g.

[0210] Yet another embodiment of the present invention relates to the nanofibrous cellulose scaffold as described herein, wherein BET surface area is measured according to ISO 9277:2022 - Determination of the specific surface area of solids by gas adsorption — BET method.

[0211] The cellulose nanofibers may be combined with other types of nanofibers to enhance the nanofibrous cellulose scaffold with new properties. This may be beneficial for specific stem cell types wherein the addition of other types of nanofibers widens the options for mimicking the local extracellular matrix of that particular tissue wherefrom the stem cells are. The additional polymers may be of natural or synthetic origin. For natural nanofibers, especially types of nanofibers traditionally present in the extracellular environment, such as collagen, may work in synergy with cellulose to mimic the ECM. Additional polymers may also be introduced to modify the mechanical properties of the material.

[0212] Therefore, an embodiment of the present invention relates to the nanofibrous cellulose scaffold as described herein, wherein the nanofibrous scaffold further comprises one or more nanofibers selected from natural polymers or synthetic polymers.

[0213] Another embodiment of the present invention relates to the nanofibrous cellulose scaffold as described herein, wherein the natural polymers are selected from the group consisting of collagen, silk fibroin, keratin, gelatin and polysaccharides, and combinations thereof.

[0214] A further embodiment of the present invention relates to the nanofibrous cellulose scaffold as described herein, wherein the synthetic polymers are selected from the group consisting of polycaprolactone (PCL), poly(lactic acid) (PLA), polystyrene, polyurethane (PU), poly(lactic-co-glycolic acid) (PLGA), poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), and poly(ethylene-co-vinylacetate) (PEVA). A simple and cost-effective method for preparing the nanofibrous cellulose scaffold has been identified. The method produces a nanofibrous cellulose scaffold with great mechanical properties and a large surface-to-volume ratio, making the scaffold a commercially attractive and readily scalable end-product for use as a support matrix for stem cell culturing.

[0215] Thus, an aspect of the present invention relates to a method of preparing a nanofibrous cellulose scaffold, said method comprising the steps of:

[0216] (i) providing an initial cellulose nanofiber material,

[0217] (ii) dividing said initial cellulose nanofiber material into a processed cellulose nanofiber material,

[0218] (iii) crosslinking the processed cellulose nanofiber material, and

[0219] (iv) drying the processed cellulose nanofiber material, thereby providing the nanofibrous cellulose scaffold.

[0220] Dividing of the initial cellulose nanofiber material is important as it ensures a homogeneous distribution of the cellulose nanofibers in the final scaffold. The shorter cellulose nanofibers also significantly reduce the risk of entanglement of the nanofibers and formation of clusters of nanofibers, which leads to less available surface area exposed to the cells and increased risk of interfering with any mechanical parts in a bioreactor, such as impellers, pumps or the like. Moreover, larger entanglements or clusters of cellulose nanofibers also render the final nanofibrous cellulose scaffold difficult to handle.

[0221] It has been found that dispersing the initial cellulose nanofiber material results in a homogeneous material that is suitable for use as a support matrix. Without being bound by theory, it is contemplated that dispersing the initial cellulose nanofiber material results in a distribution which are spaced so as to allow multiple attachment points for the cells leading to improved proliferation, hereunder reduced cell population doubling time.

[0222] Accordingly, an embodiment of the present invention relates to the method as described herein, wherein dividing said initial cellulose nanofiber material is achieved by dispersing.

[0223] Another embodiment of the present invention relates to the method as described herein, wherein dispersing is performed with a high-speed disperser.

[0224] A further embodiment of the present invention relates to the method as described herein, wherein dividing said initial nanofiber material comprises a step of cutting the initial nanofiber material with a disperser. Increasing the dispersing time reduces the mean length of the cellulose nanofibers in the nanofibrous cellulose scaffold. In particular, it is advantageous to disperse the material for at least a couple of minutes to reduce nanofiber entanglement and cluster formation. Also, without being bound by theory, it is contemplated that longer cellulose nanofibers are not as easily substituted on the hydroxyl groups, thereby leading to a lower degree of substitution (DS) of functional moieties.

[0225] Thus, an embodiment of the present invention relates to the method as described herein, wherein dispersing is performed for at least 2 min, such as at least 5 min, such as at least 10 min, such as at least 15 min, such as at least 20 min, such as at least 30 min, such as at least 40 min, such as at least 50 min, such as at least 60 min, such as at least 90 min, such as at least 120 min.

[0226] Another embodiment of the present invention relates to the method as described herein, wherein dispersing is performed for a period of time in the range of 2 min to 120 min, such as 2 min to 90 min, such as 5 min to 60 min, such as 10 min to 60 min, such as 15 min to 60 min.

[0227] A preferred embodiment of the present invention relates to the method as described herein, wherein dispersing is performed for at least 5 min.

[0228] Another preferred embodiment of the present invention relates to the method as described herein, wherein dispersing is performed for at least 15 min.

[0229] A still further preferred embodiment of the present invention relates to the method as described herein, wherein dispersing is performed for at least 60 min.

[0230] Dispersing of the initial cellulose nanofiber material for at least 15 min, and even at least 60 min, can be advantageous as it reduces entanglement and cluster formation of the cellulose nanofibers.

[0231] Preferably dispersing is performed at high speed, such as at about 18000 rpm. The speed may be adjusted depending on the type of disperser. Any type of disperser may be used, including, but not limited to, laboratory high-speed disperser, pilot-scale high-speed dispersers, and production-scale high-speed dispersers. The speed may be adjusted to produce a good vortex in the solution, and can depend on volume and viscosity of the solution. Therefore, an embodiment of the present invention relates to the method as described herein, wherein dispersing is performed at a speed in the range of about 10000 rpm to about 30000 rpm, such as at about 12000 rpm to about 25000 rpm, such as about 15000 rpm to about 20000 rpm.

[0232] During the dividing step, the cellulose nanofibers are reduced in length. It is important that the cellulose nanofibers are not too long as it will cause entanglement of the nanofibers and cluster formation. Cells are not able to penetrate and migrate into these tight clusters of entangled fibers and thereby a portion of the large surface area of the nanofibers are lost.

[0233] Therefore, an embodiment of the present invention relates to the method as described herein, wherein the mean length of the cellulose nanofibers in said processed cellulose nanofiber material is shorter than the mean length of the cellulose nanofibers in said initial cellulose nanofiber material.

[0234] Another embodiment of the present invention relates to the method as described herein, wherein the mean length of the cellulose nanofibers in said processed cellulose nanofiber material is less than 250 pm, such as less than about 200 pm, such as less than about 150 pm, such as less than about 120 pm, such as less than about 100 pm.

[0235] The dividing step may be performed under cooling to lower the ductility of the cellulose nanofibers, making them more brittle and easier to divide. The cooling may occur before or during the dividing of the initial cellulose nanofiber material. Cooling may comprise cooling of the container in which the initial cellulose nanofiber material is held during the dividing step or cooling the initial cellulose nanofiber material by exposure to a coolant, such as liquid hydrogen, liquid helium and / or liquid nitrogen, or by keeping the initial cellulose nanofiber material in a fridge or freezer immediately before the dividing step.

[0236] Thus, an embodiment of the present invention relates to the method as described herein, wherein the initial cellulose nanofiber material is cooled before or during the dividing step.

[0237] Cellulose sheets, if prepared by electrospinning, may be highly static and difficult to handle. Therefore, the initial cellulose nanofiber material may conveniently be provided as a liquid sample that is ready for processing, e.g. by dispersing.

[0238] Thus, an embodiment of the present invention relates to the method as described herein, wherein the initial cellulose nanofiber material is provided as a liquid sample. Another embodiment of the present invention relates to the method as described herein, wherein the solvent of the liquid sample comprises water and / or ethanol.

[0239] The properties of the nanofibrous cellulose scaffold e.g. after drying and moulding) may be guided by the density of cellulose nanofibers that the network comprises subsequent to removal of liquid. For example, addition of lower concentrations of cellulose nanofibers will provide a more porous network wherein the cells may easier migrate within. In contrast, higher concentrations of cellulose nanofibers result in greater mechanical strength of the material. The cellulose concentration in the liquid sample during preparation of the nanofibrous cellulose scaffold is reflective also of the concentration of cellulose nanofibers in the final scaffold (after removal of liquid). Porosity and mechanical strength are also affected by the degree of crosslinking. Some advantageous combinations of cellulose content and crosslinking degree that provides sufficient mechanical strength of the material to survive shear forces in a packed bed bioreactor and at same time provide high available surface area has been identified herein.

[0240] Thus, an embodiment of the present invention relates to the method as described herein, wherein the concentration of cellulose nanofibers in the processed cellulose nanofiber material is about 0.02 wt% to about 10 wt%, such as about 0.05 wt% to about 8 wt%, such as about 0.1 wt% to about 5 wt%, such as about 0.2 wt% to about 3 wt%, such as about 0.25 wt% to about 2 wt%, preferably about 0.3 wt% to about 1 wt%, with respect to the total weight of the liquid sample.

[0241] Another embodiment of the present invention relates to the method as described herein, wherein the concentration of cellulose nanofibers in the processed cellulose nanofiber material is about 2 wt%.

[0242] For some variants of the nanofibrous cellulose scaffold it has been found to be advantageous to retain the cellulose concentration at a low level. Maintaining a low cellulose concentration secures a high porosity of the resulting nanofibrous cellulose scaffold, thereby allowing cells to migrate into the scaffold and utilise the large exposed surface area of the nanofibers.

[0243] Therefore, a preferred embodiment of the present invention relates to the method as described herein, wherein the concentration of cellulose nanofibers in the processed cellulose nanofiber material is about 0.2 wt%. A common source of cellulose nanofibers is pulp, which may be mechanically defibrillated to generate cellulosic nanofibers. Mechanical treatment may include high-pressure homogenization, grinding and / or microfluidization. Mechanical defibrillation is commonly utilised in the pulp and paper industry, as well as the textile industry. Unfortunately, it can be difficult to precisely control cellulose nanofiber dimensions and properties when the starting material is raw pulp (that can vary in composition depending on source) which is subsequently exposed to the harsh mechanical treatment. Thus, it is preferred to avoid cellulose material derived directly from a raw plant source (e.g. without any treatment to extract and / or purify the cellulose nanofibers) as the source of the cellulose nanofibers. Raw plant sources for cellulose material may include wood, such as softwood (e.g. spruce, pine, fir, larch, or hemlock) or hardwood (e.g. birch, aspen, poplar, alder, eucalyptus or acacia), or non-wood sources such as agricultural residues, grasses or other plant substances (e.g. straw, leaves, bark, seeds, hulls, flowers, vegetables or fruits from cotton, corn, wheat, oat, rye, barley, rice, flax, hemp, manilla hemp, sisal hemp, jute, ramie, kenaf, bagasse, bamboo or reed).

[0244] Accordingly, it is advantageous to use initial cellulose nanofiber material derived from a sources, e.g. plant source, that has been treated to purify the cellulose nanofibers to ensure high quality and consistency of the cellulose nanofibers utilised for preparing the nanofibrous cellulose scaffold.

[0245] Therefore, an embodiment of the present invention relates to the method of preparing a nanofibrous cellulose scaffold as described herein, wherein the initial cellulose nanofiber material is not derived from a raw plant source.

[0246] Another embodiment of the present invention relates to the method of preparing a nanofibrous cellulose scaffold as described herein, wherein the initial cellulose nanofiber material is not prepared by mechanical treatment, such as mechanical defibrillation, such as mechanical disintegration.

[0247] It is to be understood that a raw plant source is a source which has not been treated to extract or purify the cellulose nanofibers. Extraction may include chemical treatment of a cellulose source, such as a raw plant source. The chemical treatment can comprise treating the raw plant source with acetic acid and acetic anhydride in the presence of a catalyst, such as sulfuric acid. The chemical treatment can produce a cellulose acetate solution which can be further processed, e.g. by electrospinning, to yield cellulose nanofibers with highly controllable dimensions and properties. Thus, the cellulose nanofibers may advantageously be obtained from electrospinning to allow precise control over nanofiber dimensions and quality. Electrospinning involves dissolving cellulose or its derivatives in a suitable solvent and then spinning it into nanofibers using an electric field. Electrospun nanofibers are spun as monofibers that can be aligned on a drum roll to yield a cellulose sheet that can subsequently be further processed.

[0248] Therefore, an embodiment of the resent invention relates to the method of preparing a nanofibrous cellulose scaffold as described herein, wherein the initial cellulose nanofiber material comprises monofibers.

[0249] Another embodiment of the resent invention relates to the method of preparing a nanofibrous cellulose scaffold as described herein, wherein said nanofibrous cellulose scaffold comprises monofibers.

