Nanofiber scaffolds for industrial-scale cell culture

Abstract

The present invention relates to a method for preparing a nanofibrous scaffold and its use for facilitating cell culture. In particular, the nanofibrous scaffold comprises cross-linked cellulose nanofibers that result in a continuous microcarrier material for use in industrial-scale cell culture devices.

Description

[Technical Field]

[0001] The present invention relates to a method for producing a nanofibrous scaffold and its use for facilitating cell culture. In particular, the nanofibrous scaffold comprises cross-linked cellulose nanofibers that result in a continuous microcarrier material for use in industrial-scale cell culture devices. [Background technology]

[0002] Biological products (biopharmaceuticals), such as therapeutic proteins, vaccines, or cell and gene therapy products, represent a new medical paradigm. These therapies can provide remarkable patient outcomes and have already revolutionized the treatment of many diseases across a variety of fields. Most complex biological products with specific glycosylation patterns can be produced by recombinant DNA technology in mammalian cell culture, including enzymes, synthetic hormones, and monoclonal antibodies. However, biopharmaceutical manufacturing is challenging because production of the final product relies on sensitive live host cells, which requires much more care than, for example, the chemical synthesis of small molecule drugs.

[0003] The manufacturing process of biopharmaceuticals can be divided into upstream and downstream processes, where upstream processing is defined as the entire process from the initial cell isolation and culture, through cell banking and expansion of the cells, to the final harvest, i.e., termination of the culture and collection of the viable cell batch for purification.

[0004] Therefore, cell culture is the core of the upstream process, but cell immunogenicity, adverse events, and efficacy can all be affected by even slight changes in the manufacturing process. Therefore, scaling up from research to clinical quantities is very difficult, and many promising therapies cannot be brought to market. In addition, the most significant drawbacks are low product yields, the costs associated with subsequent extensive cell line development, and limited cell viability for production. Overall, the manufacturing process is very expensive, and biopharmaceuticals are not available to all patients.

[0005] Many cells and tissues, particularly mammalian cells and tissues, require a surface or other structural support to grow. Therefore, to support upstream processes, microcarriers are routinely used to support the growth of adherent cell populations in bioreactors. Microcarriers are support matrices that allow cells to be cultured in three dimensions rather than the traditional planar configuration, greatly improving the ability to accommodate more cells in a limited volume. Because cell growth is highly dependent on the surface area available for cell attachment, this approach accelerates production yields.

[0006] Packed-bed bioreactors are an attractive method for large-scale cell culture due to the high cell density that can be achieved over a large surface area, the flexibility to operate in different batch modes, and the efficiency of performing infrequent cell passage. In a packed-bed bioreactor, microcarriers are packed into the bioreactor as a bed, while culture medium is continuously circulated through the bioreactor, transferring oxygen and nutrients to the cells seeded on the microcarriers. Cell immobilization allows for high cell densities and a natural means for separating cells from the culture medium. Products secreted into the culture medium can also be easily recovered.

[0007] Because cells in packed-bed bioreactor systems are confined inside a bed containing microcarriers, the shear forces (induced by internal flow) to which cells are exposed are significantly reduced compared to systems in which cells are freely exposed to the medium. However, because the microcarriers are confined within the bed, they are subject to stresses from the moving medium. Therefore, commercially available microcarriers for use with packed-bed bioreactors are typically in the form of robust discs that can withstand the shear forces in the bioreactor. These discs are made of nonwoven fibers such as polypropylene, polyester, and polyethylene terephthalate (PET) and are thin and relatively dense. The design constraints of existing microcarriers limit the available surface area and do not provide the in vivo-like environment required for efficient growth of many mammalian cells.

[0008] Therefore, scaling up cell growth in packed-bed bioreactors remains challenging and could be improved if any commercialization is to become feasible, as large quantities of high-quality cells are required at a cost-effective scale. There is now consensus that not only the available surface area but also the local spatial environment experienced by cells during culture is critical to the quantity and quality of the final cell product. If cells are not exposed to sufficient attachment sites and a microenvironment that mimics their natural environment, cell signaling is hindered, resulting in poor cell differentiation.

[0009] While biomanufacturing of biopharmaceuticals is rapidly advancing, scaling up production is expected to become a bottleneck in the biopharmaceutical revolution over the next few years. Effective microcarriers suitable for use with packed-bed bioreactors are at the forefront of solving this problem and will help accelerate the commercialization of biopharmaceuticals.

[0010] Therefore, there is an unmet need for the provision of new and improved microcarriers that offer high quality and scalable options for cell culture in packed bed bioreactors.

[0011] It would therefore be advantageous to provide a sustainable microcarrier material that provides an increased surface area for cells in culture, replicates the extracellular environment found in the human body, and can withstand the shear forces generated by media flow.

[0012] In particular, it would be beneficial to provide a method for producing improved microcarrier materials in a simple and scalable manner that can be used for a wide range of applications from stock culture to bioreactor production. Summary of the Invention

[0013] The microcarriers presented herein are based on a processed form of cellulose nanofibers that provide a matrix with increased surface area for cell growth. The cellulose nanofibers provide a scaffolding matrix with physical properties that mimic the collagen and elastin fiber structure that constitutes the human extracellular matrix. Thus, cells grown on the microcarriers presented herein adopt physiological properties similar to those of the native tissue from which they originate, resulting in improved cell-to-cell communication and, consequently, restoration or maintenance of in vivo function.

[0014] The microcarriers presented herein are based on a moldable and resilient material that can be incorporated into any cell culture system, and is durable and able to withstand the shear forces encountered in packed-bed bioreactor systems.

[0015] Accordingly, one object of the present invention relates to the provision of microcarriers that are suitable for use in packed bed bioreactors and have the ability to enhance cell growth.

[0016] Another object of the present invention relates to the provision of a simple method for producing improved microcarriers that is scalable for industrial production.

[0017] Thus, one aspect of the present invention relates to a method for preparing a nanofibrous cellulose scaffold, said method comprising: (i) providing an initial cellulose nanofiber material; (ii) dividing the initial cellulose nanofiber material into processed cellulose nanofiber material; (iii) cross-linking the processed cellulose nanofiber material; and (iv) drying the processed cellulose nanofiber material; Thereby, the present invention relates to a method for providing a nanofibrous cellulose scaffold.

[0018] Another aspect of the present invention relates to a nanofibrous cellulose scaffold obtainable from the method described herein.

[0019] Another aspect of the invention relates to a nanofibrous cellulose scaffold comprising a processed cellulose nanofiber material having an average cellulose nanofiber length of less than about 250 μm, wherein the processed cellulose nanofiber material is crosslinked with a crosslinking agent.

[0020] A further aspect of the present invention relates to a microcarrier comprising the nanofibrous cellulose scaffold described herein.

[0021] A still further aspect of the present invention relates to the use of the nanofibrous cellulose scaffold described herein as a microcarrier for cell culture.

[0022] Another aspect of the invention relates to a cell culture device comprising a vessel loaded with the nanofibrous cellulose scaffold or microcarriers described herein.

[0023] Another aspect of the invention is a method for culturing cells, comprising the steps of: (i) providing a cell culture device as described herein; (ii) adding a composition comprising a cell population to the cell culture device; (iii) incubating the cell population to provide an expanded cell population; and (iv) optionally extracting the expanded cell population from the cell culture device. [Brief explanation of the drawings]

[0024] [Figure 1] (A) Scanning electron microscopy (SEM) image of nanofiber cellulose scaffold. (B) FTIR spectrum of cross-linked cellulose. (C) Comparison of FTIR spectra (top to bottom) of cellulose acetate, regenerated cellulose, QA-functionalized, and cross-linked cellulose. [Figure 2](A) Comparison of conventional cut nanofiber sheets (left) and molded nanofiber cellulose scaffold microcarriers (right). (B) Nanofiber cellulose scaffold microcarriers molded to fit lab-scale bioreactors (left) and pilot-scale bioreactors (right). (C-D) Force (N) vs. deformation (%) behavior of nanofiber cellulose scaffolds (Sponge QA2%). [Figure 3] Examples of laser-cut fibers are shown. (A) Laser-cut cellulose material traps air bubbles, resulting in floating cellulose pieces. (B) Laser-cut electrospun cellulose material. The laser burns the cellulose sheet. (C) SEM image of laser-cut cellulose nanofibers. The cellulose nanofibers melt and fuse together. [Figure 4] Microscopic images of cellulose nanofibers split by blending (A-E) or dispersing (F-J) for different times are shown. [Figure 5] Differences between blending and dispersing cellulose nanofibers are shown. (A) Nanofiber length as a function of the means of splitting and duration of splitting (0.5-10 min). Nanofiber length is determined from SEM images using ImageJ software. (B) Representative SEM image of cellulose nanofibers after 10 min of blending. (C) Representative SEM image of cellulose nanofibers after 10 min of dispersion. [Figure 6] (A and B) SEM micrographs of cellulose nanofibers dispersed at 18,000 rpm for 1 hour showing the determination of cellulose nanofiber diameter. (C) Histogram showing the diameter distribution of cellulose nanofibers from five separate samples. Approximately 2,500 individual cellulose nanofibers were measured using ImageJ software. [Figure 7]Figure 1 shows the viability of cells grown on nanofiber cellulose scaffold or cellulose sheet microcarriers. (A) Experimental layout of a 24-well plate showing the sample allocation of microcarriers. (B) Viability of HEK293T cells cultured on microcarriers. Cell viability is shown as a percentage compared to live and dead cell controls. The back bar represents the 24-hour time point, and the gray bar represents the 48-hour time point. [Figure 8] Representative images of cells grown on each microcarrier are shown. (A) (From left to right) Wells representing sheet plain, sponge plain, sheet plasma, and sponge plasma 0.5%. (B) (From left to right) Wells representing sheet QA, sponge QA 0.5%, sponge QA 1.5%, and sponge CMC. [Figure 9] Figure 1 shows the configuration of microcarriers arranged as stacked layers for use in packed-bed bioreactors. (A) Traditional setup with thin microcarrier discs placed between support layers. (B) Nanofiber cellulose scaffold in the form of thick discs placed between support layers. (C) Stacked layers of microcarriers arranged on a shaft. [Figure 10] Scanning electron microscope (SEM) images of electrospun nanofibers: (A) PCL, (B) PLA, (C) PLA / PCL, and (D) cellulose. All images were captured at 600x magnification, and the scale bar is 50 μm. [Figure 11] Representative photographs (left) and SEM images (right) of different electrospun nanofibers after mixing in a blender for 5 minutes: (A and B) PCL, (C and D) PLA, and (E and F) PLA / PCL. The scale bar in the SEM images is 200 μm. [Figure 12] Representative photographs (left) and SEM images (right) of different electrospun nanofibers after 5 minutes of mixing in a disperser: (A and B) PCL, (C and D) PLA, and (E and F) PLA / PCL. The scale bar in the SEM images is 50 μm. [Figure 13]Scanning electron microscope (SEM) images of blended (left) or dispersed (right) cellulose nanofibers were mixed for different times: 1 minute (A and B), 5 minutes (C and D), 15 minutes (E and F), or 60 minutes (G and H). [Figure 14] Histograms of the size (length) distribution of cellulose nanofibers separated by (A) blending or (B) dispersion. The histograms are for samples separated for 1, 5, 15, or 60 minutes (from left to right). Nanofiber lengths are displayed as the relative frequency of the mean nanofiber length in 200 μm bins. [Figure 15] (Top) Nanofibrous cellulose scaffolds prepared with different cellulose concentrations: (A) 2%, (B) 1%, (C) 0.5%, (D) 0.25%, (E) 0.125%, (F) 0.06%, (G) 0.03%, and (H) 0.015%. (Middle) Quantification of HEK293 cell concentration over time cultured on crosslinked and functionalized nanofibrous cellulose scaffolds with different cellulose concentrations. Data are presented as averages, and each data point represents a technical replicate (n=3). (Bottom) Cross-sections of nanofibrous cellulose scaffolds displaying the spatial distribution of DAPI-stained cells within the optically clear porous scaffold at 72 hours. Scale bar is 250 µm. [Figure 16] Quantification of the concentration of HEK293 cells cultured for 72 h on different functionalized nanofibrous cellulose scaffolds with different ratios of crosslinker to cellulose: low (3.8 × 10 mol% / mol%), medium (19.0 × 10 mol% / mol%), and high (94.9 × 10 mol% / mol%). Data are presented as the average, and each data point is a technical replicate (n = 3). [Figure 17] Discs of nanofibrous cellulose scaffolds fabricated with different cellulose concentrations: (I) 0.03%, (II) 0.06%, (III) 0.125%, (IV) 0.25%, and different volumes in the mold: (A) 25 ml, (B) 17 ml, (C) 10 ml, (D) 3 ml. [Figure 18](A) Mechanical testing setup. (Left) Scaffold mounted between two support discs on a stir bar. (Center) Stir bar immersed in a 500 ml beaker. (Right) Visible failure of sample IIB (0.06% cellulose and 17 ml volume). (B) Recovery of the scaffold after stirring in the mock-up reactor. Samples were designated as (A) IA, (B) IIA, (C) IIB, (D) IIIA, (E) IIIB, (F) IIIC, (G) IVA, (H) IVB, and (I) IVC according to reference number. [Figure 19] Figure 1 shows cell growth curves and glucose consumption over 5 days of culture in a 1 L commercial bioreactor using discs of crosslinked nanofibrous cellulose scaffold. DETAILED DESCRIPTION OF THE INVENTION

[0025] definition Before outlining the present invention in more detail, a set of terms and conventions will first be defined.

[0026] nanofiber In the present context, the term "nanofiber" refers to fibers with diameters in the range of 10-2000 nm. Fibers can be produced from different types of polymers, such as cellulose.

[0027] Cellulose nanofiber material In this 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, which can then be processed as described herein to provide a nanofibrous cellulose scaffold. These methods include, but are not limited to, electrospinning, meltblowing, and drawing of cellulose nanofibers.

[0028] Preferably, the cellulose nanofiber material is prepared by electrospinning of cellulose nanofibers, which may be carried out from a solution of cellulose acetate.

[0029] container In the present context, the term "vessel" refers to any compartmented vessel suitable for culturing cells. The vessel is preferably a conventional culture vessel, including, but not limited to, a bioreactor, a cell culture plate, a cell culture bottle, a spinner flask, a shaker flask, a roller bottle, a Petri dish, a tube, etc.

[0030] Because the nanofibrous cellulose scaffolds described herein are easily scalable, the vessel can be of any volume suitable for cell culture.

[0031] dispersion In this context, the term "dispersion" refers to the process of breaking up cellulose nanofiber material using a disperser. Dispersion is preferably carried out in a liquid.

[0032] In this context, a disperser is a high-speed mixing device capable of breaking down solids, such as cellulose sheets, into smaller pieces. Dispersers comprise one or more heads that constitute a means for dividing the cellulose nanofiber material. The heads may be in the form of disk blades. Disperser heads may also comprise blades with a propeller design. Dispersers create turbulence and vortices that uniformly divide the cellulose nanofiber material into smaller pieces, i.e., cellulose nanofibers that are shorter in length than the initial cellulose nanofiber material.

[0033] Any type of disperser can be used, including, but not limited to, a laboratory high-speed disperser, a pilot-scale high-speed disperser, and a production-scale high-speed disperser. The disperser can be selected according to the batch size to be divided, and the diameter of the disperser blade is adjusted accordingly. The disperser blade can be raised and lowered to remove stratification during dispersion, and such a mechanism can be automatic. For large batches, the disperser can be a floor-standing model or a tank-mounted model, and / or a multi-axis model.

[0034] Crosslinking agent In this context, the term "crosslinker" refers to any chemical or biological molecule capable of linking cellulose nanofibers together. Preferably, the crosslinker is capable of linking cellulose nanofibers via reaction with the hydroxyl groups of the cellulose backbone to produce covalently crosslinked cellulose nanofibers.

[0035] Thus, the cross-linking agent can be a molecule that contains at least two functional groups capable of reacting with hydroxyl groups.

[0036] sensuality part In this context, the term "functional moiety" refers to a chemical or biological group or molecule disposed on the nanofibrous cellulose scaffold that interacts with cells associated with the scaffold. The functional moiety may interact with cells through interactions including, but not limited to, electrostatic interactions, affinity, and hydrophobicity / hydrophilicity. The functional moiety may promote cell attachment / adhesion to the nanofibrous cellulose scaffold, induce cell differentiation and proliferation, and / or assist in maintaining cell function in vivo.

[0037] The chemical moiety can have one or more positive or negative charges to induce electrostatic interactions with charged cell membranes. Examples include, but are not limited to, quaternary ammonium (QA), carboxymethyl (CM), and diethylaminoethyl (DEAE). In many cells, the cell membrane is negatively charged, and electrostatic interactions are induced in nanofibrous cellulose scaffolds functionalized with positively charged functional moieties such as QA or DEAE.

[0038] The biological component can be any type of biological molecule capable of ensuring cell adhesion to the nanofibrous cellulose scaffold, including, but not limited to, lipid anchors, cell adhesion molecules (CAMs), antigens, receptors, and antibodies. CAMs include integrins, immunoglobulins, cadherins, and selectins. The biological moiety can also assist cell differentiation through receptor-ligand interactions, antigen-antibody interactions, protein interactions, and / or immunostimulatory signaling.

[0039] adherent cells In the present context, the term "adherent cells" refers to any cells that require a surface or artificial substrate, such as a microcarrier, to form an adherent cell culture. Preferably, the adherent cells are derived from solid tissue.

[0040] Adherent cultures are distinguished from suspension cultures in which cells grow freely floating in suspension.

[0041] Average diameter (of cellulose nanofibers) In this context, the term "average diameter" refers to the average diameter of the cellulose nanofibers in the nanofibrous cellulose scaffold. The average diameter can be determined from SEM images of the nanofibrous cellulose scaffold. Preferably, the average diameter is determined by measuring at least 100 individual nanofibers in a sample using image analysis software such as ImageJ.

[0042] The average diameter of the cellulose nanofibers can be adjusted during the process of preparing the initial cellulose nanofiber material, for example, by varying the parameters of the electrospinning process.

[0043] Preferably, the average diameter of the cellulose nanofibers in the nanofibrous cellulose scaffold is from about 250 nm to about 750 nm, for example, from about 400 nm to about 600 nm.

[0044] Average length (of cellulose nanofibers) In this context, the term "average length" refers to the average length of the cellulose nanofibers in the nanofibrous cellulose scaffold. The average length can be measured as the volume-weighted average value (D[4,3]) measured by light scattering, for example, using a Malvern Mastersizer S. D[4,3] is also known as the De Brouckere average value.

[0045] The average fiber length within a sample can be determined using the following settings on a Malvern Mastersizer S: Range lens: 300RF mm Presentation: 3OHD Analytical model: Polydisperse Particle refractive index: (1.5295, 0.1000) Refractive index of dispersant: (1.33000) Density: 1.5000g / cm 3

[0046] Preferably, the average length of the cellulose nanofibers is about 30 μm to about 250 μm, for example, about 40 μm to about 200 μm, for example, about 50 μm to about 150 μm, and preferably about 60 μm to about 100 μm.

[0047] surface area In this context, the term "surface area" refers to the Brunauer-Emmett-Teller (BET) surface area. BET surface area is typically determined by measuring the physical adsorption of a gas, such as nitrogen, to obtain a value for a sample. The BET method can accurately determine the surface area of ​​nanofibrous cellulose scaffolds because gas molecules can travel within the nanofiber matrix and probe the internal surfaces.

[0048] Surface area is the area per unit mass (e.g., cm 2 / g) and can be measured in accordance with ISO 9277:2022 "Determination of the specific surface area of ​​solids by gas adsorption - BET method".

[0049] Degree of substitution (DS) In this 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 a β(1→4)-linked D-glucose, which contains three hydroxyl groups that can be substituted. Therefore, the theoretical maximum value of DS is 3.

[0050] The degree of substitution (DS) can be determined using the following formula: DS=(162N / (1400-cAxN)) where 162 is the molecular weight of an anhydroglucose unit (AGU), N is the nitrogen fraction, and CA is the molecular weight of the cationic reagent.

[0051] The substitution of cellulose nanofibers can also be quantified as the equivalent of charge per elementary unit mass of cellulose, given in units of meq / g, which can be determined by zeta potential measurement, pH titration, or electrokinetic chromatography.

[0052] Dead volume As used herein, the term "dead volume" refers to the volume occupied by microcarriers during cell culture. Ideally, dead volume is minimized to allow for increased cell growth per volume within the vessel used to culture the cells.

[0053] mercerization In this context, the term "mercerization" refers to the process of swelling cellulose nanofiber material in aqueous or ethanolic NaOH solution to break the hydrogen bonds within the cellulose and increase the number of available hydroxyl groups (-OH).

[0054] Microcarriers In the present context, the term "microcarrier" refers to any support matrix on which adherent cells can be grown in adherent culture.

[0055] Packed Bed Bioreactor In this context, the term "packed bed bioreactor" refers to a bioreactor in which microcarriers are immobilized in a bed within the bioreactor. Fresh medium is continuously circulated through the system. Means for providing circulation (or agitation) within the bioreactor include, but are not limited to, agitators such as impellers or pumps. Cells are seeded onto the microcarriers within the bed.

[0056] Packed bed bioreactors fall into two general categories: fixed bed bioreactors, in which the packed bed is stationary, and dynamic bed bioreactors, in which the packed bed is moving.

[0057] Dry continuous material In this context, the term "dry continuous material" refers to a dry material that can be produced as a single entity that exhibits significant elongation in three dimensions, including, but not limited to, elongation of at least about 0.1 mm in all three dimensions.

[0058] Dry continuous materials are distinguished from materials that derive only from the bulk form of individual entities added together. Examples of discontinuous materials include, but are not limited to, a dense mass of solid spherical particles or an aggregate of stacked disks.

[0059] Elasticity In this context, the term "elasticity" refers to the ability of a material to return to its original shape and size after being subjected to an external force or stress. Thus, an elastic material can absorb energy when deformed and then release that energy when the deforming force is removed, returning the material to its original shape. This property is often referred to as "elasticity" or "elastic deformation." An elastic material is compressible, yet has sufficient elasticity to allow the material to decompress and stretch back to its original shape when the force on the material is removed.

[0060] Thus, a resilient material can be forced into a container or void of smaller volume and assume the shape of that container or void, and this change in shape is essentially reversible, as it will return the material to its original shape and size when released from the smaller volume or void.

[0061] Compression strength In the present context, the term "compressive strength" refers to the ability of a material to withstand a compressive load. Compressive strength can be given by the force required to cause a certain deformation. Values ​​can be given in mm / N.

[0062] As a benchmark, the compressive strength of a material can be stated as the force required to obtain 60% deformation of the material.

[0063] Compressive strength can be measured according to ISO 604:2002 - Plastics - Determination of compressive properties.

[0064] about Whenever the term "about" is used herein in the context of an absolute or relative amount (e.g., percentage, equivalent, or ratio), such as a quantity, e.g., number, purity, weight, size, etc., a time frame, and parameters such as temperature, pressure, etc., it will be understood that such variables are approximations and, as such, can vary by ±10%, e.g., ±5%, preferably ±2% (e.g., ±1%) from the actual number specified. This is true even when such a number is initially presented as a percentage (e.g., "about 10%" may mean ±10% about the number 10, which is anywhere from 9% to 11%).

[0065] Nanofibrous cellulose scaffolds This paper describes nanofibrous cellulose scaffolds that can be used as microcarriers for cell culture. Nanofibrous cellulose scaffolds possess properties such as a large surface area and low dead volume, significantly increasing cell growth per unit culture volume. Furthermore, the mechanical properties of the scaffolds make them particularly advantageous for use in packed-bed bioreactors to withstand forces from internal flow. Importantly, nanofibrous cellulose scaffolds can be produced in a simple, cost-effective manner, resulting in commercially attractive and easily scalable end products.

[0066] Thus, one aspect of the present invention relates to a method for preparing a nanofibrous cellulose scaffold, said method comprising: (i) providing an initial cellulose nanofiber material; (ii) dividing the initial cellulose nanofiber material into processed cellulose nanofiber material; (iii) cross-linking the processed cellulose nanofiber material; and (iv) drying the processed cellulose nanofiber material; Thereby, providing a nanofibrous cellulose scaffold.

[0067] The resulting nanofibrous cellulose scaffolds mimic the extracellular matrix (ECM) and have a high surface area due to the homogeneous distribution of cellulose nanofibers. The ECM is important for cell survival, proliferation, differentiation, and migration. Therefore, microcarriers that mimic the properties of the ECM are considered a step toward in vivo-like cell culture.

[0068] The high flexibility of cellulose nanofibers allows them to efficiently utilize the space they occupy by forming a strand network, reducing the dead volume of the microcarrier that is inaccessible to cells. Thus, more surface area can be packed into a smaller volume, which is advantageous for use in cell culture vessels where only a finite volume is available.

[0069] The compaction profile of the nanofibrous cellulose scaffold has the advantage that the flexible material can be easily adapted to any cell culture platform, while the mechanical strength of the material ensures that it will not be damaged or dissolved by the internal medium flow in a packed-bed bioreactor.

[0070] Thus, the present method provides an improved microcarrier material that can be used to increase cell growth and product yield.

[0071] The cross-linking of individual cellulose nanofibers is preferably achieved by adding a cross-linking agent. The cross-linking agent is not limited to any particular type of cross-linking agent. The cellulose backbone contains hydroxyl groups that can be used to cross-link the nanofibers via the cross-linking agent. Therefore, the cross-linking agent preferably has a functional group that can react with the hydroxyl groups to form a covalent bond.

[0072] Thus, one embodiment of the present invention relates to a method as described herein, wherein the processed cellulose nanofiber material is crosslinked by the addition of a crosslinking agent.

[0073] Another embodiment of the present invention relates to the method described herein, wherein the cross-linking agent comprises at least two functional groups capable of reacting with hydroxyl groups to form covalent bonds.

[0074] A further embodiment of the present invention relates to a method as described herein, wherein the crosslinker comprises at least two functional groups selected from the group consisting of carboxylic acid, aldehyde, epoxide, halide, anhydride, silane, and azetidinium.

[0075] Yet another embodiment relates to the methods described herein, wherein the cross-linking agent comprises at least two azetidinium groups.

[0076] Several cross-linking agents have been identified as being particularly useful for cross-linking cellulose nanofibers. In particular, cross-linking with polyamide epichlorohydrin is demonstrated herein to result in nanofibrous cellulose scaffolds with high surface area and excellent mechanical properties.

[0077] Thus, one embodiment of the present invention relates to a method as described herein, wherein the processed cellulose nanofiber material is crosslinked by the addition of a crosslinking agent 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.

[0078] A preferred embodiment of the present invention relates to the method described herein, wherein the crosslinker is a polyamide epichlorohydrin resin.

[0079] Another preferred embodiment of the present invention relates to the method described herein, wherein the crosslinker is a hexanedioic acid polymer with N1-(2-aminoethyl)-1,2-ethanediamine and 2-(chloromethyl)oxirane.

[0080] Hexane diacid, also known as adipic acid, has the chemical formula (CH2)4(COOH)2. Epichlorohydrin, also known as 2-(chloromethyl)oxirane, is an organic chlorine compound and an epoxide. N1-(2-aminoethyl)-1,2-ethanediamine, also known as diethylenetriamine, is an organic compound with the formula HN(CH2CH2NH2)2. It becomes N-alkylated upon reaction with the epoxide group to form a crosslink.

[0081] Thus, a preferred embodiment of the present invention relates to the method described herein, wherein the crosslinker is adipic acid-diethylenetriamine-epichlorohydrin copolymer.

[0082] It should be understood that the copolymer may also be referred to as a resin.

[0083] The amount of crosslinker can be varied to modify the properties of the nanofibrous cellulose scaffold. For example, increasing the amount of crosslinker results in a denser and more mechanically strong material. Specific ratios of cellulose nanofiber material to crosslinker have been found to promote properties such as high available surface area and sufficient mechanical strength.

[0084] Thus, one embodiment of the present invention relates to a method as described herein, wherein the ratio of crosslinker to processed cellulose nanofiber material is in the range of about 1:34 (vol% / wt%) to about 1:1 (vol% / wt%), such as about 1:30 (vol% / wt%) to about 1:5 (vol% / wt%), for example, about 1:25 (vol% / wt%) to about 1:10 (vol% / wt%), preferably about 1:20 (vol% / wt%) to about 1:15 (vol% / wt%), more preferably about 1:17 (vol% / wt%).

[0085] Another embodiment of the present invention relates to a method as described herein, wherein the ratio of crosslinker to processed cellulose nanofiber material is in the range of about 1:136 (vol% / wt%) to about 1:2 (vol% / wt%), such as about 1:68 (vol% / wt%) to about 1:5 (vol% / wt%), for example, about 1:34 (vol% / wt%) to about 1:10 (vol% / wt%), preferably about 1:20 (vol% / wt%) to about 1:15 (vol% / wt%), and more preferably about 1:17 (vol% / wt%).

[0086] A further embodiment of the present invention relates to a method as described herein, wherein the ratio of crosslinker to processed cellulose nanofiber material is in the range of about 1:50 (vol% / wt%) to about 4:1 (vol% / wt%), such as about 1:20 (vol% / wt%) to about 2:1 (vol% / wt%), such as about 1:10 (vol% / wt%) to about 1:1 (vol% / wt%), for example about 1:5 (vol% / wt%) to about 1:2 (vol% / wt%).

[0087] A preferred embodiment of the present invention relates to the method described herein, wherein the ratio of cross-linking agent to processed cellulose nanofiber material is about 1:5 (vol% / wt%).

[0088] Another preferred embodiment of the present invention relates to the method described herein, wherein the ratio of cross-linking agent to processed cellulose nanofiber material is about 1:2.5 (vol% / wt%).

[0089] A further embodiment of the present invention relates to a method described herein, wherein the ratio of crosslinker to processed cellulose nanofiber material is less than about 1:1 (vol% / wt%), such as less than about 1:15 (vol% / wt%), or less than about 1:2 (vol% / wt%).

[0090] The amount of crosslinker can also be presented as mole % of crosslinker per mole % of processed cellulose nanofiber material. This ratio is relevant because it indicates the number of crosslinking units linking glucose units within the nanofibrous cellulose scaffold. Varying the number of crosslinkers per glucose unit alters the properties of the scaffold. Thus, a higher mole % / mole % ratio may increase mechanical strength and reduce porosity. Herein, the mole % of processed cellulose nanofiber material is given per glucose unit.

[0091] Thus, one embodiment of the present invention relates to the method described herein, wherein the processed cellulose nanofiber material has a particle size of about 2×10 -3 mol% / mol% ~ approx. 175 x 10 -3 mol% / mol%, e.g., about 5×10 -3 mol% / mol% ~ approx. 100 x 10 -3 mol% / mol%, e.g., about 10×10 -3 mol% / mol% ~ approx. 80 x 10 -3 mol% / mol%, e.g., about 15×10 -3 mol% / mol% ~ approx. 50 x 10 -3 mol% / mol%, e.g., about 25×10 -3 mol% / mol% ~ approx. 40 x 10 -3Crosslinked with a crosslinker at a ratio of crosslinker to processed cellulose nanofiber material in the range of mol% / mol%.

[0092] Another embodiment of the present invention relates to the method described herein, wherein the processed cellulose nanofiber material has a particle size of about 15×10 -3 mol% / mol% ~ approx. 80 x 10 -3 mol% / mol%, e.g., about 25×10 -3 mol% / mol% ~ approx. 70 x 10 -3 mol% / mol%, e.g., about 30×10 -3 mol% / mol% ~ approx. 50 x 10 -3 mol% / mol%, e.g., about 35×10 -3 mol% / mol% ~ approx. 40 x 10 -3 Crosslinked with a crosslinker at a ratio of crosslinker to processed cellulose nanofiber material in the range of mol% / mol%.

[0093] It has been found that for some types of nanofibrous cellulose scaffolds, it can be advantageous to reduce the ratio of cross-linking agent to the processed cellulose nanofiber material. Without being bound by theory, it is contemplated herein that a lower degree of cross-linking may aid cell migration within the scaffold and result in better cell growth.

[0094] Thus, one embodiment of the present invention relates to the method described herein, wherein the processed cellulose nanofiber material has a particle size of about 90×10 -3 mol% / mol%, e.g., less than about 75×10 -3 mol% / mol%, e.g., less than about 50×10 -3 mol% / mol%, e.g., less than about 40×10 -3 mol% / mol%, e.g., less than about 30×10 -3 mol% / mol%, e.g., less than about 25×10 -3 mol% / mol%, e.g., less than about 20×10 -3 Crosslinked by a crosslinker at a ratio of crosslinker to processed cellulose nanofiber material of less than mol% / mol%.

[0095] A preferred embodiment of the present invention relates to the method described herein, wherein the processed cellulose nanofiber material has a particle size of about 30×10 -3 mol% / mol% ~ approx. 50 x 10 -3 Crosslinked with a crosslinker at a ratio of crosslinker to processed cellulose nanofiber material in the range of mol% / mol%.

[0096] It should be understood that the mole % of the cellulose material refers to the mole % per glucose unit in the processed cellulose nanofiber material.

[0097] A further embodiment of the present invention relates to the method described herein, wherein the crosslinking step is carried out at a temperature ranging from about 15°C to about 25°C, preferably at about 20°C.

[0098] Partitioning the initial cellulose nanofiber material is important to ensure a homogeneous distribution of cellulose nanofibers in the final scaffold. Additionally, shorter cellulose nanofibers significantly reduce the risk of nanofiber entanglement and nanofiber clustering, which reduces the available surface area exposed to cells and increases the risk of clogging the bioreactor impeller. Furthermore, larger entanglements or clusters of cellulose nanofibers can also make the final nanofibrous cellulose scaffold difficult to handle and can lead to clogged tubing or blockages during pipetting. As part of the upstream biomanufacturing process, continuous pipetting or tapping from the bioreactor is performed to perform cell counting, viability, and yield results. Therefore, microcarrier solutions with even the slightest risk of clogging are not practical.