[0250] The mean diameter of the cellulose nanofibers can be guided during the preparation of the initial cellulose nanofiber material. This may be achieved by varying the parameters of e.g. the electrospinning or meltblowing process, such as the voltage or heat applied, the speed and type of injection, and / or the rotational speed of the collector drum. The mean diameter of the cellulose nanofibers may be varied depending on the application, e.g. the type of stem cells to be cultured. It has been found that for many applications, a mean diameter of about 400 nm to about 600 nm, such as about 500 nm, is advantageous.

[0251] Accordingly, an embodiment of the present invention relates to the method as described herein, wherein the initial cellulose nanofiber material is prepared by a process selected from the group consisting of electrospinning, meltblowing, drawing, self-assembly, template synthesis, and thermal-induced phase separation.

[0252] Another embodiment of the present invention relates to the method as described herein, wherein the initial cellulose nanofiber material is prepared by electrospinning.

[0253] A further embodiment of the present invention relates to the method as described herein, wherein the mean diameter of the cellulose nanofibers in said initial cellulose nanofiber material and / or processed cellulose nanofiber material is in the range of about 10 nm to about 2000 nm, such as about 50 nm to about 1500 nm, such as about 100 nm to about 1000 nm, preferably about 250 nm to about 750 nm. In a preferred embodiment of the present invention relates to the to the method as described herein, wherein the mean diameter of the cellulose nanofibers in said initial cellulose nanofiber material and / or processed cellulose nanofiber material is in the range of about 400 nm to about 600 nm, preferably about 500 nm.

[0254] Crosslinking of the individual cellulose nanofibers is preferably achieved by the addition of a crosslinking agent. The crosslinking agent is not limited to any specific type of crosslinking agent. The cellulose backbone comprises hydroxyl groups that may be used to bridge the nanofibers via the crosslinking agent. Thus, the crosslinking agent preferable has functional groups that may react with the hydroxyl groups to create covalent bonds.

[0255] Therefore, an embodiment of the present invention relates to the method as described herein, wherein the processed cellulose nanofiber material is crosslinked by addition of a crosslinking agent.

[0256] It is to be understood that crosslinking agents described elsewhere herein are suitable for use with the method.

[0257] The initial cellulose nanofiber material may be prepared from a cellulose acetate solution, e.g. by electrospinning of a cellulose acetate solution. The resulting cellulose acetate sheets are preferably regenerated to cellulose sheets in a sodium hydroxide bath before any further processing. This treatment opens up hydroxyl groups that may subsequently be used for binding of functional moieties.

[0258] Thus, an embodiment of the present invention relates to the method as described herein, wherein the initial cellulose nanofiber material is prepared by electrospinning of a cellulose acetate solution to cellulose acetate sheets.

[0259] Another embodiment of the present invention relates to the method as described herein, wherein said initial cellulose nanofiber material is regenerated in a regeneration solution comprising sodium hydroxide.

[0260] Regeneration of cellulose sheets is preferably performed in an ethanol solution comprising sodium hydroxide. It is possible to use varying amounts of ethanol, such as from 5% vol / vol to 99% vol / vol. The content of ethanol in the regeneration solution may influence the mechanical properties of the resulting nanofibrous cellulose scaffold. Mechanical properties that can be impacted by the content of ethanol includes elasticity and brittleness. Without being bound by theory, it is contemplated that a more elastic and less stiff material is beneficial for interaction with the cells and to promote proliferation.

[0261] Thus, an embodiment of the present invention relates to the method as described herein, wherein the regeneration solution is an ethanol solution comprising at least about 50% vol / vol ethanol, such as at least 60% vol / vol ethanol, such as at least 70% vol / vol ethanol, such as at least 80% vol / vol ethanol, such as at least 90% vol / vol ethanol, preferably about 90% vol / vol ethanol.

[0262] By regenerating the initial cellulose nanofiber material, e.g. by alkali treatment, the cellulose obtains a monoclinic crystal structure known as cellulose II. In contrast, native cellulose found in plants has the crystal structure cellulose I. Without being bound by theory, it is contemplated that the crystal structure cellulose II is advantageous for use as the initial cellulose nanofiber material, e.g. because it is more thermodynamically stable than other polymorphs such as cellulose I. This may be due to the antiparallel polymer chain orientation and different hydrogen bonding of cellulose II.

[0263] Accordingly, an embodiment of the present invention relates to the method of preparing a nanofibrous cellulose scaffold as described herein, wherein said initial cellulose nanofiber material comprises regenerated cellulose.

[0264] Another embodiment of the present invention relates to the method of preparing a nanofibrous cellulose scaffold as described herein, wherein the crystal structure of said initial cellulose nanofiber material is cellulose II.

[0265] The cellulose nanofibers may be functionalized with functional moieties to engraft the nanofibrous scaffold with new properties. The functional moieties substituted onto the nanofibrous cellulose scaffold may include ECM proteins, peptides, and / or charged groups to e.g. increase attachment levels of the cells to the scaffold, promote proliferation of the cells, or assist in the release and isolation of the cells from the scaffold. Functionalization of the cellulose nanofibers may be carried out before or after crosslinking of the cellulose nanofibers. Preferably, the functionalization step is performed before crosslinking. However, coating with ECM proteins, such as vitronectin, may be performed after crosslinking.

[0266] Thus, an embodiment of the present invention relates to the method as described herein further comprising a step of functionalizing the processed cellulose nanofiber strands by addition of a reagent comprising a functional moiety, said further step immediately preceding or immediately following the crosslinking step (iii).

[0267] It is to be understood that functional moieties may be any of those described elsewhere herein.

[0268] Another embodiment of the present invention relates to the method as described herein further comprising a step of coating the processed cellulose nanofiber strands with an ECM protein, such as vitronectin, said further step following the crosslinking step (iii).

[0269] Subsequent to dividing of the initial cellulose nanofiber material, the resulting processed material is preferably handled to make it ready for functionalization. Part of the treatment can include filtration, washing and re-suspension of the processed cellulose nanofiber material. Filtration ensures that excess water is removed from the processed cellulose nanofiber material. Washing assists in removal of any acetate ions still present after the previous treatment. Careful washing can assist with improving the texture of the cellulose. When re-suspending the processed cellulose nanofiber material, the concentration of cellulose nanofibers may be adjusted if desired.

[0270] Therefore, an embodiment of the present invention relates to the method as described herein, wherein the dividing step (ii) is followed by a step comprising filtration, washing and suspending the processed cellulose nanofiber material.

[0271] Prior to functionalization, the cellulose nanofibers are preferably mercerized to improve the substitution of functional moieties onto the cellulose nanofibers. Mercerization is a process in which the cellulose nanofibers are swollen by immersion in a concentrated aqueous NaOH solution, washed with water, and dried. During the mercerization process, the crystal structure of the cellulose nanofiber is transformed from cellulose I to cellulose II. Under the action of concentrated alkaline solutions chemical, physicochemical and structural modifications of cellulose occur. Upon washing and neutralisation cellulose II is formed. As a result of the penetration of the base into the lattice, internal hydrogen bonds are broken and the number of available hydroxyl groups (-OH) in the cellulose nanofiber is increased. It is therefore contemplated that mercerization improves the degree of substitution (DS). The mercerization step may be performed before or after the dividing step. Therefore, an embodiment of the present invention relates to the method as described herein, wherein said step of functionalization is preceded by a step of mercerization of said processed cellulose nanofiber material.

[0272] Another embodiment of the present invention relates to the method as described herein, wherein said mercerization step comprises addition of NaOH.

[0273] A further embodiment of the present invention relates to the method as described herein, wherein the degree of substitution of the functional moiety (DS) is in the range of about 0.01 to about 1.5, such as about 0.05 to about 1.2, such as about 0.1 to about 1, such as about 0.2 to about 0.8, such as about 0.4 to about 0.6.

[0274] Functionalization of the nanofibrous cellulose scaffold may also be quantified in terms of the ion exchange capacity of the microcarrier. The ion exchange capacity can be defined as the ability of the functional moiety coupled to the cellulose nanofibers to undergo displacement of ions which are attached to its structure by opposite charged ions available in the surrounding solution. The ion exchange capacity is given in units of mmol Cl / g and is determined via titration.

[0275] Therefore, an embodiment of the present invention relates to the method as described herein, wherein the ion exchange capacity of the nanofibrous cellulose scaffold is in the range of about 0.1 mmol Cl / g to about 1.5 mmol Cl / g, such as about 0.3 mmol Cl / g to about 1 mmol Cl / g.

[0276] Another embodiment of the present invention relates to the method as described herein, wherein the ion exchange capacity of the nanofibrous cellulose scaffold is at least about 0.1 mmol Cl / g, such as at least about 0.2 mmol Cl / g, preferably at least about 0.3 mmol Cl / g.

[0277] A further embodiment of the present invention relates to the method as described herein, wherein the functional moiety is QA and / or DEAE, and wherein the ion exchange capacity of the nanofibrous cellulose scaffold is at least about 0.3 mmol Cl / g.

[0278] After functionalization of the processed cellulose nanofiber scaffold, the material is dried to yield the nanofibrous cellulose scaffold in its final shape. Drying may be performed in two steps, such as freezing followed by lyophilization, or in a single step, such as by lyophilization. Lyophilizers work by freezing the material, then reducing the pressure and adding heat to allow the frozen water in the material to sublimate. If the water that keeps the processed cellulose nanofibers in solution would be air dried or heat dried, then the slow evaporation of water could cause the final nanofibrous cellulose scaffold collapse, thereby yielding a non-uniform and deformed material. Accordingly, lyophilization is preferred.

[0279] Thus, an embodiment of the present invention relates to the method as described herein, wherein the drying step (iv) comprises freezing and / or lyophilization of the processed cellulose nanofiber material.

[0280] Another embodiment of the present invention relates to the method as described herein, wherein the drying step (iv) comprises freezing of processed cellulose nanofiber material followed by lyophilization of the frozen processed cellulose nanofiber material.

[0281] Drying may be followed by a curing step in which the nanofibrous scaffold is cured to stabilise the crosslinking and improve the mechanical stability. Curing speeds up the process with which the crosslinking takes place.

[0282] Therefore, an embodiment of the present invention relates to the method as described herein, wherein the drying step (iv) is followed by a step comprising curing of the dried processed cellulose nanofiber material.

[0283] In one variant of the method, the drying step may be followed by a compression step to reduce the thickness of the material. After the compression step, the nanofibrous cellulose scaffold is cured as described above. By including a compression step, the production of thinner materials is facilitated as the challenge of overcoming surface tension to cover the surface of the mould with a very thin liquid layer is overcome, i.e. the nanofibrous cellulose scaffold may initially be moulded at a greater thickness and by compression be reduced in thickness. This variant of the method may be used to produce e.g. thin disks of the nanofibrous cellulose scaffold.

[0284] Thus, an embodiment of the present invention relates to the method as described herein, wherein the curing step is preceded by a compression step comprising compression of the nanofibrous cellulose scaffold to reduce the thickness of the nanofibrous cellulose scaffold.

[0285] When the nanofibrous cellulose scaffold is crosslinked and dried, the cellulose nanofibers are locked into a specific shape. The shape will depend on the container in which the solution of processed cellulose nanofiber material is held at the step of drying. In principle, a large batch of the nanofibrous cellulose scaffold can be produced by choosing a large container, and then the final dimensions / shape of the nanofibrous cellulose scaffold can be determined by cutting or punching out the desired shape.

[0286] Therefore, an embodiment of the present invention relates to the method as described herein, wherein the geometrical shape of the sample is modified after the drying step (iv).

[0287] However, it may be more convenient and efficient to directly dry the processed cellulose nanofiber material in a moulding container of the desired shape. The moulding container could be a cell culturing container as such, or a mould that produces a shape of nanofibrous cellulose scaffold that can be utilised in a cell culturing container, such as a bioreactor.

[0288] Thus, a preferred embodiment of the present invention relates to the method as described herein, wherein the processed cellulose nanofiber material is transferred to a moulding container prior to the drying step (iv).

[0289] Another embodiment of the present invention relates to the method as described herein, wherein the moulding container is selected from the group consisting of a mould, a cell culturing plate, a well plate, a bioreactor, a cell culture bottle, a spinner flask, a shaker flask, and a roller bottle, a petri dish, and a tube.

[0290] Yet another embodiment of the present invention relates to the method as described herein, wherein the mould is shaped to provide a casting of the processed cellulose nanofiber material in a geometry selected from the group consisting of a disk, cylinder, cube, sheet, and sphere.

[0291] A further embodiment of the present invention relates to the method as described herein, wherein the nanofibrous cellulose scaffold is a disk or cylinder with a thickness in the range of about 0.2 mm to about 20 mm, such as about 0.5 mm to about 15 mm, such as about 1 mm to about 10 mm, such as about 1.5 mm to about 8 mm, such as about 2 mm to about 5 mm.

[0292] Another aspect of the present invention relates to a nanofibrous cellulose scaffold obtainable from a method as described herein.