[0099] It has been found that dispersing the initial cellulose nanofiber material results in a homogenous material suitable for use as a microcarrier. Without being bound by theory, it is believed that dispersing the initial cellulose nanofiber material allows cells to be spaced apart so that they have multiple attachment points, improving proliferation.

[0100] Thus, one embodiment of the present invention relates to a method as described herein, wherein dividing the initial cellulose nanofiber material is achieved by dispersion.

[0101] Another embodiment of the present invention relates to the method described herein, wherein the dispersing is carried out in a high speed disperser.

[0102] A further embodiment of the present invention relates to a method as described herein, wherein dividing the initial nanofiber material comprises cutting the initial nanofiber material with a disperser.

[0103] Increasing the dispersion time shortens the average length of the cellulose nanofibers in the nanofibrous cellulose scaffold. In particular, it is advantageous to disperse the material for at least several minutes to reduce nanofiber entanglement and cluster formation. Also, without being bound by theory, it is believed that longer cellulose nanofibers are less easily substituted on the hydroxyl groups, thereby resulting in a lower degree of substitution (DS) of the functional moieties.

[0104] Thus, one embodiment of the present invention relates to a method as described herein, wherein the dispersion is carried out for at least 2 minutes, such as at least 5 minutes, such as at least 10 minutes.

[0105] Another embodiment of the present invention relates to a method as described herein, wherein the dispersion is carried out for at least 2 minutes, such as at least 5 minutes, for example at least 10 minutes, such as at least 15 minutes, for example at least 20 minutes, such as at least 30 minutes, for example at least 40 minutes, such as at least 50 minutes, for example at least 60 minutes, such as at least 90 minutes, for example at least 120 minutes.

[0106] A further embodiment of the present invention relates to a method as described herein, wherein the dispersion is carried out for a period ranging from 2 minutes to 120 minutes, such as from 2 minutes to 90 minutes, for example from 5 minutes to 60 minutes, such as from 10 minutes to 60 minutes, for example from 15 minutes to 60 minutes.

[0107] A preferred embodiment of the present invention relates to the method described herein, wherein the dispersion is carried out for at least 5 minutes.

[0108] Another preferred embodiment of the present invention relates to the method described herein, wherein the dispersion is carried out for at least 15 minutes.

[0109] A further embodiment of the present invention relates to a method as described herein, wherein the dispersion is carried out for at least 60 minutes.

[0110] Dispersing the initial cellulose nanofiber material for at least 15 minutes, or even at least 60 minutes, can be advantageous as it reduces entanglement and clustering of the cellulose nanofibers.

[0111] Preferably, the dispersion is carried out at a high speed, such as about 18,000 rpm. The speed can be adjusted depending on the type of disperser. Any type of disperser can be used, including, but not limited to, a laboratory high-speed disperser, a pilot-scale high-speed disperser, and a production-scale high-speed disperser. The speed can be adjusted to generate a good vortex in the solution, and can depend on the volume and viscosity of the solution.

[0112] Thus, one embodiment of the present invention relates to a method as described herein, wherein the dispersion is carried out at a speed ranging from about 10,000 rpm to about 30,000 rpm, such as from about 12,000 rpm to about 25,000 rpm, for example from about 15,000 rpm to about 20,000 rpm.

[0113] Another embodiment of the present invention relates to a method as described herein, wherein the dispersion is carried out at at least 15000 rpm, such as at least 20000 rpm, such as at least 25000 rpm.

[0114] During the splitting process, the length of the cellulose nanofibers is shortened. It is important that the cellulose nanofibers are not too long, as this causes the nanofibers to entangle and cluster. Cells are unable to penetrate and migrate into these dense clusters of entangled fibers, thereby losing part of the large surface area of ​​the nanofibers.

[0115] Thus, one embodiment of the present invention relates to a method as described herein, wherein the average length of the cellulose nanofibers in the processed cellulose nanofiber material is shorter than the average length of the cellulose nanofibers in the initial cellulose nanofiber material.

[0116] Another embodiment of the present invention relates to a method as described herein, wherein the average length of the cellulose nanofibers in said processed cellulose nanofiber material is less than 250 μm, such as less than about 200 μm, for example less than about 150 μm, such as less than about 120 μm, for example less than about 100 μm, such as less than about 80 μm.

[0117] A further embodiment of the present invention relates to a method as described herein, wherein the average length of the cellulose nanofibers in the processed cellulose nanofiber material is in the range of about 30 μm to about 250 μm, such as about 40 μm to about 200 μm, for example about 50 μm to about 150 μm, preferably about 60 μm to about 100 μm.

[0118] Yet further embodiments of the present invention relate to methods described herein, wherein the average length of the cellulose nanofibers in the processed cellulose nanofiber material ranges from about 70 μm to about 120 μm.

[0119] The splitting step can be carried out under cooling to reduce the ductility of the cellulose nanofibers, making them more brittle and easier to split. Cooling can occur before or during splitting of the initial cellulose nanofiber material. Cooling can include cooling a container in which the initial cellulose nanofiber material is held during the splitting step, or cooling the initial cellulose nanofiber material by exposing it to a coolant such as liquid hydrogen, liquid helium, and / or liquid nitrogen, or by storing the initial cellulose nanofiber material in a refrigerator or freezer immediately prior to the splitting step.

[0120] Thus, one embodiment of the present invention relates to a method as described herein, wherein the initial cellulose nanofiber material is cooled prior to or during the splitting step.

[0121] Cellulose sheets, when prepared by electrospinning, can be highly static and difficult to handle, therefore the initial cellulose nanofiber material can be conveniently provided as a liquid sample that can be readily processed, for example, by dispersion.

[0122] Thus, one embodiment of the present invention relates to a method as described herein, wherein the initial cellulose nanofiber material is provided as a liquid sample.

[0123] Another embodiment of the present invention relates to a method as described herein, wherein the solvent of the liquid sample comprises water and / or ethanol.

[0124] A further embodiment of the present invention relates to a method as described herein, wherein the solvent of the liquid sample comprises ethanol.

[0125] The properties of nanofibrous cellulose scaffolds (e.g., after drying and shaping) can be influenced by the density of cellulose nanofibers contained in the network after the liquid is removed. For example, adding lower concentrations of cellulose nanofibers provides a more porous network through which cells can migrate more easily. In contrast, the higher the concentration of cellulose nanofibers, the higher the mechanical strength of the material. These properties are also influenced by the degree of cross-linking. Several advantageous combinations of cellulose content and degree of cross-linking have been identified herein that provide the material with sufficient mechanical strength to withstand the shear forces in a packed-bed bioreactor while simultaneously providing a high available surface area.

[0126] Thus, one embodiment of the present invention relates to a method as described herein, wherein the concentration of cellulose nanofibers in the processed cellulose nanofiber material is from about 0.1 wt% to about 10 wt%, for example from about 0.15 wt% to about 8 wt%, for example from about 0.2 wt% to about 5 wt%, for example from about 0.2 wt% to about 3 wt%, for example from about 0.25 wt% to about 2 wt%, preferably from about 0.3 wt% to about 1 wt%, based on the total weight of the liquid sample.

[0127] Another embodiment of the present invention relates to a method described herein, wherein the concentration of cellulose nanofibers in the processed cellulose nanofiber material is about 0.1 wt% to about 5 wt%, for example, about 0.15 wt% to about 2.5 wt%, for example, about 0.25 wt% to about 1.5 wt%, preferably about 1 wt%, based on the total weight of the liquid sample.

[0128] In some variations of nanofibrous cellulose scaffolds, it has been found advantageous to maintain the cellulose concentration at a low level, which ensures high porosity in the resulting nanofibrous cellulose scaffold, thereby allowing cells to migrate into the scaffold and utilize the large exposed surface area of ​​the nanofibers.

[0129] Thus, one embodiment of the present invention relates to a method as described herein, wherein the concentration of cellulose nanofibers in the processed cellulose nanofiber material is between about 0.01 wt% and about 1 wt%, such as between about 0.02 wt% and about 0.5 wt%, for example between about 0.03 wt% and about 0.2 wt%.

[0130] A preferred embodiment of the present invention relates to the method described herein, wherein the concentration of cellulose nanofibers in the processed cellulose nanofiber material is between about 0.05 wt% and about 0.2 wt%, for example, between about 0.08 wt% and about 0.17 wt%, for example, between about 0.1 wt% and about 0.15 wt%.

[0131] Another preferred embodiment of the present invention relates to the methods described herein, wherein the concentration of cellulose nanofibers in the processed cellulose nanofiber material is about 0.125 wt%.

[0132] Yet another embodiment of the present invention relates to a method described herein, wherein the concentration of cellulose nanofibers in the processed cellulose nanofiber material is from about 0.25 wt% to about 2 wt% based on the total weight of the liquid sample, and the ratio of crosslinker to processed cellulose nanofiber material is in the range of from about 1:20 (vol% / wt%) to about 1:15 (vol% / wt%).

[0133] Further embodiments of the present invention relate to methods described herein, wherein the concentration of cellulose nanofibers in the processed cellulose nanofiber material is about 0.3 wt% to about 1 wt%, for example about 0.3 wt% to about 0.7 wt%, based on the total weight of the liquid sample, and the ratio of crosslinker to processed cellulose nanofiber material is in the range of about 1:20 (vol% / wt%) to about 1:15 (vol% / wt%), preferably about 1:17 (vol% / wt%).

[0134] A preferred embodiment of the present invention relates to the method described herein, wherein the concentration of cellulose nanofibers in the processed cellulose nanofiber material is about 0.05 wt % to about 0.2 wt %, for example about 0.1 wt % to about 0.15 wt %, based on the total weight of the liquid sample, and the ratio of crosslinker to processed cellulose nanofiber material is about 30×10 -3 mol% / mol% ~ approx. 50 x 10 -3 in the range of mol% / mol%.

[0135] Another preferred embodiment of the present invention relates to the method described herein, wherein the concentration of cellulose nanofibers in the processed cellulose nanofiber material is about 0.05 wt % to about 0.2 wt %, for example about 0.1 wt % to about 0.15 wt %, based on the total weight of the liquid sample, and the ratio of crosslinker to processed cellulose nanofiber material is 30×10 -3 mol% / mol% ~ approx. 50 x 10 -3 The nanofibrous cellulose scaffolds are discs with thicknesses ranging from about 5 mm to about 9 mm.

[0136] A further preferred embodiment of the present invention relates to the method described herein, wherein the concentration of cellulose nanofibers in the processed cellulose nanofiber material is about 0.125 wt % based on the total weight of the liquid sample, and the ratio of cross-linker to processed cellulose nanofiber material is about 30×10 -3 mol% / mol% ~ approx. 50 x 10 -3 The nanofibrous cellulose scaffolds are discs with a thickness of approximately 9 mm.

[0137] The average diameter of the cellulose nanofibers can be induced during the preparation of the initial cellulose nanofiber material. This can be achieved, for example, by varying the parameters of the electrospinning or meltblowing process, such as the applied voltage or heat, the rate and type of injection, and / or the rotation speed of the collector drum. The average diameter of the cellulose nanofibers can vary depending on the application, for example, the type of cells being cultured. In many applications, an average diameter of about 400 nm to about 600 nm, e.g., about 500 nm, has been found to be advantageous.

[0138] Thus, one embodiment of the present invention relates to a 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 thermally induced phase separation.

[0139] Another embodiment of the present invention relates to the method described herein, wherein the initial cellulose nanofiber material is prepared by electrospinning.

[0140] A further embodiment of the present invention relates to a method as described herein, wherein the average diameter of the cellulose nanofibers in the initial cellulose nanofiber material and / or the processed cellulose nanofiber material is in the range of from about 10 nm to about 2000 nm, such as from about 50 nm to about 1500 nm, for example from about 100 nm to about 1000 nm, preferably from about 250 nm to about 750 nm.

[0141] A preferred embodiment of the present invention relates to the method described herein, wherein the average diameter of the cellulose nanofibers in the initial cellulose nanofiber material and / or the processed cellulose nanofiber material is in the range of about 400 nm to about 600 nm, preferably about 500 nm.

[0142] Cellulose nanofibers may be combined with other types of nanofibers to enhance nanofiber-cellulose scaffolds with novel properties. This may be beneficial for specific cell types, expanding the options for mimicking the local extracellular matrix of the specific tissue in which the cells reside. Additional polymers can be of natural or synthetic origin. Natural nanofibers, particularly types traditionally present in the extracellular environment such as collagen, may work synergistically with cellulose to mimic the ECM. Additional polymers may also be introduced to modify the mechanical properties of the material.

[0143] Thus, one embodiment of the present invention relates to a method as described herein, wherein the initial cellulose nanofiber material further comprises one or more nanofibers selected from natural or synthetic polymers.

[0144] Another embodiment of the present invention relates to a method as described herein, wherein the natural polymer is selected from the group consisting of collagen, silk fibroin, keratin, gelatin and polysaccharides, and combinations thereof.

[0145] Further embodiments of the present invention relate to methods described herein, wherein the synthetic polymer is 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-vinyl acetate) (PEVA), and combinations thereof.

[0146] The initial cellulose nanofiber material can be prepared from a cellulose acetate solution, for example, by electrospinning the cellulose acetate solution. The resulting cellulose acetate sheet is preferably regenerated into a cellulose sheet in a sodium hydroxide bath before any further processing. This treatment opens the hydroxyl groups, which can then be used for the attachment of functional moieties.

[0147] Thus, one embodiment of the present invention relates to a method as described herein, wherein an initial cellulose nanofiber material is prepared by electrospinning a cellulose acetate solution into a cellulose acetate sheet.

[0148] Another embodiment of the present invention relates to a method as described herein, wherein cellulose acetate sheets are regenerated into cellulose sheets by treatment with NaOH.

[0149] Yet further embodiments of the present invention relate to methods described herein, wherein said initial cellulose nanofiber material is regenerated in a regeneration solution comprising sodium hydroxide.

[0150] Another embodiment of the present invention relates to a method as described herein, wherein the regeneration solution comprises sodium hydroxide in the range of about 0.1 M to about 1 M, such as about 0.2 M to about 0.8 M, for example, about 0.3 M to about 0.7 M, for example, about 0.4 M to about 0.6 M, preferably about 0.5 M.

[0151] The regeneration of the cellulose sheet is preferably carried out in an ethanol solution containing sodium hydroxide. Various amounts of ethanol can be used, such as 5% vol / vol to 99% vol / vol.

[0152] Accordingly, one embodiment of the present invention relates to a method described herein, wherein the regeneration solution is an ethanol solution comprising from about 5% vol / vol to about 99% vol / vol ethanol, such as from about 10% vol / vol to about 95% vol / vol ethanol, such as from about 20% vol / vol to about 90% vol / vol ethanol, such as from about 30% vol / vol to about 80% vol / vol ethanol, for example, from about 40% vol / vol to about 70% vol / vol ethanol.

[0153] The ethanol content in the regeneration solution may affect the mechanical properties of the resulting nanofibrous cellulose scaffold. Mechanical properties that may be affected by the ethanol content include elasticity and brittleness. Without being bound by theory, it is believed that a more elastic, less stiff material is beneficial for promoting cell interaction and proliferation.

[0154] Thus, one embodiment of the present invention relates to a 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, for example at least 70% vol / vol ethanol, such as at least 80% vol / vol ethanol, for example at least 90% vol / vol ethanol, preferably about 90% vol / vol ethanol.

[0155] Cellulose nanofibers can be functionalized with functional moieties to implant nanofiber scaffolds with novel properties. The functional moieties substituted onto the nanofiber cellulose scaffolds can include, for example, ECM proteins, peptides, and / or charged groups to enhance the level of cell attachment to the scaffold, promote cell differentiation, or aid in the release and isolation of cells from the scaffold. Functionalization of the cellulose nanofibers can be performed before or after crosslinking the cellulose nanofibers. Preferably, the functionalization step is performed before crosslinking.

[0156] Thus, one embodiment of the present invention relates to a method as described herein, further comprising the step of functionalizing the processed cellulose nanofiber strands by addition of a reagent comprising a functional moiety, said further step immediately preceding or following the cross-linking step (iii).

[0157] Another embodiment of the present invention relates to a method as described herein, further comprising the step of functionalizing the processed cellulose nanofiber strands by the addition of a reagent comprising a functional moiety, said further step preceding the cross-linking step (iii).

[0158] Following splitting of the initial cellulose nanofiber material, the resulting processed material is preferably treated to prepare it for functionalization. Part of the treatment can include filtering, washing, and resuspension of the processed cellulose nanofiber material. Filtration ensures that excess water is removed from the processed cellulose nanofiber material, while washing helps remove any acetate ions still present after previous treatment. Careful washing can help improve the texture of the cellulose. When resuspending the processed cellulose nanofiber material, the concentration of the cellulose nanofibers can be adjusted as needed.

[0159] Thus, one embodiment of the present invention relates to a method as described herein, wherein the dividing step (ii) is followed by steps including filtering, washing and suspending the processed cellulose nanofiber material.

[0160] Another embodiment of the present invention relates to a method as described herein, wherein said filtering comprises sieving the processed cellulose nanofiber material.

[0161] Yet another embodiment of the present invention relates to the methods described herein, wherein the suspension comprises water and / or ethanol.

[0162] 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 cellulose nanofibers are swollen by immersion in a concentrated aqueous NaOH solution, washed with water, and dried. During the mercerization process, the crystalline structure of the cellulose nanofibers is transformed from cellulose I to cellulose II. Under the action of the concentrated alkaline solution, chemical, physicochemical, and structural modifications of cellulose occur. After washing and neutralization, cellulose II is formed. As a result of base penetration into the lattice, internal hydrogen bonds are broken, increasing the number of available hydroxyl groups (-OH) in the cellulose nanofibers. Therefore, mercerization is believed to improve the degree of substitution (DS). The mercerization step can be performed before or after the splitting step.

[0163] Thus, one embodiment of the present invention relates to a method as described herein, further comprising the step of mercerizing said processed cellulose nanofiber material.

[0164] Another embodiment of the present invention relates to the process described herein, wherein the mercerization step immediately precedes or follows the dividing step (ii).

[0165] Yet another embodiment of the present invention relates to the method described herein, wherein said mercerization step comprises the addition of NaOH.

[0166] A further embodiment of the present invention relates to a method as described herein, wherein the concentration of NaOH ranges from about 0.05M to about 2M, such as from about 0.1M to about 1.5M, for example, from about 0.25M to about 0.75M, preferably from about 0.4M to about 0.6M.

[0167] A still further embodiment of the present invention relates to a method as described herein, wherein the mercerization step is carried out for a period of about 1 hour to about 3 hours, preferably about 2 hours.

[0168] A still further embodiment of the present invention relates to the methods described herein, wherein the degree of substitution (DS) of the functional moiety ranges from about 0.01 to about 2.

[0169] Another embodiment of the present invention relates to the method described herein, wherein the degree of substitution (DS) of the functional moiety 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, for example, about 0.4 to about 0.6.

[0170] The amount of substitution can also be quantified as equivalents of charge per elementary unit mass of cellulose (meq / g). For positively charged functional moieties such as QA and DEAE, a range of 0.5 to 3.5 meq / g has been found to be advantageous.

[0171] Thus, one embodiment of the present invention relates to the method described herein, wherein the functional moiety is QA and / or DEAE, and the charge equivalent per basic unit mass of cellulose on the nanofibrous cellulose scaffold is in the range of about 0.5 meq / g to about 3.5 meq / g, for example, in the range of 1 meq / g to about 2 meq / g, preferably in the range of about 1.25 meq / g to about 1.75 meq / g.

[0172] The functional moieties attached to the cellulose nanofibers can be of either chemical or biological origin. In particular, positively charged groups are advantageous because they induce electrostatic interactions between the nanofibrous cellulose scaffold and cells with negatively charged membranes, thereby increasing cell adhesion to the microcarriers. Biological moieties typically include proteins and peptides, which are essential for the interaction between cells and the extracellular environment. This interaction can further promote cell adhesion to the nanofibrous cellulose scaffold.

[0173] Thus, one embodiment of the present invention relates to a method as described herein, wherein the functional moiety is selected from a chemical moiety or a biological molecule.

[0174] Another embodiment of the present invention relates to a method described herein, wherein the chemical moiety is selected from the group consisting of a negatively charged group, a positively charged group, a zwitterionic group, a hydrophobic group, a hydrophilic group, an amphiphilic group, a ligand, and combinations thereof.

[0175] A further embodiment of the present invention relates to a method described herein, wherein the chemical moiety is selected from the group consisting of quaternary ammonium (QA), carboxymethyl (CM), diethylaminoethyl (DEAE), 4-(1-bromoethyl)benzoic acid, 4-(dimethylamino)pyridine, epoxide, and combinations thereof.

[0176] A preferred embodiment of the present invention relates to the methods described herein, wherein the chemical moiety is a quaternary ammonium (QA).

[0177] It should be understood that chemical moieties can be attached to the cellulose backbone using conventional chemical reactions. Thus, reagents such as, but not limited to, 3-chloro-2-hydroxypropyltrimethylammonium chloride (CHPTAC), 2-chloro-N,N-diethylethylamine hydrochloride (DAECH), and monochloroacetic acid (MCAA) can be used to attach QA, DEAE, and CM, respectively.

[0178] A still further embodiment of the present invention relates to a method as described herein, wherein the biological molecule is selected from the group consisting of a protein, a peptide, an antibody, an amino acid, a polypeptide, a glycoprotein, a lipoprotein, and an antigen, and combinations thereof.

[0179] After functionalization of the processed cellulose nanofiber scaffold, the material is dried to obtain the final nanofiber cellulose scaffold. Drying can be performed in two steps, such as freezing and subsequent freeze-drying, or in a single step, such as lyophilization. A freeze-dryer works by freezing the material and then reducing the pressure and applying heat to sublimate the frozen water in the material. If the water that keeps the processed cellulose nanofibers in solution is air-dried or heated, the slow evaporation of the water can cause the final nanofiber cellulose scaffold to collapse, resulting in a non-uniform and distorted material. Therefore, freeze-drying is preferred.

[0180] Thus, one embodiment of the present invention relates to a method as described herein, wherein the drying step (iv) comprises freezing and / or lyophilization of the processed cellulose nanofiber material.

[0181] Another embodiment of the present invention relates to the methods described herein, wherein the drying step (iv) comprises freezing the processed cellulose nanofiber material followed by freeze-drying the processed cellulose nanofiber material after freezing.

[0182] Drying may be followed by a curing step to harden the nanofiber scaffold to stabilize the crosslinks and improve mechanical stability. Curing accelerates the process by which crosslinking occurs.

[0183] Thus, one embodiment of the present invention relates to a method as described herein, wherein the drying step (iv) is followed by a step comprising hardening the processed cellulose nanofiber material after drying.

[0184] Another embodiment of the present invention relates to a method as described herein, wherein said curing is carried out at a temperature ranging from about 20°C to about 200°C, preferably from about 100°C to about 150°C.

[0185] A further embodiment of the present invention relates to a method as described herein, wherein said curing is carried out for a period of 2 to 4 hours.

[0186] Yet another embodiment of the present invention relates to the method described herein, wherein said curing is carried out at 120° C. for 3 hours.

[0187] In one variation of this 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 hardened as described above. Including a compression step facilitates the production of thinner materials, as it overcomes the challenge of overcoming surface tension to cover the surface of a mold with a very thin liquid layer; i.e., the nanofibrous cellulose scaffold may be initially molded at a greater thickness and then reduced in thickness by compression. This variation of the method may be used, for example, to produce thin disks of nanofibrous cellulose scaffold.

[0188] Thus, one embodiment of the present invention relates to a method as described herein, wherein the curing step is preceded by a compaction step comprising compacting the nanofibrous cellulose scaffold to reduce its thickness.

[0189] Once the nanofibrous cellulose scaffold is crosslinked and dried, the cellulose nanofibers are fixed into a specific shape. This shape depends on the container in which the solution of processed cellulose nanofiber material is held during the drying step. In principle, large quantities of nanofibrous cellulose scaffolds can be produced by selecting a large container, and the final dimensions / shape of the nanofibrous cellulose scaffold can then be determined by cutting or punching the desired shape.

[0190] Therefore, one embodiment of the present invention relates to a method as described herein, wherein the geometry of the sample is modified after the drying step (iv).

[0191] However, it may be more convenient and efficient to dry the processed cellulose nanofiber material directly in a forming vessel of the desired shape, which may be the cell culture device itself or a mold that generates the shape of the nanofiber cellulose scaffold that can be inserted into the cell culture device.

[0192] Therefore, a preferred embodiment of the present invention relates to the method described herein, wherein the processed cellulose nanofiber material is transferred to a forming vessel prior to drying step (iv).

[0193] Another embodiment of the present invention relates to a method as described herein, wherein the shaped vessel is selected from the group consisting of a mold, a cell culture plate, a bioreactor, a cell culture bottle, a spinner flask, a shaker flask, and a roller bottle, a Petri dish, and a tube, and is preferably a mold.

[0194] Yet another embodiment of the present invention relates to a method as described herein, wherein a mold is shaped to provide a casting of processed cellulose nanofiber material in a shape selected from the group consisting of a cylinder, a disk, a cube, a sheet, and a sphere.

[0195] In particular, it is preferred that the processed cellulose nanofiber material be formed into discs suitable for use in bioreactors.

[0196] Thus, a preferred embodiment of the present invention relates to the methods described herein, wherein the nanofibrous cellulose scaffold is a disc having a thickness in the range of about 3 mm to about 30 mm, such as about 3 mm to about 20 mm, for example about 3 mm to about 15 mm, preferably about 3 mm to about 10 mm.

[0197] A further embodiment of the present invention relates to a method as described herein, wherein the formed container is made from a material selected from the group consisting of metal, plastic, glass, ceramic, and composite materials, and combinations thereof.

[0198] The methods described herein provide nanofibrous cellulose scaffolds with high surface area and low dead volume that can be advantageously utilized as microcarriers for cell culture. Because the cellulose nanofibers are crosslinked, the nanofibrous cellulose scaffolds take the form of a dry, continuous material with sufficient mechanical strength for use as microcarriers in packed-bed bioreactors.

[0199] Thus, one aspect of the present invention relates to a nanofibrous cellulose scaffold obtainable from the method described herein.

[0200] Another aspect of the invention relates to a nanofibrous cellulose scaffold comprising an engineered cellulose nanofiber material having an average cellulose nanofiber length of less than about 250 μm, wherein the engineered cellulose nanofiber material is crosslinked with a crosslinking agent.

[0201] The cellulose nanofibers are connected in a three-dimensional network by a crosslinker, which may be any of the crosslinkers described herein, that provides mechanical strength to the microcarrier material.

[0202] Thus, one embodiment of the present invention relates to a nanofibrous cellulose scaffold as described herein, wherein the cellulose nanofibers of the processed cellulose nanofiber material are connected in a three-dimensional network by a cross-linking agent.

[0203] Splitting cellulose nanofibers significantly increases the surface area, resulting in a material with low dead volume. Without being bound by theory, it is believed that the shorter nanofiber strands may contribute to the mechanical strength of the nanofibrous cellulose scaffold, as shorter fiber strands generally appear stiffer than longer fiber strands. Functionalization as described herein can further provide the nanofibrous cellulose scaffold with desirable properties, such as a positive charge to induce cell attachment to the cellulose nanofibers. The functional moieties attached to the cellulose nanofibers can be as described herein.

[0204] Thus, one embodiment of the present invention relates to a nanofibrous cellulose scaffold as described herein, wherein the cellulose nanofibers have an average length in the range of about 30 μm to about 250 μm, such as about 40 μm to about 200 μm, for example about 50 μm to about 150 μm, preferably about 60 μm to about 100 μm.

[0205] Advantageous cross-linker to cellulose ratios have been identified to provide nanofibrous cellulose scaffolds that enhance cell proliferation. The selected cross-linker to processed cellulose nanofiber material ratio is advantageous in that it allows cells to move freely within the porous structure while providing sufficient mechanical strength to the microcarriers suitable for use in stirred bioreactors.

[0206] Thus, one embodiment of the present invention relates to a nanofibrous cellulose scaffold as described herein, wherein the ratio of crosslinker to processed cellulose nanofiber material is about 52×10 -3 mol% / mol% ~ approx. 175 x 10 -3 mol% / mol%, e.g., about 5×10 -3 mol% / mol% ~ approx. 100 x 10 -3 mol% / mol%, e.g., about 10×10 -3 mol% / mol% ~ approx. 80 x 10 -3 mol% / mol%, e.g., about 15×10 -3 mol% / mol% ~ approx. 50 x 10 -3mol% / mol%, e.g., about 25×10 -3 mol% / mol% ~ approx. 40 x 10 -3 in the range of mol% / mol%.

[0207] A preferred embodiment of the present invention relates to the nanofibrous cellulose scaffolds described herein, wherein the ratio of crosslinker to processed cellulose nanofiber material is about 30×10 -3 mol% / mol% ~ approx. 50 x 10 -3 in the range of mol% / mol%.

[0208] It should be understood that the mole % of the processed cellulose nanofiber material refers to the mole % per glucose unit in the cellulose material.

[0209] Another embodiment of the present invention relates to the nanofibrous cellulose scaffolds described herein, wherein the processed cellulose nanofiber material is functionalized with a functional moiety.

[0210] Nanofibrous cellulose scaffolds have the form of a dry, continuous material, which can be adapted to fit existing cell culture equipment, such as packed-bed bioreactors. The advantage of nanofibrous cellulose scaffolds is that they can be produced in any shape and size. This means that thicker discs, cylinders, or sheets of material can be formed, thereby eliminating the need to stack thin discs or sheets directly on top of each other to achieve the desired microcarrier volume. Stacking microcarrier entities on top of each other can cause variability in culture because the microcarrier homogeneity is lost at the interface between them. Overall, nanofibrous cellulose scaffolds can act as a homogenous microcarrier solution, offering a higher surface area and lower dead volume than existing solutions.

[0211] Thus, one embodiment of the present invention relates to a nanofibrous cellulose scaffold as described herein, wherein the nanofibrous cellulose scaffold is provided as a dry continuous material.

[0212] Another embodiment of the present invention relates to a nanofibrous cellulose scaffold as described herein, wherein the physical shape of the nanofibrous cellulose scaffold is a shape selected from the group consisting of a cylinder, a disk, a cube, a sheet, and a sphere.

[0213] A further embodiment of the present invention relates to a 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, for example at least about 0.3 mm, such as at least about 0.4 mm, for example at least about 0.5 mm.

[0214] A still further embodiment of the present invention relates to a nanofibrous cellulose scaffold as described herein, wherein the nanofibrous cellulose scaffold is a disc having a thickness ranging from about 0.2 mm to about 2 mm, such as from about 0.3 mm to about 1.5 mm, for example from about 0.4 mm to about 1.25 mm, preferably from about 0.5 mm to about 1 mm.

[0215] Thicker discs can be advantageous because they can better withstand shear forces within the bioreactor than smaller discs. Furthermore, thicker discs provide a large, continuous scaffold for cells to grow within without artifacts due to spaces at the interfaces of stacked discs.

[0216] Accordingly, one embodiment of the present invention relates to a nanofibrous cellulose scaffold as described herein, wherein the nanofibrous cellulose scaffold has a thickness in all three dimensions of at least about 3 mm, such as at least about 4 mm, for example at least about 5 mm, such as at least about 6 mm, for example at least about 7 mm, such as at least about 8 mm, for example at least about 9 mm, such as at least about 10 mm.

[0217] Another embodiment of the present invention relates to a nanofibrous cellulose scaffold as described herein, wherein the nanofibrous cellulose scaffold is a disc having a thickness ranging from about 3 mm to about 30 mm, such as from about 3 mm to about 20 mm, for example, from about 3 mm to about 15 mm, preferably from about 3 mm to about 10 mm.

[0218] A preferred embodiment of the present invention relates to the nanofibrous cellulose scaffolds described herein, wherein the nanofibrous cellulose scaffolds are discs having a thickness ranging from about 5 mm to about 9 mm.

[0219] Yet another embodiment of the present invention relates to a nanofibrous cellulose scaffold as described herein, wherein the nanofibrous cellulose scaffold is a disc having a diameter ranging from about 50 mm to about 500 mm, such as from about 100 mm to about 300 mm, for example, from about 150 mm to about 200 mm.

[0220] A preferred embodiment of the present invention relates to the nanofibrous cellulose scaffolds described herein, wherein the nanofibrous cellulose scaffolds are discs having a diameter ranging from about 40 mm to about 70 mm, for example, from about 50 mm to about 60 mm, preferably about 57 mm.

[0221] The discs can also have a smaller diameter and pack into the bed in a more randomly oriented manner within the bed.

[0222] Thus, one embodiment of the present invention relates to a nanofibrous cellulose scaffold as described herein, wherein the nanofibrous cellulose scaffold is a disc having a diameter in the range of about 2 mm to about 50 mm, such as about 2 mm to about 25 mm, for example about 2 mm to about 10 mm.

[0223] The flexibility of the production method means that nanofibrous cellulose scaffolds can be produced in any shape to fit existing or future cell culture devices. The material is microporous to allow cell migration and proliferation within the support matrix, yet elastic enough to withstand the shear forces in packed-bed bioreactors.

[0224] Thus, one embodiment of the present invention relates to a nanofibrous cellulose scaffold as described herein, wherein the nanofibrous cellulose scaffold is microporous.

[0225] A further embodiment of the present invention relates to a nanofibrous cellulose scaffold as described herein, wherein the nanofibrous scaffold is elastic.

[0226] The compressive strength of the nanofibrous scaffolds can be altered by adjusting the amount of cellulose nanofibers and cross-linking agent.

[0227] Accordingly, one embodiment of the present invention relates to a nanofibrous cellulosic 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.

[0228] Another embodiment of the present invention relates to a nanofibrous cellulose scaffold as described herein, wherein the nanofibrous scaffold has a compressive strength in the range of about 10 mm / N to about 40 mm / N, such as about 12 mm / N to about 35 mm / N, for example, about 15 mm / N to about 30 mm / N, such as about 15 mm / N to about 22 mm / N.