[0293] The listing or discussion of an apparently prior published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge. Preferences, options and embodiments for a given aspect, feature or parameter of the invention should, unless the context indicates otherwise, be regarded as having been disclosed in combination with any and all preferences, options and embodiments for all other aspects, features and parameters of the invention. This is especially true for the description of the method of preparing the nanofibrous cellulose scaffold and all its features, which may readily be part of the nanofibrous cellulose scaffold perse, its use for culturing stem cells, and the method for culturing stem cells, and vice versa. Embodiments and features of the present invention are also outlined in the following items.

[0294] Items

[0295] XI. A method of preparing a nanofibrous cellulose scaffold, said method comprising the steps of:

[0296] (i) providing an initial cellulose nanofiber material,

[0297] (ii) dividing said initial cellulose nanofiber material into a processed cellulose nanofiber material,

[0298] (iii) crosslinking the processed cellulose nanofiber material, and

[0299] (iv) drying the processed cellulose nanofiber material, thereby providing the nanofibrous cellulose scaffold.

[0300] X2. The method according to item XI, wherein dividing said initial cellulose nanofiber material is achieved by dispersing.

[0301] X3. The method according to any one of items XI or X2, wherein dividing said initial nanofiber material comprises a step of cutting the initial nanofiber material with a disperser.

[0302] X4. The method according to any one of items X2 or X3, wherein dispersing is performed with a high-speed disperser.

[0303] X5. The method according to any one of items X2-X4, wherein dispersing is performed for at least 2 min, such as at least 5 min, such as at least 10 min, such as at least 15 min, such as at least 20 min, such as at least 30 min, such as at least 40 min, such as at least 50 min, such as at least 60 min, such as at least 90 min, such as at least 120 min.

[0304] X6. The method according to any one of items X2-X5, wherein dispersing is performed for a period of time in the range of 2 min to 120 min, such as 2 min to 90 min, such as 5 min to 60 min, such as 10 min to 60 min, such as 15 min to 60 min. X7. The method according to any one of items X2-X6, wherein dispersing is performed for at least 5 min.

[0305] X8. The method according to any one of items X2-X7, wherein dispersing is performed for at least 15 min.

[0306] X9. The method according to any one of items X2-X8, wherein dispersing is performed for at least 60 min.

[0307] X10. The method according to any one of items X2-X9, wherein dispersing is performed at a speed in the range of about 10000 rpm to about 30000 rpm, such as at about 12000 rpm to about 25000 rpm, such as about 15000 rpm to about 20000 rpm.

[0308] XI 1. The method according to any one of the preceding items, wherein the mean length of the cellulose nanofibers in said processed cellulose nanofiber material is shorter than the mean length of the cellulose nanofibers in said initial cellulose nanofiber material.

[0309] X12. The method according to any one of the preceding items, wherein the mean length of the cellulose nanofibers in said processed cellulose nanofiber material is less than 250 pm, such as less than about 200 pm, such as less than about 150 pm, such as less than about 120 pm, such as less than about 100 pm.

[0310] X13. The method according to any one of the preceding items, wherein the mean length of the cellulose nanofibers in said processed cellulose nanofiber material is in the range of about 30 pm to about 250 pm, such as about 40 pm to about 200 pm, such as about 50 pm to about 150 pm, preferably about 60 pm to about 100 pm.

[0311] X14. The method according to any one of the preceding items, wherein the initial cellulose nanofiber material is provided as a liquid sample.

[0312] X15. The method according to item X14, wherein the solvent of the liquid sample comprises water and / or ethanol.

[0313] X16. The method according to any one of items X14 or X15, wherein the solvent of the liquid sample comprises ethanol.

[0314] X17. The method according to any one of the preceding items, wherein the concentration of cellulose nanofibers in the processed cellulose nanofiber material is about 0.02 wt% to about 10 wt%, such as about 0.05 wt% to about 8 wt%, such as about 0.1 wt% to about 5 wt%, such as about 0.2 wt% to about 3 wt%, such as about 0.25 wt% to about 2 wt%, preferably about 0.3 wt% to about 1 wt%, with respect to the total weight of the liquid sample.

[0315] X18. The method according to any one of the preceding items, wherein the concentration of cellulose nanofibers in the processed cellulose nanofiber material is about 0.1 wt% to about 5 wt%, such as about 0.15 wt% to about 2.5 wt%, such as about 0.2 wt% to about 2 wt%, with respect to the total weight of the liquid sample.

[0316] X19. The method according to any one of the preceding items, wherein the concentration of cellulose nanofibers in the processed cellulose nanofiber material is about 2 wt%.

[0317] X20. The method according to any one of the preceding items, wherein the concentration of cellulose nanofibers in the processed cellulose nanofiber material is about 0.2 wt%.

[0318] X21. The method according to any one of the preceding items, wherein the mean diameter of the cellulose nanofibers in said initial cellulose nanofiber material and / or processed cellulose nanofiber material is in the range of about 10 nm to about 2000 nm, such as about 50 nm to about 1500 nm, such as about 100 nm to about 1000 nm, preferably about 250 nm to about 750 nm.

[0319] X22. The method according to any one of the preceding items, wherein the processed cellulose nanofiber material is crosslinked by addition of a crosslinking agent.

[0320] X23. The method according to item X22, wherein crosslinking agent is selected from the group consisting of epichlorohydrin, formaldehyde dimethyl acetate (FDA), maleic anhydride (MAL), 3-glycidoxypropyltrimethoxysilane (GPS), citric acid, and 1, 2,3,4- butanetetracarboxylic acid.

[0321] X24. The method according to any one of items X22 or X23, wherein the crosslinking agent is a polyamide epichlorohydrin resin.

[0322] X25. The method according to any one of items X22-X24, wherein the crosslinking agent is a hexanedioic acid, polymer with Nl-(2-aminoethyl)-l,2-ethanediamine and 2- (chloromethyl)oxirane. X26. The method according to any one of items X22-X25, wherein the crosslinking agent is adipic acid diethylenetriamine epichlorohydrin copolymer.

[0323] X27. The method according to any one of the preceding items, wherein the processed cellulose nanofiber material is crosslinked with a crosslinking agent in a molar ratio of crosslinking agent to processed cellulose nanofiber material in the range of about 2*10’3mol / mol to about 175*10’3mol / mol, such as about 5*10’3mol / mol to about 100*10’3mol / mol, such as about 10*10’3mol / mol to about 80*10’3mol / mol, such as about 12*10’3mol / mol to about 50*10’3mol / mol, such as about 15*10’3mol / mol to about 25*10’3mol / mol.

[0324] X28. The method according to any one of the preceding items, wherein the processed cellulose nanofiber material is crosslinked with a crosslinking agent in a molar ratio of crosslinking agent to processed cellulose nanofiber material in the range of about 5*10’3mol / mol to about 50*10’3mol / mol, such as about 10*10’3mol / mol to about 40*10’3mol / mol, such as about 12*10’3mol / mol to about 30*10’3mol / mol, such as about 16*10’3mol / mol to about 24*10’3mol / mol.

[0325] X29. The method according to any one of the preceding items, wherein the processed cellulose nanofiber material is crosslinked with a crosslinking agent in a molar ratio of crosslinking agent to processed cellulose nanofiber material of about 19*10’3mol / mol.

[0326] X30. The method according to any one of the preceding items, wherein the processed cellulose nanofiber material is crosslinked with a crosslinking agent in a molar ratio of crosslinking agent to processed cellulose nanofiber material of less than about 90*10’3mol / mol, such as less than about 75*10’3mol / mol, such as less than about 50*10’3mol / mol, such as less than about 40*10’3mol / mol, such as less than about 30*10’3mol / mol, such as less than about 25*10’3mol / mol, such as less than about 20*10’3mol / mol.

[0327] X31. The method according to any one of items X22-X30, wherein the concentration of cellulose nanofibers in the processed cellulose nanofiber material is about 0.2 wt% to about 2 wt%, with respect to the total weight of the liquid sample, and the molar ratio of crosslinking agent to processed cellulose nanofiber material is in the range of about 15*10’3mol / mol to about 25*10’3mol / mol. X32. The method according to any one of the preceding items, wherein the crosslinking step is performed at a temperature in the range of about 15°C to about 25°C, preferably at about 20°C.

[0328] X33. The method according to any one of the preceding items, wherein the initial cellulose nanofiber material is prepared by a process selected from the group consisting of electrospinning, meltblowing, drawing, self-assembly, template synthesis, and thermal- induced phase separation, preferably electrospinning.

[0329] X34. The method according to any one of the preceding items further comprising a step of functionalizing the processed cellulose nanofiber strands by addition of a reagent comprising a functional moiety, said further step immediately preceding or immediately following the crosslinking step (iii).

[0330] X35. The method according to item X34, wherein the functional moiety is selected from one or more chemical moieties and / or biological molecules.

[0331] X36. The method according to item X35, wherein the chemical moieties are selected from the group consisting of negatively charged groups, positively charged groups, zwitterionic groups, hydrophobic groups, hydrophilic groups, amphiphilic groups, ligands, and combinations thereof.

[0332] X37. The method according to any one of items X35 or X36, wherein the chemical moieties are selected from the group consisting of quaternary ammonium (QA), carboxymethyl (CM), diethylaminoethyl (DEAE), 4-(l-bromoethyl)benzoic acid, 4- (dimethylamino)pyridine, epoxide, and combinations thereof.

[0333] X38. The method according to any one of items X35-X37, wherein the chemical moiety is quaternary ammonium (QA).

[0334] X39. The method according to any one of items X35-X38, wherein the biological molecules are selected from the group consisting of proteins, peptides, antibodies, amino acids, polypeptides, glycoproteins, lipoproteins, and antigens, and combinations thereof.

[0335] X40. The method according to any one of items X35-X39, wherein the biological molecule is a glycoprotein of the extracellular matrix selected from the group consisting of vitronectin, fibronectin, collagen, proteoglycans, laminin, tenascins, and integrins. X41. The method according to any one of items X35-X40, wherein the biological molecule is vitronectin.

[0336] X42. The method according to any one of items X34-X41, wherein the one or more functional moieties comprises quaternary ammonium (QA) and vitronectin.

[0337] X43. The method according to any one of items X34-X42, wherein the degree of substitution of the functional moiety (DS) is in the range of about 0.01 to about 2.

[0338] X44. The method according to any one of items X34-X43, wherein said step of functionalization is preceded by a step of mercerization of said processed cellulose nanofiber material.

[0339] X45. The method according to item X44, wherein the mercerization step is immediately before or after the dividing step (ii).

[0340] X46. The method according to any one of items X44 or X45, wherein said mercerization step comprises addition of NaOH.

[0341] X47. The method according to item X46, wherein concentration of NaOH is in the range of about 0.05 M to about 2 M, such as about 0.1 M to about 1.5 M, such as about 0.25 M to about 0.75 M, preferably about 0.4 M to about 0.6 M.

[0342] X48. The method according to any one of items X44-X47, wherein the mercerization step is performed for a period of about 1 hour to about 3 hours, preferably about 2 hours.

[0343] X49. The method according to any one of the preceding items, wherein the initial cellulose nanofiber material is prepared by electrospinning of a cellulose acetate solution to cellulose acetate sheets.

[0344] X50. The method according to any one of the preceding items, wherein said initial cellulose nanofiber material is regenerated in a regeneration solution comprising sodium hydroxide.

[0345] X51. The method according to item X50, wherein the regeneration solution comprises sodium hydroxide in the range of about 0.1M to about 1 M, such as about 0.2 M to about 0.8 M, such as about 0.3 M to about 0.7 M, such as about 0.4 M to about 0.6 M, preferably about 0.5 M. X52. The method according to any one of items X50 or X51, wherein the regeneration solution is an ethanol solution comprising about 5% vol / vol to about 99% vol / vol ethanol, such as about 10% vol / vol to about 95% vol / vol ethanol, such as about 20% vol / vol to about 90% vol / vol ethanol, such as about 30% vol / vol to about 80% vol / vol ethanol, such as about 40% vol / vol to about 70% vol / vol ethanol.

[0346] X53. The method according to any one of items X50-X52, wherein the regeneration solution is an ethanol solution comprising at least about 50% vol / vol ethanol, such as at least 60% vol / vol ethanol, such as at least 70% vol / vol ethanol, such as at least 80% vol / vol ethanol, such as at least 90% vol / vol ethanol, preferably about 90% vol / vol ethanol.

[0347] X54. The method according to any one of the preceding items, wherein the dividing step (ii) is followed by a step comprising filtration, washing and suspending the processed cellulose nanofiber material.

[0348] X55. The method according to item X54, wherein said filtration comprises sieving of the processed cellulose nanofiber material.

[0349] X56. The method according to any one of items X54 or X55, wherein the suspension comprises water and / or ethanol.

[0350] X57. The method according to any one of the preceding items, wherein the drying step (iv) comprises freezing and / or lyophilization of the processed cellulose nanofiber material.

[0351] X58. The method according to any one of the preceding items, wherein the drying step (iv) comprises freezing of processed cellulose nanofiber material followed by lyophilization of the frozen processed cellulose nanofiber material.

[0352] X59. The method according to any one of the preceding items, wherein the drying step (iv) is followed by a step comprising curing of the dried processed cellulose nanofiber material.

[0353] X60. The method according to item X59, wherein said curing is performed at a temperature in the range of about 20°C to about 200°C, preferably about 100°C to about 150°C.

[0354] X61. The method according to any one of items X59 or X60, wherein said curing is performed for a period of 2-4 hours. X62. The method according to any one of items X59-X61, wherein said curing is performed at 120°C for 3 hours.

[0355] X63. The method according to any one of the preceding items, wherein the processed cellulose nanofiber material is transferred to a moulding container prior to the drying step (iv).

[0356] X64. The method according to item X63, wherein the moulding container is selected from the group consisting of a mould, a cell culturing plate, a well plate, a bioreactor, a cell culture bottle, a spinner flask, a shaker flask, and a roller bottle, a petri dish, and a tube.

[0357] X65. The method according to item X64, wherein the mould is shaped to provide a casting of the processed cellulose nanofiber material in a geometry selected from the group consisting of a disk, cylinder, cube, sheet, and sphere.

[0358] X66. The method according to any one of items X64 or X65, wherein the mould is shaped to provide a casting of the processed cellulose nanofiber material in the form of a disk or cylinder, preferably a disk.

[0359] X67. The method according to any one of items X64-X66, wherein the nanofibrous cellulose scaffold is a disk or cylinder with a thickness in the range of about 0.2 mm to about 20 mm, such as about 0.5 mm to about 15 mm, such as about 1 mm to about 10 mm, such as about 1.5 mm to about 8 mm, such as about 2 mm to about 5 mm.

[0360] X68. The method according to any one of items X63-X67, wherein the moulding container is made from a material selected from the group consisting of metal, plastic, glass, ceramic, and composite, and combinations thereof.

[0361] X69. The method according to any one of the preceding items further comprising a step of coating the nanofibrous cellulose scaffold with a glycoprotein of the extracellular matrix selected from the group consisting of vitronectin, fibronectin, collagen, proteoglycans, laminin, tenascins, and integrins.

[0362] X70. The method according to any one of the preceding items further comprising a step of coating the nanofibrous cellulose scaffold with vitronectin. X71. The method according to any one of the preceding items, wherein the initial cellulose nanofiber material is not derived from a raw plant source.

[0363] X72. The method according to any one of the preceding items, wherein the initial cellulose nanofiber material is not prepared by mechanical treatment, such as mechanical defibrillation, such as mechanical disintegration.

[0364] X73. The method according to any one of the preceding items, wherein said initial cellulose nanofiber material comprises regenerated cellulose.

[0365] X74. The method according to any one of the preceding items, wherein the crystal structure of said initial cellulose nanofiber material is cellulose II.

[0366] Yl. A nanofibrous cellulose scaffold obtainable from a method according to items X1-X74.

[0367] Zl. A nanofibrous cellulose scaffold comprising cellulose nanofibers with a mean length of less than about 250 pm, and wherein the cellulose nanofibers are crosslinked with a crosslinking agent.

[0368] Z2. The nanofibrous cellulose scaffold according to item Zl, wherein the cellulose nanofibers are connected by the crosslinking agent in a three-dimensional network.

[0369] Z3. The nanofibrous cellulose scaffold according to any one of items Zl or Z2, wherein the crosslinking agent is selected from the group consisting of epichlorohydrin, formaldehyde dimethyl acetate (FDA), maleic anhydride (MAL), 3-glycidoxypropyltrimethoxysilane (GPS), citric acid, and 1,2,3,4-butanetetracarboxylic acid.

[0370] Z4. The nanofibrous cellulose scaffold according to any one of items Z1-Z3, wherein the crosslinking agent is a polyamide epichlorohydrin resin.

[0371] Z5. The nanofibrous cellulose scaffold according to any one of items Z1-Z4, wherein the molar ratio of crosslinking agent to cellulose nanofibers is in the range of about 2*10-3mol / mol to about 175*10-3mol / mol, such as about 5*10-3mol / mol to about 100*10-3mol / mol, such as about 10*10-3mol / mol to about 80*10-3mol / mol, such as about 12*10’3mol / mol to about 50*10’3mol / mol, such as about 15*10’3mol / mol to about 25*10’3mol / mol. Z6. The nanofibrous cellulose scaffold according to any one of items Z1-Z5, wherein the cellulose nanofibers have a mean length in the range of about 30 m to about 250 pm, such as about 40 pm to about 200 pirn, such as about 50 pirn to about 150 pirn, preferably about 60 pm to about 100 pm.

[0372] Z7. The nanofibrous cellulose scaffold according to any one of items Z1-Z6, wherein the cellulose nanofibers have a mean diameter in the range of about 10 nm to about 2000 nm, such as about 50 nm to about 1500 nm, such as about 100 nm to about 1000 nm, preferably about 250 nm to about 750 nm.

[0373] Z8. The nanofibrous cellulose scaffold according to any one of items Z1-Z7, wherein the cellulose nanofibers are functionalized with one or more functional moieties.

[0374] Z9. The nanofibrous cellulose scaffold according to item Z8, wherein the one or more functional moieties are selected from chemical moieties and / or biological molecules.

[0375] Z10. The nanofibrous cellulose scaffold according to item Z9, wherein the chemical moieties are selected from the group consisting of negatively charged groups, positively charged groups, zwitterionic groups, hydrophobic groups, hydrophilic groups, amphiphilic groups, ligands, and combinations thereof.

[0376] Zll. The nanofibrous cellulose scaffold according to any one of items Z9 or Z10, wherein the chemical moieties are positively charged groups.

[0377] Z12. The nanofibrous cellulose scaffold according to any one of items Z9-Z11, wherein the chemical moieties are selected from the group consisting of quaternary ammonium (QA), carboxymethyl (CM), diethylaminoethyl (DEAE), 4-(l-bromoethyl)benzoic acid, 4- (dimethylamino)pyridine, epoxide, and combinations thereof.

[0378] Z13. The nanofibrous cellulose scaffold according to any one of items Z9-Z12, wherein the chemical moiety is quaternary ammonium (QA).

[0379] Z14. The nanofibrous cellulose scaffold according to any one of items Z9-Z13, wherein the biological molecules are selected from the group consisting of proteins, peptides, antibodies, amino acids, polypeptides, glycoproteins, lipoproteins, and antigens, and combinations thereof. Z15. The nanofibrous cellulose scaffold according to any one of items Z9-Z14, wherein the biological molecule is a glycoprotein of the extracellular matrix selected from the group consisting of vitronectin, fibronectin, collagen, proteoglycans, laminin, tenascins, and integrins, preferably vitronectin.

[0380] Z16. The nanofibrous cellulose scaffold according to any one of items Z9-Z15, wherein the one or more functional moieties comprises quaternary ammonium (QA) and vitronectin.

[0381] Z17. The nanofibrous cellulose scaffold according to any one of items Z1-Z16, wherein the cellulose nanofibers are electrospun cellulose nanofibers.

[0382] Z18. The nanofibrous cellulose scaffold according to any one of items Z1-Z17, wherein the cellulose nanofibers comprise monofibers.

[0383] Z19. The nanofibrous cellulose scaffold according to any one of items Z1-Z18, wherein the cellulose nanofibers are derived from regenerated cellulose.

[0384] Z20. The nanofibrous cellulose scaffold according to any one of items Z1-Z19, wherein the cellulose nanofibers have the cellulose II crystal structure.

[0385] Z21. The nanofibrous cellulose scaffold according to any one of items Z1-Z20, wherein the cellulose nanofibers are not derived from a raw plant source.

[0386] Z22. The nanofibrous cellulose scaffold according to any one of items Z1-Z21, wherein the cellulose nanofibers are not obtained by mechanical treatment, such as mechanical defibrillation, such as mechanical disintegration.

[0387] Z23. The nanofibrous cellulose scaffold according to any one of items Z1-Z22, wherein said nanofibrous cellulose scaffold is not a hydrogel.

[0388] Z24. The nanofibrous cellulose scaffold according to any one of items Z1-Z23, wherein the nanofibrous cellulose scaffold is provided as a dry continuous material.

[0389] Z25. The nanofibrous cellulose scaffold according to any one of items Z1-Z24, wherein the physical form of the nanofibrous cellulose scaffold is a geometry selected from the group consisting of a disk, cylinder, cube, sheet, and sphere. Z26. The nanofibrous cellulose scaffold according to any one of items Z1-Z25, wherein the nanofibrous cellulose scaffold has a thickness in all three dimensions of at least about 0.1 mm, such as at least about 0.2 mm, such as at least about 0.3 mm, such as at least about 0.4 mm, such as at least about 0.5 mm.

[0390] Z27. The nanofibrous cellulose scaffold according to any one of items Z1-Z26, wherein the nanofibrous cellulose scaffold has a thickness in all three dimensions of at least about 0.5 mm, such as at least about 1 mm, such as at least about 1.5 mm, such as at least about

[0391] 2 mm, such as at least about 3 mm, such as at least about 4 mm, such as at least about

[0392] 5 mm, such as at least about 6 mm, such as at least about 7 mm, such as at least about

[0393] 8 mm, such as at least about 9 mm, such as at least about 10 mm.

[0394] Z28. The nanofibrous cellulose scaffold according to any one of items Z1-Z27, wherein the nanofibrous cellulose scaffold is a disk or cylinder with a thickness in the range of about 0.2 mm to about 20 mm, such as about 0.5 mm to about 15 mm, such as about 1 mm to about 10 mm, such as about 1.5 mm to about 8 mm, such as about 2 mm to about 5 mm.

[0395] Z29. The nanofibrous cellulose scaffold according to any one of items Z1-Z28, wherein nanofibrous cellulose scaffold is a disk with a diameter in the range of about 8 mm to about 70 mm, such as about 10 mm to about 60 mm, such as about 10 mm to about 40 mm, such as about 10 mm to about 30 mm, such as about 10 mm to about 20 mm.

[0396] Z30. The nanofibrous cellulose scaffold according to any one of items Z1-Z29, wherein nanofibrous cellulose scaffold is a disk with a diameter in the range of about 8 mm to about 16 mm, such as about 10 mm to about 14 mm, preferably about 12 mm.

[0397] Z31. The nanofibrous cellulose scaffold according to any one of items Z1-Z30, wherein nanofibrous cellulose scaffold is a disk with a diameter in the range of about 40 mm to about 70 mm, such as about 50 mm to about 60 mm, preferably about 57 mm.

[0398] Z32. The nanofibrous cellulose scaffold according to any one of items Z1-Z31, wherein the nanofibrous scaffold is provided as a lyophilized material.

[0399] Z33. The nanofibrous cellulose scaffold according to any one of items Z1-Z32, wherein the nanofibrous scaffold is resilient.

[0400] Z34. The nanofibrous cellulose scaffold according to any one of items Z1-Z33, wherein the nanofibrous cellulose scaffold has a BET surface area of at least about 40000 cm2 / g, such as at least about 50000 cm2 / g, such as at least about 55000 cm2 / g, such as at least about 60000 cm2 / g, such as at least about 70000 cm2 / g, such as at least about 80000 cm2 / g.

[0401] Z35. The nanofibrous cellulose scaffold according to any one of items Z1-Z34, wherein the nanofibrous cellulose scaffold is microporous.

[0402] Z36. The nanofibrous cellulose scaffold according to any one of items Z1-Z35, wherein the nanofibrous cellulose scaffold has a density in the range of about 0.0005 g / cm3to about 0.5 g / cm3, such as about 0.001 g / cm3to about 0.1 g / cm3, such as about 0.001 g / cm3to about 0.040 g / cm3.

[0403] Z37. The nanofibrous cellulose scaffold according to any one of items Z1-Z36, wherein the nanofibrous scaffold has a compressive strength in the range of about 0.1 mm / N to about 30 mm / N.

[0404] Z38. The nanofibrous cellulose scaffold according to any one of items Z1-Z37, wherein the nanofibrous scaffold further comprises one or more nanofibers selected from natural polymers or synthetic polymers.

[0405] Z39. The nanofibrous cellulose scaffold according to item Z38, wherein the natural polymers are selected from the group consisting of collagen, silk fibroin, keratin, gelatin and polysaccharides, and combinations thereof.

[0406] Z40. The nanofibrous cellulose scaffold according to any one of items Z38 or Z39, wherein the synthetic polymers are selected from the group consisting of polycaprolactone (PCL), poly(lactic acid) (PLA), polystyrene, polyurethane (PU), poly(lactic-co-glycolic acid) (PLGA), poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), and poly(ethylene-co- vinylacetate) (PEVA).