[0229] Yet another embodiment of the present invention relates to a nanofibrous cellulose scaffold as described herein, wherein the compressive strength is measured according to ISO 604:2002 - Plastics - Determination of compressive properties.

[0230] Nanofibrous cellulose scaffolds have a low density. Without being bound by theory, it is believed that the low density is caused by the homogeneous distribution of processed cellulose nanofibers in the material, which ensures that large entanglements and clusters of nanofibers are avoided. Thus, nanofibrous cellulose scaffolds have a significantly reduced density compared to the density of pure cellulose in sheet form, which is 1.5 g / cm. 3 Despite their low density, nanofibrous cellulose scaffolds offer excellent accessible surface area that is advantageous for the culture of adherent cells.

[0231] Thus, one embodiment of the present invention relates to a nanofibrous cellulose scaffold as described herein, wherein the nanofibrous cellulose scaffold has a pore size of about 0.0005 g / cm 3 ~about 0.5g / cm 3 , for example, about 0.001 g / cm 3 ~about 0.1g / cm 3 , for example, about 0.001 g / cm 3 ~Approx. 0.040g / cm 3 The density is in the range of

[0232] Another embodiment of the present invention relates to a nanofibrous cellulose scaffold as described herein, wherein the nanofibrous cellulose scaffold has a fiber densitometry of at least about 40,000 cm 2 / g, e.g., at least about 50,000 cm 2 / g, e.g., at least about 55,000 cm 2 / g, e.g., at least about 60,000 cm 2 / g, e.g., at least about 70,000 cm 2 / g, e.g., at least about 80,000 cm 2 / g BET surface area.

[0233] Another embodiment of the present invention relates to a nanofibrous cellulose scaffold as described herein, wherein the nanofibrous cellulose scaffold has a fiber densitometry of at least about 25,000 cm 2 / g, e.g., at least about 30,000 cm 2 / g, e.g., at least about 40,000 cm 2 / g, e.g., at least about 50,000 cm 2 / g, e.g., at least about 60,000 cm 2 / g, e.g., at least about 70,000 cm 2 / g BET surface area.

[0234] Yet another embodiment of the present invention relates to the nanofibrous cellulose scaffold as described herein, wherein the BET surface area is measured according to ISO 9277:2022 - Determination of the specific surface area of ​​solids by gas adsorption - BET method.

[0235] The properties of the nanofibrous cellulose scaffolds can be further modified by incorporating additional types of nanofibers into the cellulose nanofiber material, which can be any of the natural or synthetic polymers described herein.

[0236] Thus, one embodiment of the present invention relates to a nanofibrous cellulosic scaffold as described herein, wherein the nanofibrous scaffold further comprises one or more nanofibers selected from natural or synthetic polymers.

[0237] Despite the fact that bacterial, yeast, and insect cell expression systems are capable of overexpressing recombinant proteins, mammalian cell culture remains the foundation of biopharmaceutical biomanufacturing due to its ability to propagate human viruses, express monoclonal antibodies, and incorporate post-translational modifications such as glycosylation, which are essential for the production of effective biopharmaceuticals. Among the most utilized mammalian cell lines are human embryonic kidney (HEK) 293 cells, which have been engineered to produce high levels of therapeutic proteins and antibodies. However, HEK 293 cells and other adherent mammalian cells are highly dependent on an appropriate support matrix for cell growth and viability. One preferred system for culturing adherent mammalian cells is the packed-bed bioreactor. Therefore, microcarriers suitable for use with packed-bed bioreactors are key products that continue to make biomanufacturing more cost-effective.

[0238] The nanofibrous cellulose scaffolds described herein possess all the necessary properties (high surface area, low dead volume, sufficient mechanical strength, customizable) to be used as efficient microcarriers for loading in packed-bed bioreactors. Importantly, nanofibrous cellulose scaffolds can be easily scaled up for industrial applications when used in large quantities without compromising the cost and applicability of microcarriers.

[0239] Accordingly, one aspect of the present invention relates to a microcarrier comprising the nanofibrous cellulose scaffold described herein.

[0240] Another aspect of the present invention relates to the use of the nanofibrous cellulose scaffolds described herein as microcarriers for cell culture.

[0241] Yet another aspect of the present invention relates to a cell culture device comprising a vessel loaded with the nanofibrous cellulose scaffold or microcarriers described herein.

[0242] The container of the cell culture device is not limited to any particular container as long as it is a container suitable for culturing cells.

[0243] Thus, one embodiment of the present invention relates to a cell culture device as described herein, wherein the vessel is selected from the group consisting of a bioreactor, a cell culture plate, a cell culture bottle, a spinner flask, a shaker flask, a roller bottle, a Petri dish, and a tube, and is preferably a bioreactor.

[0244] Bioreactors are particularly important in large-scale industrial production. They may hold larger volumes than laboratory or pilot research equipment and are used in upstream processes to expand and scale up cell cultures for production. A typical process involves initial expansion of cells in smaller vessels, followed by continuous expansion into larger culture vessels. Once culture volume and density are optimized, the cells are transferred to a production bioreactor, which provides a controlled microenvironment and nutrient supply, controlling cell growth and differentiation and improving standardization and reproducibility. The nanofibrous cellulose scaffolds described herein are suitable for use at any point in this process and are particularly advantageous at production scales using packed-bed bioreactors, where other microcarriers may be too expensive or lack sufficient mechanical strength for viable commercial use and therefore unsuitable.

[0245] Thus, one embodiment of the present invention relates to a cell-cultivation device as described herein, wherein the vessel is a bioreactor, preferably a packed-bed bioreactor.

[0246] Packed-bed bioreactors fall into two main categories: one in which the bed is fixed within the reactor, commonly referred to as a fixed-bed bioreactor; and one in which the bed moves within the reactor, also referred to as a dynamic-bed or moving-bed bioreactor. In the latter type of bioreactor, the bed may be connected to one or more shafts that move within the bioreactor, such as by rotation. The nanofiber cellulose scaffold can be used with any type of packed-bed bioreactor.

[0247] Thus, one embodiment of the present invention relates to a cell-cultivation device as described herein, wherein the bioreactor is a fixed-bed bioreactor.

[0248] Another embodiment of the present invention relates to a cell-cultivation device as described herein, wherein the bioreactor is a dynamic bed bioreactor.

[0249] Many existing solutions employ stacked discs or sheets of microcarrier material. The stacked discs or sheets are spaced apart by support layers (see Figure 9A). The support layers may be plastic and are macroporous to allow the flow medium to pass through the packed bed. The nanofibrous cellulose scaffolds described herein may be used with these types of solutions and are advantageous in that they can form larger continuous discs or sheets, allowing the bed to be packed with more microcarrier material per volume. Among other things, this is achieved by requiring fewer support layers per volume (see Figure 9B).

[0250] Thus, one embodiment of the present invention relates to a cell culture device as described herein, wherein the vessel is loaded with a plurality of nanofibrous cellulose scaffold layers.

[0251] Another embodiment of the present invention relates to a cell culture device as described herein, wherein each nanofibrous cellulose scaffold layer is spaced apart by a support layer.

[0252] Yet another embodiment of the present invention relates to a cell culture device as described herein, wherein each nanofibrous cellulose scaffold layer is disposed between two support layers.

[0253] A further embodiment of the present invention relates to a cell-cultivating device as described herein, wherein the support layer is macroporous.

[0254] Yet another embodiment of the present invention relates to a cell-culture device as described herein, wherein the support layer is made from a material selected from the group consisting of plastic, metal ceramic, and composite materials, and combinations thereof.

[0255] Yet another embodiment of the present invention relates to a cell culture device as described herein, wherein the nanofibrous cellulose scaffold layers and support layers are arranged in an alternating stack.

[0256] Another embodiment of the present invention relates to a cell culture device as described herein, wherein the laminate is covered / terminated at each end by a support layer.

[0257] The stacked layers can be placed on another shaft within the bioreactor (see Figure 9C). In the case of a dynamic bed bioreactor, the shaft can move (e.g., by rotation) during cultivation.

[0258] The shape and dimensions of the nanofibrous cellulose scaffold can be as described herein. Nanofibrous cellulose scaffolds can be produced by the simple and cost-effective methods described herein, and are inexpensive materials in terms of surface area per volume compared to commercially available microcarriers. Therefore, nanofibrous cellulose scaffolds are easily scalable for industrial-scale production and can be used in large-volume bioreactors.

[0259] The nanofibrous cellulose scaffold is suitable for all applications, from pilot scale / product development (1 L-100 L) to production scale (100 L-1000 L). If necessary, this material can also be used as a microcarrier in even larger bioreactors.

[0260] Therefore, one embodiment of the present invention relates to a cell culture device as described herein, wherein the volume of the vessel is in the range of about 1 L to about 1000 L, for example, about 100 L to about 1000 L.

[0261] Thus, one embodiment of the present invention relates to a cell culture device according to item VV, wherein the volume of the vessel is at least 10 L, such as at least 50 L, for example at least 100 L, such as at least 200 L, for example at least 500 L, for example at least 1000 L.

[0262] The vessel is not limited to any particular material, but may be, for example, stainless steel or a disposable material, the latter providing flexibility and reducing downtime due to the need to clean and sterilize the culture equipment.

[0263] The nanofibrous cellulose scaffold can be utilized as a microcarrier in conventional cell culture methods. The nanofibrous cellulose scaffold can be provided as part of a cell culture device with a container loaded with microcarriers. The nanofibrous cellulose scaffold is believed to be suitable for use in culturing any cell line that may benefit from interaction with a support matrix during culture. It is understood that the nanofibrous cellulose scaffold can be used in any conventional scale-up process for cell culture, i.e., first adding microcarriers and cells to a small container (e.g., a flask), then transferring the cell population to a larger container, and finally transferring the cell culture to a bioreactor.

[0264] Thus, one aspect of the present invention relates to a method of culturing cells, comprising: (i) providing a cell culture device as described herein; (ii) adding a composition comprising a cell population to the cell culture device; (iii) incubating the cell population to provide an expanded cell population; and (iv) optionally, extracting the expanded cell population from the cell culture device.

[0265] One embodiment of the present invention relates to a method of culturing cells as described herein, wherein the composition comprises a solvent.

[0266] Another embodiment of the invention relates to a method of culturing cells as described herein, wherein the solvent comprises cell culture medium.

[0267] This cell culture method is particularly advantageous for culturing adherent cells, which grow while adhering to a culture vessel. Ideally, cultured cells are cultured in a manner that reflects the conditions in which they exist in vivo. Adherent cells are cells that adhere to tissues under natural conditions.

[0268] Thus, one embodiment of the present invention relates to a method of culturing cells as described herein, wherein the cell population comprises adherent cells.

[0269] Methods for culturing cells can be utilized to achieve many different end results, including, but not limited to, the production of vaccines, recombinant therapeutic molecules, or stem cells. Other growing areas that could benefit from the advantages of nanofibrous cellulose scaffolds include cellular agriculture production, such as the production of existing agricultural products like milk and (cultured) meat from cells. Cellular agriculture is considered a means to achieve animal-free agriculture. Therefore, it is contemplated herein that nanofibrous cellulose scaffolds can be used in cellular agriculture. Depending on the desired outcome, different types of cells and cell lines can be cultured.

[0270] Thus, one embodiment of the present invention relates to a method of culturing the cells described herein, wherein the origin of the cell population is selected from the group consisting of mammalian cells, human cells, yeast cells, bacterial cells, insect cells and plant cells, preferably mammalian cells.

[0271] Another embodiment of the invention relates to a method of culturing cells as described herein, wherein the cell population comprises a cell type selected from the group consisting of stem cells, kidney cells, lung cells, liver cells, pancreatic cells, cardiac cells, ovarian cells, hybridoma cells, and immortalized cells.

[0272] Yet another embodiment of the present invention relates to a method of culturing the cells described herein, wherein the cell population comprises a cell type selected from the group consisting of myocytes, myoblasts, and adipocytes.

[0273] For biomanufacturing, some cell lines are preferred due to properties such as ease of handling, ability to propagate human viruses, or implementation of preferred glycosylation patterns. One example is HEK293 cells, which can be used for packaging and amplifying recombinant adenoviruses.

[0274] Accordingly, one embodiment of the present invention relates to a method of culturing the cells described herein, wherein the cell population comprises a cell line selected from the group consisting of HEK293 cells, ARN8 cells, Vero cells, MRC-5 cells, WI-38 cells, CHO cells, BHK-21 cells, Sf9 cells, mesenchymal stem cells, pluripotent stem cells, and MDCK cells.

[0275] Further embodiments of the present invention relate to methods of culturing cells as described herein, wherein the cell population comprises stem cells.

[0276] Another embodiment of the invention relates to a method of culturing the cells described herein, wherein the cell population comprises a HEK293 cell line.

[0277] The method of culturing cells is preferably carried out while agitating the cell culture in the vessel to facilitate better transport of nutrients and oxygen to the cells. The method of culturing cells may be carried out in a packed-bed bioreactor. In this type of bioreactor, typical agitation means include, but are not limited to, agitation by an impeller and / or a circulation pump.

[0278] Thus, one embodiment of the present invention relates to a method of culturing cells as described herein, wherein the incubation is carried out under agitation.

[0279] Another embodiment of the present invention relates to the methods described herein, wherein the agitation is achieved by mechanical agitation, a circulation pump, and / or pneumatic means.

[0280] A further embodiment of the present invention relates to the methods described herein, wherein the agitating comprises stirring.

[0281] In particular in packed-bed bioreactors, agitation creates a flow of solvent within the bioreactor that continuously supplies fresh nutrients and oxygen to the cells attached to the microcarriers within the bed. The direction of the flow can vary depending on the type of bioreactor.

[0282] Thus, one embodiment of the present invention relates to a method as described herein, wherein there is a flow of solvent through the cell culture device.

[0283] Another embodiment of the present invention relates to the methods described herein, wherein the solvent flow is longitudinal and / or radial.

[0284] 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.

[0285] Preferences, options, and embodiments of a given aspect, feature, or parameter of the invention should be considered as disclosed in combination with any and all preferences, options, and embodiments of all other aspects, features, and parameters of the invention, unless the context dictates otherwise. This is particularly true for the description of methods for preparing a nanofibrous cellulose scaffold and all its features (which may be part of the nanofibrous cellulose scaffold itself), or uses or methods of using it for cell culture. Embodiments and features of the invention are also outlined in the following sections.

[0286] item X1. A method for preparing a nanofibrous cellulose scaffold, comprising: (i) providing an initial cellulose nanofiber material; (ii) dividing the initial cellulose nanofiber material into processed cellulose nanofiber material; (iii) cross-linking the processed cellulose nanofiber material; and (iv) drying the processed cellulose nanofiber material; Thereby, providing a nanofibrous cellulose scaffold. X2. The method according to item X1, wherein the processed cellulose nanofiber material is crosslinked by the addition of a crosslinking agent 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. X3. The method according to item X1 or X2, wherein the crosslinking agent is a polyamide epichlorohydrin resin. X4. The method according to any one of items X1 to X3, wherein the ratio of crosslinking agent to processed cellulose nanofiber material is in the range of about 1:34 (vol% / wt%) to about 1:1 (vol% / wt%), for example, about 1:30 (vol% / wt%) to about 1:5 (vol% / wt%), for example, about 1:25 (vol% / wt%) to about 1:10 (vol% / wt%), preferably about 1:20 (vol% / wt%) to about 1:15 (vol% / wt%), and more preferably about 1:17 (vol% / wt%). X5. The method according to any one of items X1 to X4, wherein the crosslinking step is carried out at a temperature in the range of about 15°C to about 25°C, preferably about 20°C. X6. The method according to any one of items X1 to X5, wherein the splitting of the initial cellulose nanofiber material is achieved by dispersion. X7. The method according to item X6, wherein the dispersion is carried out using a high-speed disperser. X8. The method according to item X6 or X7, wherein the dispersion is carried out for at least 2 minutes, such as at least 5 minutes, for example at least 10 minutes. X9. The method according to any one of items X6 to X8, wherein the dispersion is carried out at a speed in the range of about 10,000 rpm to about 30,000 rpm, for example, about 12,000 rpm to about 25,000 rpm, for example, about 15,000 rpm to about 20,000 rpm. X10. The method according to any one of items X1 to X9, wherein the average length of the cellulose nanofibers in the processed cellulose nanofiber material is shorter than the average length of the cellulose nanofibers in the initial cellulose nanofiber material. X11. The method according to any one of items X1 to X10, wherein the average length of the cellulose nanofibers in the processed cellulose nanofiber material is less than 250 μm, for example less than about 200 μm, for example less than about 150 μm, for example less than about 120 μm, for example less than about 100 μm, for example less than about 80 μm. X12. The method according to any one of Items X1 to X11, wherein the average length of the cellulose nanofibers in the processed cellulose nanofiber material is in the range of about 30 μm to about 250 μm, for example, about 40 μm to about 200 μm, for example, about 50 μm to about 150 μm, preferably about 60 μm to about 100 μm. X13. The method of any one of items X1 to X12, wherein the initial cellulose nanofiber material is provided as a liquid sample. X14. The method according to item X13, wherein the solvent of the liquid sample comprises water and / or ethanol. X15. The method according to any one of items X1 to X14, wherein the concentration of cellulose nanofibers in the processed cellulose nanofiber material is about 0.1 wt% to about 10 wt%, for example, about 0.15 wt% to about 8 wt%, for example, about 0.2 wt% to about 5 wt%, for example, about 0.2 wt% to about 3 wt%, for example, about 0.25 wt% to about 2 wt%, preferably about 0.3 wt% to about 1 wt%, based on the total weight of the liquid sample. X16. The method according to any one of Items X1 to X15, wherein the average diameter of the cellulose nanofibers in the initial cellulose nanofiber material and / or the processed cellulose nanofiber material is in the range of about 10 nm to about 2000 nm, for example, about 50 nm to about 1500 nm, for example, about 100 nm to about 1000 nm, preferably about 250 nm to about 750 nm. X17. The method of any one of items X1 to X16, wherein the initial cellulose nanofiber material further comprises one or more nanofibers selected from natural or synthetic polymers. X18. The method according to item X17, wherein the natural polymer is selected from the group consisting of collagen, silk fibroin, keratin, gelatin and polysaccharides, and combinations thereof. X19. The method according to item X17 or X18, wherein the synthetic polymer is 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-vinyl acetate) (PEVA), and combinations thereof. X20. The method of any one of items X1 to X19, 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 thermally induced phase separation. X21. The method according to any one of items X1 to X20, further comprising a step of functionalizing the processed cellulose nanofiber strands by adding a reagent comprising a functional moiety, said further step being immediately before or after the cross-linking step (iii). X22. The method according to item X21, wherein the functional moiety is selected from a chemical moiety or a biological molecule. X23. The method according to item X22, wherein the chemical moiety is selected from the group consisting of a negatively charged group, a positively charged group, a zwitterionic group, a hydrophobic group, a hydrophilic group, an amphiphilic group, a ligand, and combinations thereof. X24. The method according to item X22 or X23, wherein the chemical moiety is selected from the group consisting of quaternary ammonium (QA), carboxymethyl (CM), diethylaminoethyl (DEAE), 4-(1-bromoethyl)benzoic acid, 4-(dimethylamino)pyridine, epoxide, and combinations thereof. X25. The method of any one of items X22 to X24, wherein the chemical moiety is a quaternary ammonium (QA). X26. The method according to any one of items X22 to X25, wherein the biological molecule is selected from the group consisting of proteins, peptides, antibodies, amino acids, polypeptides, glycoproteins, lipoproteins, and antigens, and combinations thereof. X27. The method according to any one of items X21 to X26, wherein the degree of substitution (DS) of the functional moiety is in the range of about 0.01 to about 2. X28. The method according to any one of items X21 to X27, wherein the functionalization step is preceded by a step of mercerizing the processed cellulose nanofiber material. X29. The method according to item X28, wherein the mercerization step is immediately before or after the division step (ii). X30. The method according to item X28 or X29, wherein the mercerization step comprises the addition of NaOH. X31. The method according to item X30, wherein the concentration of NaOH is in the range of about 0.05M to about 2M, for example, about 0.1M to about 1.5M, for example, about 0.25M to about 0.75M, preferably about 0.4M to about 0.6M. X32. The method according to any one of items X28 to X31, wherein the mercerization step is carried out for a period of about 1 hour to about 3 hours, preferably about 2 hours. X33. The method according to any one of items X1 to X32, wherein the dividing step (ii) is followed by a step comprising filtering, washing and suspending the processed cellulose nanofiber material. X34. The method according to item X33, wherein the filtering comprises screening the processed cellulose nanofiber material. X35. The method according to item X33 or X34, wherein the suspension comprises water and / or ethanol. X36. The method according to any one of items X1 to X35, wherein the drying step (iv) comprises freezing and / or lyophilization of the processed cellulose nanofiber material. X37. The method according to any one of items X1 to X36, wherein the drying step (iv) comprises freezing the processed cellulose nanofiber material, followed by freeze-drying the frozen processed cellulose nanofiber material. X38. The method according to any one of items X1 to X37, wherein the drying step (iv) is followed by a step comprising hardening the processed cellulose nanofiber material after drying. X39. The method according to item X38, wherein the curing is carried out at a temperature ranging from about 20°C to about 200°C, preferably from about 100°C to about 150°C. X40. The method according to any one of items X38 or X39, wherein the curing is carried out for a period of 2 to 4 hours. X41. The method according to any one of items X38 to X40, wherein the curing is carried out at 120°C for 3 hours. X42. The method according to any one of items X1 to X41, wherein the processed cellulose nanofiber material is transferred to a forming vessel before the drying step (iv). X43. The method according to item X42, wherein the molded vessel is selected from the group consisting of a mold, a cell culture plate, a bioreactor, a cell culture bottle, a spinner flask, a shaker flask, and a roller bottle, a Petri dish, and a tube, and is preferably a mold. X44. The method according to item X43, wherein the mold is shaped to provide a casting of the processed cellulose nanofiber material in a shape selected from the group consisting of a cylinder, a disk, a cube, a sheet, and a sphere. X45. The method according to any one of items X42 to X44, wherein the molded container is made from a material selected from the group consisting of metal, plastic, glass, ceramic, and composite materials, and combinations thereof. Z1. A nanofibrous cellulose scaffold obtainable by the method described in items X1 to X45. Y1. A nanofibrous cellulose scaffold comprising an processed cellulose nanofiber material having an average cellulose nanofiber length of less than about 200 μm, wherein the processed cellulose nanofiber material is crosslinked with a crosslinking agent. Y2. The nanofibrous cellulose scaffold according to item Y1, wherein the cellulose nanofibers of the processed cellulose nanofiber material are connected in a three-dimensional network by a crosslinker. Y3. The nanofibrous cellulose scaffold of any one of paragraphs Y1 or Y2, wherein the crosslinker 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. Y4. The nanofibrous cellulose scaffold according to any one of items Y1 to Y3, wherein the crosslinker is a polyamide epichlorohydrin resin. Y5. A nanofibrous cellulose scaffold according to any one of items Y1 to Y4, wherein the ratio of crosslinker to processed cellulose nanofiber material is in the range of about 1:34 (vol% / wt%) to about 1:1 (vol% / wt%), for example, about 1:30 (vol% / wt%) to about 1:5 (vol% / wt%), for example, about 1:25 (vol% / wt%) to about 1:10 (vol% / wt%), preferably about 1:20 (vol% / wt%) to about 1:15 (vol% / wt%), and more preferably about 1:17 (vol% / wt%). Y6. The nanofibrous cellulose scaffold according to any one of items Y1 to Y5, wherein the cellulose nanofibers have an average length in the range of about 30 μm to about 250 μm, for example, about 40 μm to about 200 μm, for example, about 50 μm to about 150 μm, preferably about 60 μm to about 100 μm. Y7. The nanofibrous cellulose scaffold according to any one of items Y1 to Y6, wherein the cellulose nanofibers have an average diameter in the range of about 10 nm to about 2000 nm, for example, about 50 nm to about 1500 nm, for example, about 100 nm to about 1000 nm, preferably about 250 nm to about 750 nm. Y8. The nanofibrous cellulose scaffold of any one of paragraphs Y1 to Y7, wherein the cellulose nanofiber material is electrospun, meltblown or drawn, preferably electrospun. Y9. The nanofibrous cellulose scaffold of any one of paragraphs Y1-Y8, wherein the processed cellulose nanofiber material is functionalized with a functional moiety. Y10. The nanofibrous cellulose scaffold according to item Y9, wherein the functional moiety is selected from a chemical moiety or a biological molecule. Y11. The nanofibrous cellulose scaffold according to item Y10, wherein the chemical moiety is selected from the group consisting of a negatively charged group, a positively charged group, a zwitterionic group, a hydrophobic group, a hydrophilic group, an amphiphilic group, a ligand, and combinations thereof. Y12. The nanofibrous cellulose scaffold according to item Y10 or Y11, wherein the chemical moiety is selected from the group consisting of quaternary ammonium (QA), carboxymethyl (CM), diethylaminoethyl (DEAE), 4-(1-bromoethyl)benzoic acid, 4-(dimethylamino)pyridine, epoxide, and combinations thereof. Y13. The nanofibrous cellulose scaffold according to any one of paragraphs Y10 to Y12, wherein the chemical moiety is a quaternary ammonium (QA). Y14. The nanofibrous cellulose scaffold according to any one of items Y10 to Y13, wherein the biological molecule is selected from the group consisting of proteins, peptides, antibodies, amino acids, polypeptides, glycoproteins, lipoproteins, and antigens, and combinations thereof. Y15. The nanofibrous cellulose scaffold according to any one of paragraphs Y10 to Y14, wherein the degree of substitution (DS) of the functional moiety ranges from about 0.01 to about 2. Y16. The nanofibrous cellulose scaffold according to any one of paragraphs Y1 to Y15, wherein the nanofibrous cellulose scaffold is provided as a dry continuous material. Y17. The nanofibrous cellulose scaffold according to any one of paragraphs Y1 to Y16, wherein the physical shape of the nanofibrous cellulose scaffold is a shape selected from the group consisting of a cylinder, a disc, a cube, a sheet, and a sphere. Y18. The nanofibrous cellulose scaffold according to any one of paragraphs Y1 to Y17, 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, for example at least about 0.3 mm, such as at least about 0.4 mm, for example at least about 0.5 mm. Y19. The nanofibrous cellulose scaffold according to any one of items Y1 to Y18, wherein the nanofibrous cellulose scaffold is a disc having a diameter in the range of about 50 mm to about 500 mm, such as about 100 mm to about 300 mm, for example about 150 mm to about 200 mm. Y20. The nanofibrous cellulose scaffold according to any one of paragraphs Y1 to Y19, wherein the nanofibrous cellulose scaffold is provided as a freeze-dried material. Y21. The nanofibrous cellulose scaffold according to any one of paragraphs Y1 to Y20, wherein the nanofibrous scaffold is elastic. Y22. The nanofibrous cellulose scaffold has a cross section of at least about 40,000 cm 2 / g, e.g., at least about 50,000 cm 2 / g, e.g., at least about 55,000 cm 2 / g, e.g., at least about 60,000 cm 2 / g, e.g., at least about 70,000 cm 2 / g, e.g., at least about 80,000 cm 2 The nanofibrous cellulose scaffold according to any one of items Y1 to Y21, having a BET surface area of ​​1 / g. Y23. The nanofibrous cellulose scaffold according to any one of paragraphs Y1 to Y22, wherein the nanofibrous cellulose scaffold is microporous. Y24. Nanofiber cellulose scaffolds have a density of approximately 0.0005 g / cm 3 ~about 0.5g / cm 3 , for example, about 0.001 g / cm 3 ~about 0.1g / cm 3 , for example, about 0.001 g / cm 3 ~Approx. 0.040g / cm 3 The nanofibrous cellulose scaffold according to any one of items Y1 to Y23, having a density in the range of Y25. The nanofibrous cellulose scaffold according to any one of paragraphs Y1 to Y24, wherein the nanofibrous scaffold has a compressive strength in the range of about 0.1 mm / N to about 30 mm / N. Y26. The nanofibrous cellulosic scaffold according to any one of paragraphs Y1 to Y25, wherein the nanofibrous scaffold further comprises one or more nanofibers selected from natural or synthetic polymers. Y27. The nanofibrous cellulose scaffold according to item Y26, wherein the natural polymer is selected from the group consisting of collagen, silk fibroin, keratin, gelatin and polysaccharides, and combinations thereof. Y28. The nanofibrous cellulose scaffold according to any one of items Y26 or Y27, wherein the synthetic polymer is 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-vinyl acetate) (PEVA). A1. A microcarrier comprising a nanofibrous cellulose scaffold according to any one of items Y1 to Y28 or Z1. U1. Use of a nanofibrous cellulose scaffold according to items Y1 to Y28 or Z1 as a microcarrier for cell culture. V1. A cell culture device comprising a container loaded with the nanofiber cellulose scaffold according to any one of items Y1 to Y28 or Z1 or the microcarrier according to item A1. V2. The cell culture device according to item V1, wherein the vessel is selected from the group consisting of a bioreactor, a cell culture plate, a cell culture bottle, a spinner flask, a shaker flask, and a roller bottle, a Petri dish, and a tube, and is preferably a bioreactor. V3. The cell-cultivation device according to any one of paragraphs V1 or V2, wherein the vessel is a bioreactor, preferably a packed-bed bioreactor. V4. The cell-culture device of any one of paragraphs V2 or V3, wherein the bioreactor is a dynamic bed bioreactor. V5. The cell culture device according to any one of items V1 to V4, wherein the volume of the vessel 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. V6. The cell culture device of any one of items V1 to V5, wherein the container is loaded with a plurality of nanofibrous cellulose scaffold layers. V7. The cell culture device of item V6, wherein each nanofibrous cellulose scaffold layer is spaced apart by a support layer. V8. The cell-culture device of any one of paragraphs V6 or V7, wherein the support layer is macroporous. V9. The cell culture device according to any one of items V6 to V8, wherein the support layer is made from a material selected from the group consisting of plastic, metal ceramic, and composite materials, and combinations thereof. V10. The cell culture device according to any one of items V6 to V9, wherein the nanofiber cellulose scaffold layer and the support layer are arranged in an alternating stack. V11. The cell-cultivation device of item V10, wherein the stack is covered / terminated at each end by a support layer. V12. The cell culture device according to any one of items V6 to V11, wherein the physical shape of the nanofiber cellulose scaffold layer is a shape selected from the group consisting of a cylinder, a disk, a cube, a sheet, and a sphere, preferably a disk. V13. The cell culture device of any one of paragraphs V1 to V12, wherein the nanofibrous cellulose scaffold layer has a thickness in all three dimensions of at least about 0.1 mm, such as at least about 0.2 mm, for example at least about 0.3 mm, such as at least about 0.4 mm, for example at least about 0.5 mm. V14. The cell culture device according to any one of items V1 to V13, wherein the nanofiber cellulose scaffold layer is a disk having a diameter in the range of about 25 mm to about 500 mm, for example, about 50 mm to about 400 mm, for example, about 100 mm to about 300 mm, for example, about 150 mm to about 200 mm. V15. The cell culture device according to any one of items V1 to V14, wherein the cell culture device comprises a solvent. V16. The cell culture device according to item V15, wherein the solvent is a cell culture medium. T1. A method for culturing cells, comprising: (i) providing a cell culture device according to any one of items V1 to V16; (ii) adding a composition comprising a cell population to the cell culture device; (iii) incubating the cell population to provide an expanded cell population; and (iv) optionally extracting the expanded cell population from the cell culture device. T2. The method of item T1, wherein the composition comprises a solvent. T3. The method of item T2, wherein the solvent comprises cell culture medium. T4. The method of any one of paragraphs T1 to T3, wherein the cell population comprises adherent cells. T5. The method of any one of items T1 to T4, wherein the source of the cell population is selected from the group consisting of mammalian cells, human cells, yeast cells, bacterial cells, insect cells and plant cells, preferably mammalian cells. T6. The method of any one of paragraphs T1-T5, wherein the cell population comprises a cell type selected from the group consisting of stem cells, kidney cells, lung cells, liver cells, pancreatic cells, cardiac cells, ovarian cells, hybridoma cells, and immortalized cells. T7. The method of any one of paragraphs T1 to T6, wherein the cell population comprises a cell line selected from the group consisting of HEK293 cells, ARN8 cells, Vero cells, MRC-5 cells, WI-38 cells, CHO cells, BHK-21 cells, Sf9 cells, mesenchymal stem cells, pluripotent stem cells, and MDCK cells. T8. The method of any one of paragraphs T1 to T7, wherein the incubation is carried out under agitation. T9. The method according to item T8, wherein the stirring is performed by mechanical stirring, a circulation pump, and / or pneumatic means. T10. The method of any one of items T8 and T9, wherein the stirring includes stirring. T11. The method of any one of paragraphs T1-T10, wherein there is a flow of solvent through the cell culture device. T12. The method according to item T11, wherein the solvent flow is longitudinal and / or radial.

[0287] The invention will now be described in further detail in the following non-limiting examples. [Example]

[0288] Example 1: Preparation of nanofibrous cellulose scaffolds This example provides a non-limiting demonstration of how nanofibrous cellulose scaffolds can be prepared. Core properties of the material, such as surface area, degree of substitution, and compression profile, were characterized.

[0289] method Preparation of initial cellulose nanofiber materials A 19% cellulose acetate solution was prepared by adding cellulose acetate (6.38 g) to 12 ml of acetone in an Erlenmeyer flask, followed by 12 ml of DMF and 6 ml of 96% ethanol. The solution was stirred on a magnetic stirrer overnight at room temperature.