[0407] Z41. The nanofibrous cellulose scaffold according to any one of items Z1-Z40, wherein the nanofibrous cellulose scaffold is coated with a glycoprotein of the extracellular matrix selected from the group consisting of vitronectin, fibronectin, collagen, proteoglycans, laminin, tenascins, and integrins.

[0408] Z42. The nanofibrous cellulose scaffold according to any one of items Z1-Z41, wherein the nanofibrous cellulose scaffold is coated with vitronectin. Z43. The nanofibrous cellulose scaffold according to any one of items Z14-Z42, wherein the molar ratio of vitronectin to cellulose nanofibers is in the range of about 5*10-7mol / mol to about 75*10-6mol / mol, such as about IO-6mol / mol to about 50*10-6mol / mol, such as about 2*10-6mol / mol to about 25*10-6mol / mol, such as about 4*10-6mol / mol to about 12*io-6mol / mol.

[0409] Wl. A support matrix comprising a nanofibrous cellulose scaffold according to items Y1 or Z1-Z43.

[0410] QI. A method for culturing of stem cells, said method comprising the steps of: providing a container comprising a support matrix;

[0411] - seeding stem cells on said support matrix; and incubating said stem cells; wherein said support matrix comprises cellulose nanofibers.

[0412] Q2. The method according to item QI, wherein the cellulose nanofibers have a mean length of less than about 250 pm, and wherein the cellulose nanofibers are crosslinked with a crosslinking agent.

[0413] Q3. The method according to any one of items QI or Q2, wherein the cellulose nanofibers have a mean length in the range of about 30 pm to about 250 pm, such as about 40 pm to about 200 pm, such as about 50 pm to about 175 pm, such as about 60 pm to about 150 pm, preferably about 70 pm to about 120 pm.

[0414] Q4. The method according to any one of items Q1-Q3, wherein the cellulose nanofibers are electrospun cellulose nanofibers.

[0415] Q5. The method according to any one of items Q1-Q4, wherein the cellulose nanofibers have a mean diameter in the range of about 10 nm to about 2000 nm, such as about 50 nm to about 1500 nm, such as about 100 nm to about 1000 nm, preferably about 250 nm to about 750 nm.

[0416] Q6. The method according to any one of items Q2-Q5, wherein the crosslinking agent is selected from the group consisting of epichlorohydrin, formaldehyde dimethyl acetate (FDA), maleic anhydride (MAL), 3-glycidoxypropyltrimethoxysilane (GPS), citric acid, and 1,2,3,4-butanetetracarboxylic acid. Q7. The method according to any one of items Q2-Q6, wherein the molar ratio of crosslinking agent to cellulose nanofibers is in the range of about 2*10-3mol / mol to about 175*10-3mol / mol, such as about 5*10-3mol / mol to about 100*10-3mol / mol, such as about 1O*1O-3mol / mol to about 80*10-3mol / mol, such as about 12*10-3mol / mol to about 5O*io-3mol / mol, such as about 15*10-3mol / mol to about 25*10-3mol / mol.

[0417] Q8. The method according to any one of items Q1-Q7, wherein the cellulose nanofibers comprise one or more functional moieties selected from chemical moieties and / or biological molecules.

[0418] Q9. The method according to item Q8, wherein the chemical moieties are selected from the group consisting of quaternary ammonium (QA), carboxymethyl (CM), diethylaminoethyl (DEAE), 4-(l-bromoethyl)benzoic acid, 4-(dimethylamino)pyridine, epoxide, and combinations thereof, preferably quaternary ammonium (QA).

[0419] Q10. The method according to any one of items Q8 or Q9, wherein the biological molecules are selected from the group consisting of proteins, peptides, antibodies, amino acids, polypeptides, glycoproteins, lipoproteins, and antigens, and combinations thereof.

[0420] Qll. The method according to any one of items Q8-Q10, wherein the biological molecule is a glycoprotein of the extracellular matrix selected from the group consisting of vitronectin, fibronectin, collagen, proteoglycans, laminin, tenascins, and integrins, preferably vitronectin.

[0421] Q12. The method according to any one of items Q8-Q11, wherein the one or more functional moieties comprises quaternary ammonium (QA) and vitronectin.

[0422] Q13. The method according to any one of items Q1-Q12, wherein the stem cells are selected from the group consisting of mesenchymal stem cells (MSCs), induced pluripotent stem cells (iPSCs), embryonic stem cells (ESCs), hematopoietic stem cells (HSCs), neural stem cells (NSCs), cardiac stem cells, amniotic fluid stem cells, epidermal stem cells, adipose derived stem cells (ASC) and endothelial progenitor cells (EPCs).

[0423] Q14. The method according to any one of items Q1-Q13, wherein the stem cells are mesenchymal stem cells (MSCs).

[0424] Q15. The method according to any one of items Q1-Q14, wherein the stem cells are of human origin. Q16. The method according to any one of items Q1-Q15, wherein the container comprises a solvent.

[0425] Q17. The method according to item Q16, wherein the solvent comprises a cell culturing medium.

[0426] Q18. The method according to any one of items Q1-Q17, wherein an expansion medium is added to the container at a time between about 24 hours and about 72 hours after seeding of said stem cells, such as between about 36 hours and 60 hours after seeding of said stem cells, preferably about 48 hours after seeding of said stem cells.

[0427] Q19. The method according to any one of items Q1-Q18, wherein the step of incubating the stem cells is followed by extracting the proliferated stem cell population from the container.

[0428] Q20. The method according to any one of items Q1-Q19, wherein said container is a bioreactor.

[0429] Q21. The method according to item Q20, wherein said bioreactor comprises a compartment containing the support matrix.

[0430] Q22. The method according to any one of items Q1-Q21, wherein said container is a packed bed bioreactor, such as a fixed bed bioreactor or a dynamic bed bioreactor.

[0431] Q23. The method according to any one of items Q1-Q22, wherein incubating said stem cells is performed under agitation.

[0432] Q24. The method according to item Q23, wherein agitation is effected by mechanical agitation, circulation pump, and / or pneumatic means.

[0433] Q25. The method according to any one of items Q23-Q24, wherein said agitation comprises perfusion of said solvent through the container.

[0434] Q26. The method according to item Q25, wherein said perfusion of solvent is longitudinal and / or radial with respect to the container. Q27. The method according to any one of items Q1-Q26, wherein the cellulose nanofibers are coated with a glycoprotein of the extracellular matrix selected from the group consisting of vitronectin, fibronectin, collagen, proteoglycans, laminin, tenascins, and integrins.

[0435] Q28. The method according to any one of items Q1-Q27, wherein the cellulose nanofibers are coated with vitronectin.

[0436] Q29. The method according to any one of items Q11-Q28, wherein the molar ratio of vitronectin to cellulose nanofibers is in the range of about 5*10-7mol / mol to about 75*10"6mol / mol, such as about 10"6mol / mol to about 50*10"6mol / mol, such as about 2*10"6mol / mol to about 25*10"6mol / mol, such as about 4*10"6mol / mol to about 12*10"6mol / mol.

[0437] Q30. The method according to any one of items Q1-Q29, wherein said container comprises a support matrix according to item Wl.

[0438] Q31. The method according to any one of items Q1-Q30, wherein the doubling time of said stem cells is less than about 60 hours, such as less than about 55 hours, preferably less than 50 hours.

[0439] Ul. Use of a nanofibrous cellulose scaffold according to items Y1 or Z1-Z43 as a support matrix for culturing of stem cells.

[0440] The invention will now be described in further details in the following non-limiting examples.

[0441] Examples

[0442] Example 1: Preparation of the nanofibrous cellulose scaffold

[0443] In this example is given a non-limiting demonstration of how the nanofibrous cellulose scaffold can be prepared. The core properties of the material, such as surface area, degree of substitution, and compression profile, were characterized.

[0444] Method

[0445] Preparation of initial cellulose nanofiber material

[0446] A solution of 19% cellulose acetate was prepared by addition of cellulose acetate (6.38 g) to 12 ml of acetone in an Erlenmeyer flask, followed by addition of 12 ml DMF and 6 ml 96% ethanol. The solution was stirred using a magnetic stirrer overnight at room temperature.

[0447] The 19% cellulose acetate solution was electrospun to prepare cellulose acetate nanofiber sheets. The cellulose nanofibers were electrospun (Fluidnatek LE50) using a drum speed of 200 rpm and a flow of 10 ml / hour at 18 kV+ (emitter) and 10 kV- (collector). The cellulose acetate nanofibers were collected on an aluminium substrate, with temperature being 23°C and at a relative humidity of 63%. After the electrospinning process completed, the cellulose acetate sheet comprising cellulose nanofibers was removed from the drum.

[0448] The cellulose acetate sheets were regenerated to cellulose by submergence in a 0.5 M NaOH solution in ethanol. The cellulose acetate sheets were left in the solution for 6 hours followed by transfer to a sieve and washing with copious volumes of distilled water. The washed sheets were put in the oven at 80°C for 12 hours to provide dry cellulose sheets. The cellulose sheets were weighed.

[0449] Processing of initial cellulose nanofiber material

[0450] The dry cellulose sheets were cut into rough pieces of approximately 2x2 cm squares using scissors. The size of the pieces does not have to be exact, but larger pieces should be avoided as they may hamper the dividing step. The rough pieces of cellulose were fully submerged in water and dispersed using a high-speed disperser (IKA T25 digital Ultra Turrax) at 18,000 rpm for 60 min (with intermittent stops to cool the disperser). The processed cellulose nanofiber material was transferred to a sieve, washed with water to remove any remaining acetate ions, and drained to remove excess water. Cellulose nanofiber material was transferred to a flask and fresh water was added to provide a cellulose concentration of 2 wt%.

[0451] Samples with a cellulose concentration of 0.33 wt%, 0.5 wt%, 0.66 wt%, and 1.5 wt% were also prepared. The following steps are described for 2 wt% cellulose, but equally applies to other cellulose concentrations.

[0452] Functionalization of processed cellulose nanofiber material

[0453] The processed cellulose nanofiber material was functionalized with different chemical moieties according to the processes below.

[0454] Quaternary ammonium (QA):

[0455] 0.4 g NaOH was dissolved in 20 ml water. 0.4 g regenerated cellulose nanofiber material was then added to the solution. Mercerization continued for 2 hours at room temperature. The temperature of the cellulose suspension was increased to 80°C and 3.4 ml of 60% 3- chloro-2-hydroxypropyltrimethyl ammonium chloride (CHPTAC) was added dropwise. The reaction was continued at 80°C for 4 hours. Then the reaction mixture was filtered after cooling down to room temperature and washed repeatedly with water to remove any unreacted CHPTAC or NaOH. Finally, the filtered processed and functionalized cellulose nanofiber material was mixed with water to make a 2% suspension (or 0.2%, 0.25%, 0.5%, 1.0% or 1.5%) again for further experimentation.

[0456] Crosslinking of processed cellulose nanofiber material

[0457] The processed cellulose nanofiber material was subjected to crosslinking using polyamide epichlorohydrin (Kymene GHP20 (Solenis), molecular weight of 341.83 g / mol, hereafter "Kymene") as a crosslinking agent. The processed cellulose nanofiber material was poured into distilled water and a calculated amount of Kymene was added dropwise and mixed mechanically for 45 s at 18000 rpm to make suspensions of varying molar ratios of Kymene to cellulose (2.8*10-3, 5.6*103, 9.5*103, 19.0*103, 47.4*103mol / mol

[0458] Kymene:cellulose). The resultant suspensions were poured into moulds of desired dimensions. Examples of dimensions of the moulds include a height of 60 mm, 30 mm, 10 mm or 4 mm and inner diameter of 12, mm, 19 mm and 57 mm.

[0459] Drying

[0460] Moulds containing the cellulose nanofiber material suspension (crosslinked material) was transferred to a freezer and left for 24 hours at -84°C. The moulds containing the frozen cellulose nanofiber material was then transferred to a lyophilizer and processed for 24 hours at a pressure of <0.001 bar and a temperature of -84°C to yield a dry continuous material.

[0461] Curing

[0462] The dry continuous material was extracted from the mould and cured in the oven for 3 hours at 120°C.

[0463] The dry continuous and cured material is also referred to as "cured and dry" herein.

[0464] Scanning electron microscopy

[0465] Morphological studies of the nanofibrous cellulose scaffolds were carried out using scanning electron microscopy (SEM). The samples were dried and sputter coated with gold before carrying out the analysis. The micrographs were obtained in secondary electron (SE) imaging mode on a Hitachi SU3500 at an accelerating voltage of 5 kV and a working distance of 7 mm at varying magnifications.

[0466] FTIR spectroscopy

[0467] FTIR analysis was performed on solid dried samples at room temperature using a Perkin- Elmer Spectrum Two instrument provided with Universal ATR in the range of 450-3600 cm . The samples were dried at 80°C for 2 hours before carrying out the analysis.