[0290] A 19% cellulose acetate solution was electrospun to prepare cellulose acetate nanofiber sheets. Cellulose nanofibers were electrospun (Fluidnatek LE50) at 18 kV+ (emitter) and 10 kV- (collector) with a drum speed of 200 rpm and a flow rate of 10 ml / hr. The cellulose acetate nanofibers were collected on an aluminum substrate at 23°C and 63% relative humidity. After the electrospinning process was completed, the cellulose acetate sheet containing the cellulose nanofibers was removed from the drum.

[0291] Cellulose acetate sheets were regenerated into cellulose by immersion in a 0.5 M NaOH solution in ethanol. After leaving the cellulose acetate sheets in the solution for 6 hours, they were transferred to a sieve and washed with a large amount of distilled water. The washed sheets were then dried in an oven at 80 °C for 12 hours to obtain dried cellulose sheets. The weight of the cellulose sheets was measured.

[0292] Processing of initial cellulose nanofiber materials The dried cellulose sheet was cut with scissors into coarse pieces approximately 2 x 2 cm square. The size of the pieces did not need to be precise, but large pieces should be avoided as they could interfere with the splitting process. The coarse cellulose pieces were completely immersed in water and dispersed using a high-speed disperser (IKA T25 Digital Ultra Turrax) at 18,000 rpm for 60 minutes (with intermittent stops to cool the disperser). The processed cellulose nanofiber material was transferred to a sieve, washed with water to remove residual acetate ions, and drained to remove excess water. The cellulose nanofiber material was transferred to a flask and fresh water was added to achieve a cellulose concentration of 2 wt%.

[0293] Samples with cellulose concentrations 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 apply equally to other cellulose concentrations.

[0294] Functionalization of engineered cellulose nanofiber materials The processed cellulose nanofiber material was functionalized with different chemical moieties according to the following process.

[0295] Quaternary Ammonium (QA): 0.4g of NaOH was dissolved in 20ml of water. Then, 0.4g of regenerated cellulose nanofiber material was added to the solution. Mercerization was continued for 2 hours at room temperature.

[0296] The temperature of the cellulose suspension was raised to 80°C, and 3.4 ml of 60% 3-chloro-2-hydroxypropyltrimethylammonium chloride (CHPTAC) was added dropwise. The reaction was continued at 80°C for 4 hours. Next, the reaction mixture was cooled to room temperature, filtered, and repeatedly washed with water to remove any unreacted CHPTAC or NaOH. Finally, the filtered processed and functionalized cellulose nanofiber material was mixed with water to prepare a 2% (or 0.5% or 1.5%) suspension again for further experiments.

[0297] Combining this functionalization protocol and cross-linking resulted in a nanofibrous cellulose scaffold called "Sponge QA."

[0298] Carboxymethyl (CM): 0.6 g of NaOH was dissolved in 25 ml of ethanol. Then, 0.5 g of regenerated cellulose nanofiber material was added to the solution. Mercerization was continued for 1 hour at room temperature.

[0299] The temperature of the cellulose suspension was raised to 60°C, and 0.14 g of monochloroacetic acid (MCAA) dissolved in 1.2 ml of ethanol was added dropwise. The reaction was continued for 2 hours at 60°C. Filtration and washing were carried out as in the QA functionalization.

[0300] Combining this functionalization protocol and cross-linking resulted in a nanofibrous cellulose scaffold called "sponge CMC."

[0301] Crosslinking of engineered rose nanofiber materials The processed cellulose nanofiber material was crosslinked using polyamide epichlorohydrin (Kymene GHP20 (Solenis), hereafter referred to as "Kymene") as a crosslinking agent. The processed cellulose nanofiber material was poured into distilled water, and a calculated amount of Kymene was added dropwise. The mixture was mechanically mixed at 18,000 rpm for 45 seconds to produce suspensions with various Kymene to cellulose ratios (1:34, 1:17, 1:10, 1:5, and 1:2 vol% / wt% Kymene:cellulose). The resulting suspensions were poured into molds of the desired dimensions. Examples of dimensions produced include heights of 60 mm, 30 mm, or 10 mm and an inner diameter of 19 mm.

[0302] Drying The mold containing the cellulose nanofiber material suspension (crosslinked material) was transferred to a freezer and left at -84°C for 24 hours. The mold containing the frozen cellulose nanofiber material was then transferred to a freeze-drying device and treated at a pressure of less than 0.001 bar and a temperature of -84°C for 24 hours to obtain a dry continuous material.

[0303] hardening The dry continuous material was removed from the mold and cured in an oven at 120°C for 3 hours.

[0304] The dry, continuous, and hardened material is also referred to herein as a "sponge."

[0305] Scanning electron microscopy Morphological studies of the nanofibrous cellulose scaffolds were performed using scanning electron microscopy (SEM). Samples were dried and sputter-coated with gold before analysis. Micrographs were acquired at various magnifications in secondary electron (SE) imaging mode on a Hitachi SU3500 microscope, with an accelerating voltage of 5 kV and a working distance of 7 mm.

[0306] FTIR spectroscopy FTIR analysis was performed from 450 to 3600 cm -1The analysis was performed on solid, dried samples at room temperature using a PerkinElmer Spectrum Two instrument equipped with a universal ATR in the range of 1000 Hz to 1000 Hz. Samples were dried at 80°C for 2 hours before analysis.

[0307] Surface area measurement The Brunauer-Emmett-Teller (BET) model is 0.162 nm 2 Nitrogen molecules with a molecular cross-section of 0.05 mm were used to measure the specific surface area. The dried samples were degassed under vacuum for 6 h before measuring the BET surface area. Nitrogen adsorption and desorption isotherms were acquired at 77.3 K on an ASAP 2020 M analyzer (Micromeritics).

[0308] Elemental analysis and degree of substitution Elemental analysis of the nanofibrous cellulose scaffolds was performed using a 5 mg sample on a Thermo Fischer Scientific FlashEA1112 elemental analyzer. The sample was thoroughly dried to remove adsorbed water before analysis.

[0309] The degree of substitution (DS) of cellulose functionalized with QA (i.e., sponge QA 2%) was calculated using the following equation: DS=(162N / (1400-cA×N)) where 162 is the molecular weight of an anhydroglucose unit (AGU), N is the nitrogen fraction, and CA is the molecular weight of the cationic reagent. In the nanofibrous cellulose scaffold of the present invention, the cationic reagent is CHPTAC, which has a molecular weight of 188.1.

[0310] Compression Analysis Compression analysis was performed on a Zwick testing machine (inv. no. BX32358) using a 50 N load cell (inv. no. BX32803) with a preload of 0.002 N and a test speed of 1 mm / min. Prior to analysis, the nanofibrous cellulose scaffolds were cut to a height of 15 mm.

[0311] Compressive modulus measures the stiffness of a material, or its ability to resist a change in length when subjected to a compressive load. The higher the compressive modulus, the stiffer the material.

[0312] result Nanofibrous cellulose scaffolds with crosslinkers and different types of functional moieties were prepared, and the dry continuous material was imaged by SEM (Figure 1A). The resulting nanofibrous cellulose scaffolds are highly homogeneous, in the sense that there are no large entanglements or clusters of nanofibers. The homogeneous distribution of nanofibers in the material ensures optimal exposure of surface area for cell adhesion and interaction. The crosslinker Kymene is seen to be trapped between the cellulose nanofibers (Figure 1A), imparting mechanical strength to the nanofibrous cellulose scaffolds.

[0313] To confirm the formation of bonds in the crosslinked cellulose, FTIR analysis was performed. -1 The band centered around 1743 cm was attributed to the stretching vibration of the carbonyl (C=O) group incorporated into the cellulose backbone after Kymene crosslinking (Figure 1B). Comparison of the FTIR spectra of cellulose acetate, regenerated cellulose, cellulose functionalized with QA, and crosslinked cellulose (Figure 1C) indicates that cellulose acetate was converted to crosslinked cellulose via the QA functionalization. The band at 1743 cm, which belongs to the carbonyl (C=O) group of cellulose acetate, was attributed to the stretching vibration of the carbonyl (C=O) group of cellulose acetate. -1 The disappearance of the absorbance band at 3345 cm, which is attributed to the hydroxyl groups (OH) of cellulose, -1 The appearance of a band at 865 cm, which is associated with the quaternary ammonium functional group, indicated the successful regeneration of cellulose acetate to form cellulose nanofibers. -1 The absorbance band at 1633 cm confirmed the successful formation of QA-functionalized cellulose. -1 The appearance of an additional absorbance band at 1000 nm indicated that cross-linking had occurred in the cellulose architecture.

[0314] The elemental composition and degree of substitution (DS) of a nanofibrous cellulose scaffold functionalized with QA (sponge QA 2%) were evaluated. The carbon (C) content was 37.71%, the hydrogen (H) content was 6.59%, and the nitrogen (N) content was 1.28%. The DS was calculated based on the N content to be 0.18. The amount of substituted QA may affect cell proliferation because its positive charges promote electrostatic interactions with negatively charged cell membranes.

[0315] Surface area measurements reveal a surprisingly large 60,000 cm 2 We demonstrated that a BET surface area of ​​1000 nm / g was obtained for a nanofibrous cellulose scaffold (Sponge QA 2%). Without being bound by theory, it is believed that the short cellulose strands result in a material with an increased degree of exposed surface. The large surface area is significantly greater than that of commercially available microcarriers currently on the market.

[0316] Successful crosslinking of cellulose nanofibers provided a material with the necessary mechanical strength to obtain a dry, continuous material that could be molded and shaped to fit the desired application (Figure 2A-B). This was true for all nanofibrous cellulose scaffolds produced using various amounts of cellulose and crosslinker.

[0317] Compression tests were used to evaluate the behavior of nanofibrous cellulose scaffolds under uniaxial compressive load. The change in length (or height), dL, was measured by applying a compressive load to the nanofibrous cellulose scaffold, and the behavior of the material was recorded (Figure 2C-D). The force required to produce a specific deformation (dL / Fmax) was 0.2 mm / N for the nanofibrous cellulose scaffold (sponge QA 2%, 1:10 vol% / wt% Kymene and cellulose). In particular, the force required to obtain 60% deformation of the material was measured to be 9.8 N.

[0318] Similar measurements were performed on α with a cellulose concentration of 0.5 wt% (sponge QA 0.5%, Kymene and cellulose at 1:10 vol% / wt%), with a dL / Fmax of 17 mm / N and a force of 0.4 N to obtain 60% deformation for the material.

[0319] Similar compression profile data were obtained with nanofibrous cellulose scaffolds with cellulose to Kymene ratios varying from 34:1 to 2:1.

[0320] The physical properties of nanofibrous cellulose scaffolds have surprisingly resulted in versatile materials that can return to their original shape after undergoing deformation. This resilient behavior is advantageous for molding and adapting nanofibrous cellulose scaffolds to existing and future cell culture platforms.

[0321] conclusion This example demonstrates that several different variants of nanofibrous cellulose scaffolds can be fabricated in a simple manner that is readily scalable for industrial applications. The nanofibrous cellulose scaffolds have a large surface area available to cells and do not contain collapsed or tangled nanofibers that prevent cells from accessing the surface.

[0322] This flexible material can be easily integrated into all current cell culture solutions, such as packed-bed bioreactors, from R&D to clinical manufacturing scale, allowing customers to maintain the same cell culture solution throughout process development (thus reducing risk and costs).

[0323] Example 2: Processing of initial cellulose nanofiber materials In this example, various methods of partitioning the initial cellulose nanofiber material were tested and their effect on the nanofibrous cellulose scaffold was evaluated.

[0324] method The preparation of the initial cellulose nanofiber material was carried out as described in Example 1.

[0325] Processing protocol Various methods for partitioning the initial cellulose nanofiber material were evaluated. Therefore, samples were prepared as follows.

[0326] Mechanical cutting (with scissors): 2.5 g of cellulose sheet was cut into 10 × 10 mm pieces with scissors and analyzed directly as fragmented cellulose sheet.

[0327] blend: 2.5 g of cellulose sheet was cut into 20 × 20 mm pieces with scissors and added to a laboratory mixer (LB20, Waring Laboratory) along with 250 ml of water. The cellulose nanofiber material was processed at 7000 rpm for different durations (0.5, 1, 2.5, 5, and 10 min).

[0328] Laser Cutting: The cellulose sheets were cut into three different sizes (0.75 × 0.75 mm, 1.5 × 1.5 mm, and 3 × 3 mm) using a laser cutter (Epilog laser, Zing24). The cut sheets were analyzed as they were.

[0329] Dispersion: 2.5 g of cellulose sheet was cut into 20 × 20 mm pieces with scissors and added to an Erlenmeyer flask with 250 ml of water. The samples were processed using a disperser (IKA T25 Digital Ultra-Turrax and S25 NB-25G disperser tool) at 18000 rpm for different durations (0.5, 1, 2.5, 5, 10, 15, and 60 min).

[0330] Microscopy A sample of nanofibrous cellulose scaffold (diluted to 0.2 wt% cellulose) was added to a glass slide. Images of the cellulose nanofiber network in the sample were captured with an optical microscope (Leica) using 40x magnification.

[0331] Scanning Electron Microscopy (SEM): Samples of the processed cellulose nanofiber material were diluted 1000-10000 times with water, and a droplet of the sample was applied to an SEM fixture. The sample was dried and sputter-coated with gold, after which images were captured using a Hitachi SU3500. Images were captured at different magnifications.

[0332] Fiber length measurement The fiber length of cellulose nanofibers was determined by SEM (Hitachi SU3500) or light scattering (Malvern Mastersizer S).

[0333] SEM images were manually evaluated by visual inspection to ensure that both ends of all measured fibers were visible. Fiber length was determined using ImageJ software. To obtain a larger data set, fiber length was determined from several SEM images.

[0334] Fiber length was also determined using light scattering. Briefly, 1 ml of sample was added to a Malvern Mastersizer S in water, and measurements were performed using the settings described under the definition of "mean length." Sample was added to the sample container until the obscuration value was between 15 and 20%. From each sample, a fiber length histogram displaying the fiber length distribution was generated. Distribution statistics were calculated from the results using an internationally agreed-upon method for defining the mean and other moments of the derived diameter D[m,n], i.e., 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 are smaller than this length and 50% are greater than this length. This value, when used for particles, is also known as the mass median diameter (MMD). D(v,0.1) is the fiber length at which 10% of the sample are less than this length. D(v,0.9) gives the fiber length below which 90% of the sample is. The volume-weighted average fiber length D[4,3] was also determined.

[0335] Pipetting / Floatation Test Samples of the processed cellulose nanofiber material were tested for their ability to be pipetted. 1 ml of sample was pipetted from the sample container and subsequently expelled through the pipette tip into a tube containing water. Ease of pipetting was assessed, including the tendency of the processed cellulose nanofiber material to clog the pipette.

[0336] The processed cellulose nanofiber material was then transferred to a tube containing water and evaluated for its tendency to float: the sample was shaken vigorously and visually inspected to determine whether the material floated immediately after shaking and after 24 hours.

[0337] result Pipetting tests of scissors-cut and laser-cut cellulose sheets showed that the cut fragments were too large for pipetting because they clogged the pipette tip. Furthermore, cellulose nanofiber materials processed in this way produce fragments large enough to trap air bubbles and float in solution (Figure 3A). Furthermore, laser cutting is not an appropriate technique for splitting cellulose nanofibers because the cellulose nanofibers melt, burn, and stick together (Figure 3B-C).

[0338] Cellulose nanofiber material processed in a blender (Figure 4A-E) produced a more densely entangled sample with less homogeneously distributed cellulose nanofibers than cellulose nanofiber material processed in a disperser (Figure 4F-J). Cellulose nanofiber material dispersed for at least 2.5 minutes appeared to be more homogeneously distributed throughout the sample, with fewer long cellulose nanofiber strands (Figure 4F-J). Therefore, dispersing for a minimum of 2 minutes may be advantageous to avoid excessive entanglement and clustering of cellulose nanofibers.

[0339] SEM images of the processed cellulose nanofiber materials confirmed that the samples processed by blending produced longer ( Figure 5A ) and more entangled ( Figure 5B–C ) cellulose nanofibers compared to the processed cellulose nanofiber materials processed by dispersion.

[0340] The dispersion time affected the average length of the cellulose nanofibers in the cellulose nanofiber material. Longer dispersion times resulted in shorter fiber lengths. Shorter fibers can be advantageous because they are less likely to form clusters and entanglements, thereby reducing the amount of inaccessible surface area and the risk of clogging. A summary of the determined average fiber lengths is shown in Table 1. [Table 1] Table 1. Fiber length measurements from a Malvern Mastersizer S.

[0341] SEM measurements were used to determine the diameter of the cellulose nanofibers (Figure 6A-B). Over 2,000 individual cellulose nanofibers were measured using ImageJ software, resulting in an average cellulose nanofiber diameter of 500 nm (Figure 6C).

[0342] None of the cellulose nanofiber materials treated by dispersing trapped air bubbles tended to float in solution. Pipetting of these samples was easiest for samples that had been dispersed for longer periods of time. Therefore, dispersing for at least 10 minutes was preferred to improve flow through the pipette.

[0343] conclusion This example demonstrates that not all methods for dividing the initial cellulose nanofiber material are equally effective and suitable for preparing nanofibrous cellulose scaffolds. In particular, dispersion is advantageous because it divides the cellulose nanofiber material homogeneously without creating entanglements or clusters.