[0468] Surface area measurements

[0469] The Brunauer-Emmett-Teller (BET) model was employed to measure the specific surface areas using a molecule of nitrogen bearing molecular cross-sectional area of 0.162 nm2. Dried samples were degassed under vacuum for 6 hours before the BET surface area was measured. Nitrogen adsorption and desorption isotherms were acquired on an ASAP 2020 M analyzer (Micromeritics) at 77.3 K.

[0470] Elemental analysis and Degree of substitution

[0471] Elemental analysis of the nanofibrous cellulose scaffold was performed on an elemental analyzer FlashEA 1112 (Thermo Fischer Scientific) using 5 mg of sample. The samples were well dried before the analysis to remove any adsorbed moisture.

[0472] Degree of substitution (DS) of QA functionalized cellulose ( / .e. cured and dry QA 2%) was calculated using the following formula:

[0473] DS = (162N / (1400-CAxN)) where 162 is the molecular weight of the anhydrous glucose unit (AGU); N is the percentage of nitrogen; CA is the molecular weight of the cationic reagent. For the present nanofibrous cellulose scaffold the cationic reagent is CHPTAC with a molecular weight of 188.1.

[0474] Compression analysis

[0475] Compression analysis was performed on a Zwick test machine (inv. No. BX32358) using a 50 N load cell (inv. No. BX32803), a preload of 0,002 N was set and the test speed was 1 mm / min. The nanofibrous cellulose scaffolds were cut to a height of 15 mm before carrying out the analysis.

[0476] The compression modulus measures the stiffness of the material or the ability of the material to withstand changes in length when subjected to compressive loads. The higher the compression modulus, the stiffer the material. Results

[0477] Nanofibrous cellulose scaffolds with a crosslinker and different types of functional moieties were prepared and the dry continuous material was imaged by SEM (Figure 1A). The resulting nanofibrous cellulose scaffold is highly homogenous in the sense that no large degree of entanglement or clusters of nanofibers are present. The homogenous distribution of nanofibers in the material ensures optimal exposure of the surface area for cell attachment and interaction. The crosslinking agent Kymene could be seen trapped between the cellulose nanofibers (Figure 1A) adding mechanical strength to the nanofibrous cellulose scaffold.

[0478] FTIR analysis was carried out to confirm the formation of linkages in the crosslinked cellulose. The band centered around 1633 cm1was attributed to stretching vibrations of carbonyl (C=O) groups incorporated into the cellulosic framework after crosslinking of Kymene (Figure IB). A comparison of FTIR spectra of cellulose acetate, regenerated cellulose, QA functionalized cellulose and crosslinked cellulose (Figure 1C) shows the successful transformation of cellulose acetate to crosslinked cellulose through QA functionalized cellulose. Disappearance of the absorbance band at 1743 cm1belonging to the carbonyl group (C=O) of cellulose acetate and appearance of a band at 3345 cm1belonging to hydroxyl groups (OH) of cellulose indicated success of regeneration of cellulose acetate to form cellulose nanofibers. Furthermore, the absorbance band at 865 cm1associated with quaternary ammonium functionality corroborated the successful formation of QA functionalized cellulose. Appearance of an additional absorbance band at 1633 cm1indicated that crosslinking has occurred in the cellulose architecture.

[0479] Elemental composition and degree of substitution (DS) was assessed for the nanofibrous cellulose scaffold functionalized with QA (cured and dry QA 2%). The content of carbon (C) was 37.71%, the content of hydrogen (H) was 6.59%, and the content of nitrogen (N) was 1.28%. Based on the content of N, the DS was calculated to 0.18. The amount of substituted QA may influence the cell growth as the positive charge facilitates electrostatic interactions with negatively charged cell membranes.

[0480] Surface area measurements demonstrated that a surprisingly large BET surface area of 60000 cm2 / g was obtained for the nanofibrous cellulose scaffold (cured and dry QA 2%). Without being bound by theory, it is contemplated that the short cellulose strands yield a material with increased degree of exposed surface. The large surface area is significantly higher than commercially available support matrix (microcarriers) currently on the market The successful crosslinking of the cellulose nanofibers provided the material with the mechanical strength necessary to yield a dry continuous material that can be moulded and formed to fit the desired application (Figure 2A-B). This was true for all the nanofibrous cellulose scaffold produced with varying amounts of cellulose and crosslinker.

[0481] Compression testing was used to evaluate the behaviour of the nanofibrous cellulose scaffold under a uniaxial compressive load. Change of length (or height), dL, was measured by subjecting the nanofibrous cellulose scaffold to compressive loads and the behaviour of the material was recorded (Figure 2C-D). The force required to bring in a specific deformation (dL / Fmax) was 0.2 mm / N for the nanofibrous cellulose scaffold (cured and dry QA 2%, 9.5*10-3mol / mol Kymene:cellulose). In particular, the force to obtain 60% deformation for the material was measured to be 9.8 N.

[0482] Similar measurements was performed for a with a cellulose concentration of 0.5 wt% (cured and dry QA 0.5%, 9.5*10-3mol / mol Kymene:cellulose), yielding a dL / Fmax of 17 mm / N and a force to obtain 60% deformation for the material of 0.4 N.

[0483] Compression profiles were obtained with nanofibrous cellulose scaffolds with molar ratios of Kymene:cellulose varying from 2.4*10-3mol / mol to 47.4*10-3mol / mol and resulted in the same shape.

[0484] The physical properties of the nanofibrous cellulose scaffold surprisingly yielded a versatile material that revert to its original form after being subjected to deformation. This resilient behaviour is advantageous for moulding and adapting the nanofibrous cellulose scaffold to existing and future cell culture platforms.

[0485] Conclusion

[0486] This example demonstrates that it is possible to produce different variants of the nanofibrous cellulose scaffold in a simple manner which is readily scalable for industrial usage. The nanofibrous cellulose scaffold has a large surface area which is available to the cells and does not comprise nanofibers collapsed in clusters or entangled in a fashion that exclude cells from gaining access to the surface.

[0487] The resilient material can easily be integrated into all current cell culture solutions from R&D to clinical manufacturing scale, such as packed bed bioreactors allowing customers to stay with the same cell culturing solution throughout their process development (thus reducing risks and costs). Example 2: Evaluation of functionalization of nanofibrous cellulose scaffold for culturing of stem cells

[0488] In this example different types of functionalization of the nanofibrous cellulose scaffold were tested to improve proliferation of stem cells. The stability of the internal structure of the scaffold was also probed.

[0489] Method:

[0490] Investigating coating of the nanofibrous cellulose scaffold

[0491] Nanofibrous cellulose scaffolds with a cellulose concentration of 0.2% were prepared following the protocol of Example 1 but adjusting the cellulose concentration to 0.2%.

[0492] Moreover, the following adjustments to the protocol (bold) of Example 1 was used;

[0493] - 3 g NaOH was dissolved in 50 ml water. 1 g regenerated cellulose nanofiber material was then added to the solution.

[0494] - The temperature of the cellulose suspension was increased to 80°C and 2.5 ml of 60% 3-chloro-2-hydroxypropyltrimethyl ammonium chloride (CHPTAC) was added dropwise.

[0495] - The molar ratio of Kymene to cellulose was 19.0*10'3mol / mol.

[0496] Scaffolds with and without QA functionalization were crosslinked with 0.2 pl crosslinker / mg of cellulose. 500 pl of each scaffold mixture was subsequently added to 48-well molds (maximum fill volume 0.5 ml, diameter 12 mm, height 4 mm). The molds were frozen for 2 hours at -80°C and thereafter freeze-dried for 16 hours. Finally, the scaffolds were cured on an aluminium tray placed in the oven at 120°C for 3 hours to generate scaffolds in the form of disks. The cured disks had a thickness of approx. 2 mm.

[0497] Prior to cell culture experiments, all the scaffolds (in form of disks) were washed twice in Dulbecco's Phosphate-Buffered Saline (DPBS), sterilized for 2 hours in ethanol 70% and washed in DPBS three more times. A selection of the scaffolds was coated with vitronectin diluted in DPBS (2.5 pg / ml from 500 pg / ml stock solution) for 2 hours corresponding to a molar ratio of vitronectin to cellulose of 8*10-6mol / mol.

[0498] Following overnight incubation, the scaffolds were transferred to ultra-low attachment 6- well plates at a density of 2 scaffolds per well. Cryopreserved human mesenchymal stem cells (hMSCs) were thawed in T-flasks (T175) and seeded at a density of 2,200 cells / cm2, expanded for 4 days and then harvested using TrypLE enzymatic dissociation. Cell seeding was performed by seeding hMSCs at a density of 10,000 cells per scaffold (for a total of 20,000 cells per well) in an inoculum volume of 2 ml and the culture plates were placed on an orbital shaker (19 mm) at 50 rpm. 5 hours post-inoculum, additional 2 ml of culture medium was added and the final culture volume of 4 ml was reached. On day 4 postinoculum, a half-medium change supplemented with 2% feedstock was performed. Live cell count and viability were assessed every 2 days from day 4 post-inoculum using NucleoCounter NC-202.

[0499] Investigating molar ratios of crosslinking agent to cellulose

[0500] For evaluating the effect of varying crosslinking concentrations, the protocol was further adjusted to prepare scaffolds with a cellulose concentration of 0.25% (concentration of cellulose suspension), and molar ratio of crosslinking agent to cellulose of either low (3.8*10-3mol / mol), medium (19.0*10-3mol / mol), or high (94.9*10-3mol / mol). The low, medium and high samples corresponded to addition of 0.04, 0.2, and 1 pl crosslinking agent per mg cellulose material, respectively. The crosslinking agent to cellulose ratio is given as mol / mol. The moles of the cellulose material corresponds to per glucose unit. Thus, the *10-3mol / mol is a measure of the number of crosslinking molecules per 1000 glucose units. The scaffolds were transferred to ultra-low attachment 24-well plates (1 scaffold per well), in a culture volume of 1 ml per well. Cell seeding was performed using 10,000 cells per scaffold in an inoculum volume of 500 pl and the culture plate was placed on an orbital shaker (19 mm) at 50 rpm. 5 hours post-inoculum, additional 500 pl of culture medium was added and the final culture volume of 1 ml was reached. On day 2 post-inoculum, half-medium change was performed. Live cell count and viability were assessed at day 6 post-inoculum using NucleoCounter NC-202.

[0501] Results:

[0502] Scaffold conditions were assessed for their ability to support and enhance cell proliferation. hMSCs adhered to all the scaffolds within 5 hours post-inoculum (Figure 3A, left) and subsequently proliferated (Figure 3B, right). Vitronectin coating improved cell proliferation on scaffold with and without QA-functionalization (Figure 3B). Low concentration of crosslinking agent increased material porosity and cell accessibility (Figure 3C). However, maintaining medium to high crosslinker concentrations may be preferred to secure mechanical stability of the scaffolds with low cellulose concentration, e.g. in larger bioreactor setups, cf. mechanical testing of discs with fixed amount of crosslinking agent (19.0*10-3mol / mol) presented in Example 8 (Figure 16B).

[0503] Conclusion:

[0504] The nanofibrous cellulose scaffolds create a supportive three-dimensional environment for hMSCs growing under shear forces on an orbital shaker. Vitronectin coating of the scaffold offers a superior solution for promoting hMSC proliferation. Low amount of crosslinker improves cell proliferation, but higher amounts of crosslinking agent may be preferred for some bioreactor applications to ensure sufficient mechanical stability of the scaffolds.

[0505] Example 3: Growth of mesenchymal stem cells (MSCs) on nanofibrous cellulose scaffold

[0506] In this example the feasibility of the nanofibrous cellulose scaffold for large-scale production of stem cells was tested and key performance indicators were determined.

[0507] Method:

[0508] Nanofibrous cellulose scaffolds with QA-functionalization and a cellulose concentration of 0.2% were prepared as described in Example 2. Varying molar ratios of vitronectin to cellulose of 8*10-6mol / mol, 16*10-6mol / mol and 31*10-6mol / mol (corresponding to 2.5 pg / ml, 5 pg / ml and 10 pg / ml concentrations of vitronectin coating were tested to assess their influence on cell growth. The molar ratio of crosslinking agent to cellulose was kept constant at 19.0*10-3mol / mol. Experiments were performed using hMSCs.

[0509] Giemsa staining was conducted on scaffolds fixed with 4% paraformaldehyde to visualize the adhesion and distribution of the hMSCs within the scaffolds, as well as for morphological assessment using an inverted microscope.

[0510] Results:

[0511] By day 8 post-inoculum, a fold change of 18.5 ± 0.7 (mean ± SEM) in live cell yield over inoculum density was reached in 6-well plate formats (Figure 4A), with sustained high cell viability throughout the culture period (Figure 4B). The population doubling time (DT) can be calculated by the following equation:

[0512] DT = ln(2) / r, wherein r is the growth rate given as: r = ln(Nt / No) / t, wherein Nt is the number of cells at time t (in hours) and No is the numbers of cells at inoculum.

[0513] The doubling time was calculated to 45.6 hours, which is a significant reduction compared to benchmark data on commercially available products, such as Fibra-Cel. Importantly, the data presented herein, obtained in a well plate format with milliliter volumes, demonstrates superior results compared to benchmark data generated in multi-liter bioreactors. Notably, these results were achieved without the benefit of volume-scaling effects and controlled cell culture parameters using bioreactor settings. All vitronectin concentrations used resulted in a high fold change over the seeded cells, with the lowest concentration proving to be the most effective. This indicates that low vitronectin concentrations can be used to achieve optimal results, thereby reducing costs (Figure 4C).

[0514] Giemsa staining allowed microscopic visualization of cell adhesion and growth within the scaffold (Figure 4D) and confirmed the prolific interaction between cells and the scaffold.

[0515] Conclusion:

[0516] The nanofibrous cellulose scaffold significantly enhances cell proliferation over time and maintains high cell viability. In particular, the use of the nanofibrous cellulose scaffold as a support matrix significantly reduces the doubling time compared to commercial products. Low concentrations of vitronectin are sufficient and preferred for optimal cell growth.

[0517] Example 4: Evaluation of nanofibrous cellulose scaffolds of varying densities

[0518] In this example the influence of varying cellulose concentration on the proliferation of hMSCs was tested.

[0519] Method:

[0520] QA-functionalised and vitronectin coated (molar ratio of vitronectin to cellulose of 8*10-6mol / mol) nanofibrous cellulose scaffolds were prepared as described in Example 2 but with varying amounts of cellulose (2.0%, 1.0%, 0.5%, 0.2%). The molar ratio of crosslinking agent to cellulose was kept constant at 19.0*10-3mol / mol. Experiments were performed using hMSCs.

[0521] In brief, experiments were performed as follows. Following the overnight incubation in culture medium, all the scaffolds (disks) were transferred to ultra-low attachment 6-well plates at a density of 4 scaffolds per well. Cell seeding was performed using 10,000 cells per scaffold (for a total of 40,000 cells per well) in an inoculum volume of 2 ml and the culture plates were placed on an orbital shaker (19 mm) at 50 rpm. 5 hours post-inoculum, additional 2 ml of culture medium was added and the final culture volume of 4 ml was reached. On day 2 post-inoculum, a 2% feedstock addition was performed, followed by a half-medium change at day 6 post-inoculum. Live cell count and viability were assessed every 2 days from day 4 post-inoculum using NucleoCounter NC-202.

[0522] Results:

[0523] Nanofibrous cellulose scaffolds efficiently facilitated cell proliferation across all cellulose concentrations (0.2% to 2.0%) (Figure 5A) and cells retained high viability (Figure 5B). For cells with large diameter such as hMSCs, cell proliferation on scaffolds of lower cellulose concentrations, e.g. 0.2% cellulose, is favoured due to the increased porosity of the scaffold.

[0524] Conclusion:

[0525] Cells displayed similar proliferation rates ed on scaffolds of varying cellulose concentrations. It is possible to retrieve cells for cell count from scaffolds of all cellulose concentrations, despite the thicker nature of scaffolds with higher cellulose concentrations. Scaffolds of lower cellulose concentrations allow for improved cell proliferation for cells with larger diameters.

[0526] Example 5: Validation of the quality of stem cells cultured on the nanofibrous cellulose scaffold

[0527] In this example the quality and identity of stem cells grown on the nanofibrous cellulose scaffold was tested by their proliferation following passaging.

[0528] Method:

[0529] A QA-functionalised and vitronectin coated (molar ratio of vitronectin to cellulose of 8*10"6mol / mol) nanofibrous cellulose scaffold with a cellulose concentration of 0.2% were prepared as described in Example 2. The molar ratio of crosslinking agent to cellulose was 19.0*10-3mol / mol. Experiments were performed using hMSCs.

[0530] Cells retrieved from scaffolds at day 8 post-inoculum using TripLE enzymatic dissociation were seeded into two-dimensional (2D) cultures (2,200 cells / cm2) in T-flasks (T175). Their proliferation rate, viability and morphology were then compared to those of 2D culture from the same passage that had not been cultured on nanofibrous cellulose scaffold, using NucleoCounter NC-202 and microscopy. Cells retrieved from the scaffolds were also analysed by flow-cytometry for key surface markers (CD34, CD45, CDllb, CD19, HLA- DR, CD73, CD90, CD105, CD44) to assess retained sternness and these results were compared to those of 2D culture from the same passage that had not been cultured on nanofibrous cellulose scaffold.

[0531] Results: hMSCs were efficiently retrieved from the scaffolds using enzymatic dissociation (Figure 6A-B) and seeded back into 2D culture, exhibiting the same proliferation rate (Figure 6C) and high viability (Figure 6D) as cells that have not formerly been cultured on the nanofibrous cellulose scaffolds suggesting retention of hMSC identity. hMSCs passaged into 2D culture following culture on the nanofibrous cellulose scaffold have the conventional fibroblast-like morphology observed in conventional 2D cultures 4 days postseeding (Figure 6E). Flow-cytometry analysis of hMSC grown for 8 days on nanofibrous cellulose scaffolds confirmed that the cells consistently expressed high levels of CD73, CD105, CD44 and CD90, which are critical markers for mesenchymal stem cells and lacked markers associated with hematopoietic stem cells or immune cells (CD34, CD45, CDllb, CD19, HLA-DR) comparable with the expression levels of cells from the same passage that had not been cultured on nanofibrous cellulose scaffold (Figure 6F).

[0532] Conclusion:

[0533] Cells grown on nanofibrous cellulose scaffold behave as the 2D control (same fold change) when passaged onto 2D culturing and are able to reestablish monolayer cultures with the same morphology and proliferative capability as prior to culturing on the nanofibrous cellulose scaffold, indicating that the quality and stem cell identity is preserved on the scaffold. The sternness of hMSCs cultured on the scaffold was retained as confirmed with flow-cytometry analysis for key surface markers and was unchanged compared to cells grown in 2D.

[0534] Example 6: Processing of the initial cellulose nanofiber material

[0535] In this example various methods of dividing the initial cellulose nanofiber material were tested and their influence on the nanofibrous cellulose scaffold was assessed.

[0536] Method

[0537] Preparation of the initial cellulose nanofiber material was performed as described in Example 1, with the exception that different means of dividing the fibers were tested.

[0538] Processinq / dividinq protocols

[0539] Different methods of dividing the initial cellulose nanofiber material were evaluated. Thus, samples were prepared according to the following:

[0540] Mechanical cutting (by scissor):

[0541] 2.5 g cellulose sheets were cut up in 10x10 mm pieces with a pair of scissors and assayed directly as fragmented cellulose sheets.

[0542] Laser cutting:

[0543] Cellulose sheets were cut with a laser cutter (Epilog laser, Zing 24) in three different sizes (0.75x0.75mm, 1.5x1.5mm and 3x3mm). The cut sheets were assayed as is.

[0544] Blending: 2.5 g cellulose sheets were cut up in 20x20 mm pieces with a pair of scissors and added together with 250 ml water to a lab blender (LB20, Waring Laboratory). The cellulose nanofiber material was processed at 7000 rpm for 1 (Bl), 5 (B5), 15 (B15) or 60 (B60) minutes.

[0545] Dispersing:

[0546] 2.5 g cellulose sheets were cut up in 20x20 mm pieces with a pair of scissors and added together with 250 ml water to an Erlenmeyer flask. The sample was processed using a disperser (IKA T25 digital Ultra-Turrax with S25 NB - 25 G disperser tool) at 18000 rpm for 1 (DI), 5 (D5), 15 (D15) or 60 (D60) minutes.

[0547] Fiber length measurements

[0548] Fiber length of the cellulose nanofibers were determined either by SEM (Hitachi SU3500) or by light scattering (Malvern Mastersizer S).

[0549] Samples of processed cellulose nanofiber material were diluted 1000-10000X in water and a droplet of sample was applied to a SEM fixture. The samples were dried and sputter coated with gold before capturing images on a Hitachi SU3500. Images were captured at different magnifications.

[0550] SEM images were evaluated manually by visual inspection to ensure that all measured fibers had both endings visible. The fiber lengths were determined by use of ImageJ software. Fiber lengths from several SEM images were determined to get a larger dataset.

[0551] Fiber lengths were also determined using light scattering. Briefly, 1 ml of sample was added in water to the Malvern Mastersizer S and measurements were performed with the settings described under the definition of "mean length". Sample was added to the sample container until the obscuration value was between 15-20%. From each sample a fiber length histogram displaying the fiber length distribution was generated. The statistics of the distribution are calculated from the results using the derived diameters D[m,n] - an internationally agreed method of defining the mean and other moments of particle size. D(v, 0.5), D(v, 0.1) and D(v, 0.9) are standard "percentile" readings from the analysis. D(v, 0.5) is the fiber length at which 50% of the sample is smaller and 50% is larger than this length. This value is also known as the Mass median diameter (MMD) when used for particles. D(v, 0.1) is the fiber length for which 10% of the sample is below this length. D(v, 0.9) gives a fiber length for which 90% of the sample is below this length. The volume weighted mean fiber length D[4,3] was also determined. Pipettinq / floatinq test

[0552] Samples of processed cellulose nanofiber material were tested for their ability to be pipetted. 1 ml of sample was pipetted out of the sample container and subsequently expelled from the pipette tip into a tube with water. The ease of pipetting was evaluated, including the propensity for processed cellulose nanofiber material to clog the pipette.

[0553] Processed cellulose nanofiber material transferred to a tube containing water was then assessed for its propensity to float. Samples were vigorously shaken and it was observed by visual inspection whether the material floated immediately after and 24 hours after shaking.

[0554] Results

[0555] The pipetting test of scissor cut and laser cut cellulose sheets showed that these cut pieces are too large to pipette as they will clog the pipette tip. Also, cellulose nanofiber material processed in this manner generates pieces of dimensions that trap air bubbles and float in solution (Figure 7A). Furthermore, cutting with a laser makes the cellulose nanofibers melt, burn and stick together and is therefore not a suitable operation for dividing the cellulose nanofibers (Figure 7B-C).

[0556] None of the cellulose nanofiber materials processed by dispersing trapped air bubbles or had any tendency to float in solution. Pipetting of these samples was least challenging for samples that had been dispersed for longer durations of time. As such it was preferred to disperse for at least 10 min to improve flow through the pipette.

[0557] SEM measurements were used to determine cellulose nanofiber diameter (Figure 8A-B) and length (Figure 9A-H + Figure 10).

[0558] More than 2000 individual cellulose nanofibers were measured using ImageJ software and gave a mean cellulose nanofiber diameter of 500 nm (Figure 8C).

[0559] It is clear from the SEM images (Figure 9A-H) that the disperser quickly provides a homogenous nanofiber population with relatively few long nanofibers and without any significant entanglement. While large chunks of uncut nanofibers are present in the samples after only 1 min of dividing (Figure 9A-B), the disperser presents a finer population of nanofibers with only few smaller chunks of entangled nanofibers already after 5 min of dispersing (Figure 9D). After 15 and 60 min of dispersing the presence of entangled nanofibers is almost completely eliminated (Figure 9F and 9H). In contrast, the blended samples comprise large chunks of entangled fibers even after 60 min of blending (Figure 9G).

[0560] The data are summarised for each of the samples in Figure 10A-B. It is clear that the frequency of long nanofibers is higher in the blended samples compared to the dispersed samples. Furthermore, there is a tendency towards longer processing times producing less long nanofibers.

[0561] This relative trend is supported by measurements of the nanofibers using light diffraction. These data are summarised in Table 1. Shorter fiber lengths were obtained for longer dispersing times.

[0562] Table 1. Fiber length measurements of cellulose nanofibers prepared by blending (Bl, B5, B15, B60) and dispersing (DI, D5, D15, D60). Measurements were performed on a Malvern Mastersizer S.

[0563] Overall, it is desired that the mean length of the cellulose nanofibers is reduced. It is also preferred that the population of nanofibers does not contain many long nanofibers as these may act as nucleus for larger clusters of nanofibers and entanglements. These clusters or entanglements of nanofibers are undesirable in the nanofibrous scaffold as large fractions of the surface area becomes inaccessible to the cells seeded thereupon and increase the risk of clogging process equipment. Moreover, an inhomogeneous population of nanofibers comprising clusters and entanglements are undesirable because the variability between batches of the nanofibrous scaffold becomes inconsistent and therefore unreliable for cell growth.

[0564] Conclusion This example demonstrates that not every method for dividing the initial cellulose nanofiber material is equally effective and suitable for preparation of the nanofibrous cellulose scaffold. In particular, dispersing is advantageous because it rapidly removes longer fibers and yields a homogeneous population of cellulose nanofiber material without generation of entanglements or clusters.

[0565] Example 7: Processing nanofibers of material different from cellulose

[0566] In this example the processing of a variety of nanofibers different from cellulose were assessed with the aim of identifying other nanofibers suitable for preparation of nanofibrous scaffolds for growing cell cultures on. The electrospun nanofibers were characterized by scanning electron microscopy (SEM) and by visual inspection (following dividing of the nanofibers).

[0567] Method

[0568] Electrospinning

[0569] Nanofibers of polycaprolactone (PCL), poly-L-lactic acid (PLA), a mixture of PCL and PLA (PLA / PCL) and cellulose were electrospun. Electrospinning was performed on a Fluidnatek LE50 apparatus as described in Example 1.

[0570] PCL fibers were obtained by dissolving 8% Polycaprolactone pellets (Sigma Aldrich, MW 80000), in chloroform: methanol, 1: 1 solution. Needle to collector distance was set to 20 cm, flow rate to 3ml / h, and voltage to 18kV.

[0571] PLA fibers were obtained by dissolving 8% PLLA pellets (Goodfellow, MFR:24), in chloroform: methanol, 3:2. Needle to collector distance was set to 24cm, flow rate to 4.5 ml / h and voltage to 35kV.

[0572] PLA / PCL fibers were obtained by dissolving PLA pellets (Goodfellow, MFR: 65) and PCL pellets (Sigma Aldrich, MW 80000) 1 :2 to an 8% polymer solution in chloroform: methanol, 3:2. Needle to collector distance was set to 20cm, flow rate to 3 ml / h and voltage to 18kV

[0573] 1 ml of polymer solution was spun for each sheet of fibers.

[0574] Scanning electron microscopy

[0575] The electrospun material of PCL, PLA, mixed PCL / PLA and cellulose were imaged on a Hitachi SU3500 as described in Example 1. Images of the nanofiber materials were obtained for both undivided and divided nanofibers. Processing of PCL, PLA and PCL / PLA nanofiber material

[0576] Electrospun sheets of PCL, PLA and mixed PLA / PCL were cut into smaller lOxlOmm pieces and mixed with two different mixing tools for 5 minutes. The cut sheets were mixed either with a blender at 7000 rpm (LB20E Laboratory blender, Waring) or with a disperser at 18000 rpm (IKA T25 digital Ultra Turrax).

[0577] Results

[0578] Nanofibers could be electrospun from all materials (PCL, PLA, PLA / PCL, and cellulose) and formed nanofibrous sheets (see Figure 11A-D).

[0579] The sheets made from nanofibers different from cellulose were subjected to two individual modes of dividing the nanofibers, namely blending (Figure 12A-F) and dispersing (Figure 13A-F). The tests showed that it was not possible to uniformly blend or disperse either PCL, PLA, or PLA / PCL nanofibers into a homogenous mixture of short strand nanofibers.

[0580] Both the PCL nanofibers (Figure 12A-B and Figure 13A-B) and the PLA / PCL nanofibers (Figure 12E-F and Figure 13E-F) melted or deformed during the process. In particular, the nanofibers melted together to form either large pieces of solid polymer or large entangled clusters of nanofibers with semi-melted nanofibers.

[0581] The PLA nanofibers (Figure 12C-D and Figure 13C-D) did not melt in the same way as the PCL fibers, but all materials did easily get stuck in the mixing tool or got entangled on the blades of the blender and halted the processing.

[0582] Conclusion

[0583] This example demonstrates that it is not possible to uniformly blend or disperse nanofibers of all materials. Accordingly, not all nanofiber materials can be easily transformed into shorter strands followed by formation of a nanofibrous scaffold as described herein. Thus, it is preferred to use cellulose nanofibers for the preparation of the nanofibrous scaffolds.

[0584] Example 8: Mechanical stability of nanofibrous cellulose scaffold

[0585] In this example the influence of cellulose concentration (concentration of the cellulose suspension during preparation) on the integrity of the nanofibrous cellulose scaffold was assessed. The samples were probed with the aim of evaluating its suitability in an industrial setup including shear stress from agitation.

[0586] An overview of the samples prepared is given in Table 2.

[0587] Table 2. Cellulose concentration used for preparation of nanofibrous cellulose scaffolds.

[0588] Method

[0589] Preparation of nanofibrous cellulose scaffolds

[0590] A first series of nanofibrous cellulose scaffolds was prepared to find the optimal porosity range of the scaffold, i.e. large enough pores for the cells to penetrate the scaffold and have access to large a surface area. Scaffolds were produced by crosslinking with a fixed amount of crosslinking agent to various cellulose concentrations. The procedure as described in Example 1 was followed with the following changes to concentrations.

[0591] Nanofibrous cellulose scaffolds with different porosities were prepared by crosslinking various concentrations of QA functionalized cellulose (2.0%, 1.0%, 0.5%, 0.25%, 0.125%, 0.06%, 0.03% and 0.015% cellulose suspension). 0.5 pl crosslinking agent (Kymene) / ml cellulose suspension was added to each concentration of functionalized cellulose. 500 pl of suspension mixed with crosslinking agent was subsequently added to a 48-well mold (maximum fill volume 0.5 ml, diameter 12 mm, height 4 mm). The molds were frozen to -80°C and thereafter freeze-dried. Finally, the scaffolds were cured in the oven at 120°C for 3 hours on an aluminium tray.

[0592] Furthermore, larger disks of nanofibrous cellulose scaffold were produced with diameters of 57 mm to fit in a bioreactor. The stability was tested based on the ability of the disks to be removed from the mold without breaking and the ability of the disks to withstand the shear stress applied in a mock-up reactor (Figure 16A).

[0593] Disks of nanofibrous cellulose scaffold with different porosities were prepared by crosslinking various concentrations of QA functionalized cellulose (0.25%, 0.125%, 0.06% and 0.03%) as described above. Nanofibrous cellulose scaffolds with higher cellulose concentrations than 0.25% were projected to be stabile and therefore not tested herein. 0.2 pl crosslinking agent per mg cellulose (19.0*10-3mol / mol Kymene:cellulose) was added to each concentration of functionalized cellulose. Different thicknesses of the scaffolds were obtained by filling the molds with different volumes (3, 10, 17 and 25 ml) of cellulose suspension. The molds had an inner diameter of 57 mm, a maximum fill height of 10 mm and a total volume of 25 ml. After filling the molds with different volumes, they were frozen at -80°C and thereafter freeze dried. Finally, the scaffolds were cured in the oven at 120°C for 3 hours on an aluminium tray.

[0594] The mechanical stability for each scaffold was tested in a mock-up bioreactor. Each scaffold was placed between two perforated support disks attached to a stirring rod (Figure 16A, left). The stirring rod was attached to a Nano Star 7.5 Digital stirrer (IKA) and immersed into a beaker with 500 ml of PBS to expose the disks to agitation as under operation of a bioreactor (Figure 16A, middle). The stirring speed was set to 50 rpm and stability was observed over time. Figure 16A (right) illustrates a broken scaffold (sample IIB) due to the shear forces applied during agitation.

[0595] Results

[0596] Nanofibrous cellulose scaffolds prepared from cellulose concentrations ranging from 0.015% to 2% were prepared (Figure 14). There was a clear tendency towards scaffolds of lower cellulose concentration (higher porosity) loosing shape. Both the 0.015% (Figure 14H) and 0.03% (Figure 14G) samples were challenging to handle due to the lightweight nature and fragility, leading to a propensity to break.

[0597] A wide range of nanofibrous cellulose scaffolds with different thicknesses and densities / porosities were prepared (Figure 15(I)-(IV)) for testing mechanical strength under shear stress. From visual inspection it was clear that the scaffolds prepared from the highest cellulose concentrations, e.g. 0.25% (Figure 15(IV)) and 0.125% (Figure 15(111)), produced the most coherent disks. An overview of the measured thickness of the scaffolds is presented in table 3.

[0598]

[0599] There was a tendency for scaffolds prepared from less than 0.125% cellulose concentration to collapse upon freeze-drying leading to decreased thickness compared to the more dense scaffolds. It was not possible to remove the thinnest scaffolds made from a volume suspension of only 3 ml from the mold without breakage. At higher suspension volumes it was easier to handle the thicker scaffolds.

[0600] The test in the mock-up bioreactor revealed that only scaffolds prepared from cellulose concentrations of 0.013% and (17 ml (partly), 25 ml) and 0.25% (10 ml, 17 ml, and 25 ml) were capable of withstanding the shear stress of the mock-up bioreactor (figure 16B).

[0601] Conclusion

[0602] This example demonstrates that it is possible to produce nanofibrous cellulose scaffolds with sufficient mechanical strength to resist breakage when used as a support matrix in a bioreactor in operation. The scaffolds are preferably prepared from a cellulose concentration of at least 0.125%, or more preferable slightly higher concentrations to avoid breakage, and may advantageously have a thickness in the range of 6 mm to 10 mm. More dense scaffolds may be prepared at a thickness of 3 mm and still preserve structural integrity.

Claims

Claims1. A method for culturing of stem cells, said method comprising the steps of: providing a container comprising a support matrix;- seeding stem cells on said support matrix; and- culturing said stem cells; wherein said support matrix comprises cellulose nanofibers.

2. The method according to claim 1, wherein the cellulose nanofibers have a mean length of less than about 250 pm, and wherein the cellulose nanofibers are crosslinked with a crosslinking agent.

3. The method according to any one of claims 1 or 2, wherein the cellulose nanofibers have a mean length in the range of about 30 pm to about 250 pm, such as about 40 pm to about 200 pm, such as about 50 pm to about 175 pm, such as about 60 pm to about 150 pm, preferably about 70 pm to about 120 pm.

4. The method according to any one of the preceding claims, wherein the cellulose nanofibers are electrospun cellulose nanofibers.

5. The method according to any one of the preceding claims, wherein the cellulose nanofibers are monofibers.

6. The method according to any one of the preceding claims, wherein the cellulose nanofibers are derived from regenerated cellulose.

7. The method according to any one of the preceding claims, wherein the cellulose nanofibers have the cellulose II crystal structure.

8. The method according to any one of claims 2-7, wherein the crosslinking agent is a polyamide epichlorohydrin resin.

9. The method according to any one of claims 2-8, wherein the molar ratio of crosslinking agent to cellulose nanofibers is in the range of about 2*10-3mol / mol to about 175*10-3mol / mol, such as about 5*10-3mol / mol to about 100*10-3mol / mol, such as about 10*10’3mol / mol to about 80*10’3mol / mol, such as about 12*10’3mol / mol to about 50*10’3mol / mol, such as about 15*10’3mol / mol to about 25*10’3mol / mol.

10. The method according to any one of the preceding claims, wherein the cellulose nanofibers comprise one or more functional moieties selected from chemical moieties and / or biological molecules.

11. The method according to claim 10, wherein the chemical moieties are positively charged groups.

12. The method according to any one of claims 10 or 11, wherein the biological molecule is a glycoprotein of the extracellular matrix selected from the group consisting of vitronectin, fibronectin, collagen, proteoglycans, laminin, tenascins, and integrins, preferably vitronectin.

13. The method according to any one of claims 10-12, wherein the one or more functional moieties comprises quaternary ammonium (QA) and vitronectin.

14. The method according to any one of the preceding claims, wherein the microcarrier is provided as a dry material.

15. The method according to any one of the preceding claims, wherein the stem cells are selected from the group consisting of mesenchymal stem cells (MSCs), induced pluripotent stem cells (iPSCs), embryonic stem cells (ESCs), hematopoietic stem cells (HSCs), neural stem cells (NSCs), cardiac stem cells, amniotic fluid stem cells, epidermal stem cells, adipose derived stem cells (ASC) and endothelial progenitor cells (EPCs).

16. The method according to any one of the preceding claims, wherein the stem cells are mesenchymal stem cells (MSCs).

17. The method according to any one of the preceding claims, wherein said container is a bioreactor comprising a compartment containing the support matrix.

18. The method according to any one of the preceding claims, wherein the doubling time of said stem cells is less than about 60 hours, such as less than about 55 hours, preferably less than 50 hours.

19. A nanofibrous cellulose scaffold comprising cellulose nanofibers with a mean length of less than about 250 pm, wherein the cellulose nanofibers are crosslinked with a crosslinking agent, wherein the cellulose nanofibers comprise quaternary ammonium (QA) and vitronectin.

20. The nanofibrous cellulose scaffold according to claim 19, wherein the nanofibrous cellulose scaffold has a BET surface area of at least about 40000 cm2 / g, such as at least about 50000 cm2 / g, such as at least about 55000 cm2 / g, such as at least about 60000 cm2 / g, such as at least about 70000 cm2 / g, such as at least about 80000 cm2 / g.

21. The nanofibrous cellulose scaffold according to any one of claims 19 or 20, wherein the physical form of the nanofibrous cellulose scaffold is a disk.

22. Use of a nanofibrous cellulose scaffold according to any one of claims 19-21 as a support matrix for culturing of stem cells.

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