[0344] Example 3: Cell viability on nanofibrous cellulose scaffolds In this example, the viability and adhesion of cells seeded onto nanofibrous cellulose scaffolds was tested.

[0345] method cell culture HEK293T cells were cultured at 75 cm at 37°C and 5% CO 2 The cells were maintained in T75 culture flasks and split approximately every 2–4 days when they reached 80–90% confluency. This was done until the day the cells were seeded onto microcarriers in 24-well plates. HEK293T cells were cultured in RPMI-1640 (culture medium) supplemented with 10% FBS and 1% penicillin / streptomycin throughout all experiments.

[0346] Nanofibrous cellulose scaffolds ("sponges") and reference cellulose sheets ("sheets") were tested as microcarriers in a 24-well plate setup according to Figure 7A. The cellulose sheets were manually cut to fit the wells. The nanofibrous cellulose scaffolds were molded to fit the wells. The microcarrier samples tested are listed in Table 2.

[0347] Plasma treatment was performed by placing the sample in a vacuum chamber (Diener Electronic ATTO Plasma) and adjusting the pressure to 0.15 mbar. Oxygen was pumped in to a pressure of 0.25 mbar, and the plasma generator was turned on at 30% power for 5 minutes. Air was introduced into the plasma chamber, and the sample was removed. [Table 2] Table 2. Samples for viability testing. Percentages correspond to the amount of cellulose nanofiber material used in the preparation of nanofibrous cellulose scaffolds.

[0348] 500 μl of microcarriers were loaded into the wells and pre-incubated with 1.75 ml of PBS at 37°C for 2 hours. Afterwards, the PBS was removed and 750 μl of culture medium was added to the wells. Next, 1 x 10 cells were cultured in 100 μl of culture medium. 6 HEK293T cells were seeded onto each microcarrier. After cell seeding, the 24-well plate was incubated overnight to allow cell attachment to the microcarriers.

[0349] After overnight incubation, 1 ml of culture medium was added to each well and the plates were incubated on a shaker (120 rpm) at 37°C and 5% CO. Half-medium changes were performed every morning and afternoon throughout the experiment to ensure adequate glucose / nutrient levels.

[0350] After 24 and 48 hours of incubation, each well was analyzed for cell attachment and viability using microscopy and a lactate dehydrogenase (LDA) cytotoxicity kit (Abcam). At 48 hours, 25 μl of trypan blue (TB) was added to the well, and the nanofiber inserts were studied microscopically from both the top and bottom.

[0351] Microscopy To analyze cell adhesion to the microcarriers, each well of the plate was examined microscopically (10x magnification). At 24 hours, the microcarriers were visualized from the bottom without TB. At 48 hours, the wells were first analyzed from the bottom, and 25 μl of TB was added to each well. The microcarriers were then disassembled to allow for examination of cell adhesion from the top of the microcarriers. For every well and time point, three images were captured.

[0352] Cell viability analysis To examine the viability of cells in microcarriers, an LDA cytotoxicity kit was used. Since LDA present in cells is released upon cell death, viability was determined from the amount of free LDA in the medium.

[0353] Briefly, 10 μl of supernatant from each well was transferred to a flat-bottom 96-well plate. Three technical replicates were performed per well. In addition, live and dead cell controls were used. Live cell controls were HEK293T cells cultured in a 12-well culture plate, and dead cell controls were generated by adding 10% lysis solution (from the LDA toxicity kit) to the wells of the 12-well plate and incubating for 30 minutes. After transferring the supernatant, 100 μl of LDA reaction mixture was added to each well of the assay plate. The plate was incubated at room temperature for 30 minutes, after which the absorbance was measured at 450 nm using a SpectraMax i3x. The absorbance measurement results were converted to the percentage of live cells using the following formula: 100 - (Test_ABS - Live CTRL_ABS) / (Dead CTRL_ABS - Live CTRL_ABS) x 100

[0354] result When examined under a microscope, cells adhered to all microcarriers (Figure 8A-B). Furthermore, no decrease in viability was observed for cells grown on any of the microcarriers (Figure 7B). In fact, viability increased slightly (100-105%) at 48 hours compared to control live cells cultured without microcarriers. This indicates that microcarriers improve viability when compared to traditional cell culture in two dimensions. Microcarriers with 0.33 wt% and 0.66 wt% cellulose, respectively, yielded similar results, i.e., they did not result in a decrease in viability (data not shown).

[0355] conclusion This example demonstrates that the viability of cells seeded on nanofibrous cellulose scaffolds is high, making them suitable for use as microcarriers.

[0356] Example 4: Processing of nanofibers of materials other than cellulose In this example, the processing of various nanofibers different from cellulose was evaluated with the aim of identifying suitable auxiliary nanofibers for the preparation of nanofiber scaffolds for growing cell cultures. Electrospun nanofibers were characterized by scanning electron microscopy (SEM) and visual inspection (after nanofiber sectioning).

[0357] method Electrospinning Nanofibers of polycaprolactone (PCL), poly-L-lactic acid (PLA), a blend of PCL and PLA (PLA / PCL), and cellulose were electrospun using the Fluidnatek LE50 apparatus described in Example 1.

[0358] PCL fibers were obtained by dissolving 8% polycaprolactone pellets (Sigma Aldrich, MW 80000) in a chloroform:methanol (1:1) solution. The needle-to-collector distance was set to 20 cm, the flow rate to 3 ml / h, and the voltage to 18 kV.

[0359] PLA fibers were obtained by dissolving 8% PLLA pellets (Goodfellow, MFR:24) in a chloroform:methanol (3:2) solution. The needle-to-collector distance was set to 24 cm, the flow rate to 4.5 ml / h, and the voltage to 35 kV.

[0360] PLA / PCL fibers were obtained by dissolving PLA pellets (Goodfellow, MFR: 65) and PCL pellets (Sigma Aldrich, MW 80000) in an 8% polymer solution of chloroform:methanol (3:2) at a ratio of 1:2. The distance between the needle and the collector was set to 20 cm, the flow rate to 3 ml / h, and the voltage to 18 kV.

[0361] For each fiber sheet, 1 ml of polymer solution was spun.

[0362] Scanning electron microscopy Electrospun materials consisting 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 unsplit and split nanofibers.

[0363] Processing of PCL, PLA, and PCL / PLA nanofiber materials Electrospun sheets of PCL, PLA, and blended PLA / PCL were cut into 10 × 10 mm pieces and mixed for 5 min using two different mixing tools: a 7000 rpm blender (LB20E laboratory blender, Waring) or an 18000 rpm disperser (IKA T25 Digital Ultra Turrax).

[0364] result Nanofibers could be electrospun from all materials (PCL, PLA, PLA / PCL, and cellulose) to form nanofiber sheets (see Figures 10A-D).

[0365] Sheets made from nanofibers other than cellulose were subjected to two distinct modes of nanofiber partitioning: blending (Figures 11A-F) and dispersion (Figures 12A-F). Tests showed that either PCL, PLA, or PLA / PCL nanofibers could not be uniformly blended or dispersed into a homogenous mixture of short-chain nanofibers.

[0366] Both the PCL nanofibers (Figures 11A-B and 12A-B) and the PLA / PCL nanofibers (Figures 11E-F and 12E-F) melted or deformed during processing. Specifically, the nanofibers melted together to form either large pieces of solid polymer or large entangled clusters of nanofibers and semi-molten nanofibers.

[0367] Although the PLA nanofibers (Figures 11C-D and Figures 12C-D) did not melt as well as the PCL fibers, all materials easily clogged the mixing tool or became entangled in the blender blades, halting processing.

[0368] conclusion This example demonstrates that nanofibers of all materials cannot be uniformly blended or dispersed. Therefore, not all nanofiber materials can be easily converted into shorter strands and subsequently formed into nanofiber scaffolds as described herein. Therefore, it is preferred to use cellulose nanofibers for the preparation of nanofiber scaffolds.

[0369] Example 5: Means for splitting initial cellulose nanofibers In this example, the effect of nanofiber splitting mode on cellulose nanofiber length was evaluated. Nanofibers were split either by blending or dispersion, and the lengths of representative nanofiber fractions were measured by individual analysis of SEM images or by laser diffraction in solution.

[0370] method QA-functionalized cellulose nanofibers were prepared for further testing. Cellulose acetate sheets were prepared by electrospinning as described in Example 1.

[0371] Cellulose regeneration Cellulose was obtained by regenerating (deacetylating) electrospun cellulose acetate sheets. Briefly, 25 g of cellulose acetate sheets were cut into smaller pieces (2 x 2 cm) and added to a beaker with 1.25 L of 0.5 M NaOH in 95% ethanol solution. The cellulose fibers were allowed to regenerate at room temperature for 24 h. After the reaction period, the regenerated cellulose nanofibers were filtered through a Buchner filter, immersed in 500 mL of dH2O for 1 min, and then filtered again. This process was repeated until the conductivity of the final washing step was 0 μS / cm, ensuring no residual NaOH or acetate remained. Finally, the nanofibers were dried overnight in an oven at 60 °C.

[0372] Cellulose nanofiber splitting A 1% cellulose suspension was prepared by adding 5 g of regenerated cellulose to 500 ml of distilled water. This solution was added to a disperser and mixed at 18,000 rpm (IKA T25 Digital Ultra Turrax) for various time points. 100 mL of labeled samples were removed after 1 minute (D1), 5 minutes (D5), 15 minutes (D15), and 60 minutes (D60) of mixing. This procedure was replicated in a blender, but the speed was set to 7,000 rpm (LB20E Laboratory Blender, Waring) (labeled samples B1, B5, B15, and B60). After mixing, the fibers were dried overnight in an oven at 60 °C. The particle size distribution of the nanofibers was analyzed by SEM and dynamic light scattering (DLS). SEM and DLS measurements were performed as described in Example 2. The length of individual cellulose nanofibers was estimated from SEM images (four 10 μL droplets at each time point). In the light scattering experiments, 10,000 fibers were measured per sample.

[0373] Functionalization of cellulose nanofibers Samples of blended or dispersed regenerated cellulose fibers at various time points were functionalized with quaternary ammonium salts (QAs). Briefly, 1 g of regenerated cellulose from each time point was resuspended in 50 ml of 1.5 M NaOH solution and mercerized for 2 hours with stirring at room temperature. After the mercerization step, the temperature was raised to 80 °C, and 2.5 ml of CHPTAC was added dropwise. The reaction continued at 80 °C for 4 hours. After the reaction, the mixture was filtered through a Buchner filter, immersed in 200 mL of dH2O for 1 minute, and then filtered again. This process was repeated until the conductivity of the final washing step was 0 μS / cm, ensuring no CHPTAC or NaOH remained. Finally, the filtered fibers were resuspended in water to a 2% solution and lyophilized for further experiments.

[0374] result From the SEM images (Figures 13A-H), it is clear that the disperser quickly provides a homogenous nanofiber population with relatively few long nanofibers and without any significant entanglements. While large clumps of uncut nanofibers are present in the sample after only 1 minute of splitting (Figures 13A-B), the disperser already presents a finer nanofiber population with only a few small clumps of entangled nanofibers after 5 minutes of dispersion (Figure 13D). After 15 and 60 minutes of dispersion, the presence of entangled nanofibers is almost completely eliminated (Figures 13F and 13H). In contrast, the blend sample contains large clumps of entangled fibers even after 60 minutes of blending (Figure 13G).

[0375] The data for each sample is summarized in Figures 14A-B. It is clear that the mixed samples had a higher frequency of long nanofibers compared to the dispersed samples. Furthermore, longer treatment times tended to produce fewer long nanofibers.

[0376] This relative trend is supported by measurements of the nanofibers using optical diffraction, and these data are summarized in Table 3. [Table 3] Table 3. Fiber length measurements of cellulose nanofibers prepared by blends (B1, B5, B15, B60) and dispersions (D1, D5, D15, D60). Measurements were performed on a Malvern Mastersizer S.

[0377] Overall, it is desirable to reduce the average length of cellulose nanofibers. However, it is also important that the nanofiber population does not contain many long nanofibers, as these can act as nuclei for larger clusters and entanglements of nanofibers. These clusters or entanglements of nanofibers are undesirable in nanofibrous scaffolds because they leave a large portion of the surface area inaccessible to cells seeded thereon. Furthermore, a heterogeneous nanofiber population containing clusters and entanglements is undesirable because it leads to inconsistent batch-to-batch variability in nanofibrous scaffolds and therefore unreliable cell growth.

[0378] conclusion This example demonstrates that the means of fractionating cellulose nanofiber material affects the nanofiber size distribution, particularly the proportion of longer fibers. Dispersing the nanofibers is preferred because it rapidly removes the longer fibers and results in the most homogeneous population of cellulose nanofibers.

[0379] Example 6: Nanofibrous cellulose scaffolds containing various amounts of cellulose and crosslinker In this example, the effect of cellulose concentration and cross-linker to cellulose ratio on nanofibrous cellulose scaffolds was evaluated through scaffold integrity and cell growth. A summary of the prepared samples is shown in Table 4. [Table 4] Table 4. Cellulose concentrations and crosslinker to cellulose ratios used in the preparation of nanofibrous cellulose scaffolds. The crosslinker to cellulose ratios are given as both vol% / wt% and mol% / mol%. The mole% of cellulose material corresponds per glucose unit. Therefore, x 10 -3 Mol% / mol% is a measure of the number of cross-linked molecules per 1000 glucose units.

[0380] method Preparation of nanofibrous cellulose scaffolds The first series of nanofibrous cellulose scaffolds was prepared to find the optimal porosity range for the scaffold, i.e., pores large enough to allow cells to penetrate the scaffold and access a large surface area. The scaffolds were produced by crosslinking various cellulose concentrations with a fixed amount of crosslinker. The concentrations were varied as follows, following the procedure described in Example 1:

[0381] Nanofibrous cellulose scaffolds with different porosities were prepared by crosslinking functionalized cellulose with various concentrations of QA (2.0, 1.0, 0.5, 0.25, 0.125, 0.06, 0.03, and 0.015% cellulose). A cellulose suspension containing 0.5 μl of crosslinker (Kymene) / ml was added to each concentration of functionalized cellulose. Subsequently, 500 μl of the crosslinker-mixed suspension was added to a 48-well mold (maximum fill volume 0.5 ml, diameter 12 mm, height 10 mm). The mold was frozen at -80°C and then lyophilized. Finally, the scaffolds were cured in an oven at 120°C for 3 hours on an aluminum tray.

[0382] A second series of nanofibrous cellulose scaffolds was prepared to evaluate the ratio of crosslinker to cellulose for a subset of cellulose concentrations (0.06%, 0.125%, and 0.25%) that were deemed beneficial. The amount of crosslinker (Kymene) added to the cellulose suspension was low (3.8 × 10 -3 mol% / mol%), medium (19.0 × 10 -3 mol% / mol%, or high (94.9 × 10-3 Samples were prepared with a cross-linker to cellulose ratio of 0.04, 0.2, and 1 μl per mg of cellulose material (see Table 4). Low, medium, and high samples corresponded to the addition of 0.04, 0.2, and 1 μl of cross-linker per mg of cellulose material, respectively.

[0383] Cell growth on nanofibrous cellulose scaffolds Cell growth experiments on nanofibrous cellulose scaffolds were performed in a 24-well plate format using HEK293T cells (ATCC CTRL-3126) at passage 8 for the first series (92% viability on the day of scaffold inoculation) or passage 10 for the second series (97.5% viability on the day of scaffold inoculation).

[0384] The scaffolds were washed three times with DPBS. One cellulose scaffold was placed in each well of a 24-well plate containing 1 ml of cell culture medium (DMEM with high glucose, GlutaMAX supplement, and pyruvate, 10% heat-inactivated FBS, and 1% penicillin-streptomycin). 300,000 cells were seeded onto each scaffold and placed in an incubator (37°C, 5% CO2) on an orbital shaker (16 mm orbital) at 35 rpm overnight.

[0385] The following morning, 0.5 ml of medium was added to each well to reach a final volume of 1.5 ml per well. The rpm of the orbital shaker (16 mm orbital) was increased to 65 rpm. At 24, 48, and 72 hours after seeding, three replicate scaffolds were transferred with tweezers to 15 ml tubes, and cell density was assessed using enzymatic cell dissociation in combination with the microcarrier program on a NucleoCounter (NC-202). The resulting mean values, corresponding to the total cell concentration (within one well) per scaffold, were determined and captured as histograms.

[0386] Fluorescence microscopy of DAPI-stained, optically clear samples was performed at 72 hours using a confocal microscope.

[0387] result Nanofibrous cellulose scaffolds were prepared from cellulose concentrations ranging from 0.015% to 2% (Figure 15, top). There was a clear tendency for scaffolds with lower cellulose concentrations (higher porosity) to lose shape.

[0388] Cell growth was particularly enhanced on scaffolds prepared from cellulose concentrations ranging from 0.03% to 0.125% (Figure 15, center). In particular, scaffolds prepared from a 0.125% cellulose concentration showed increased growth and low variability between replicates. Both the 0.015% and 0.03% samples were difficult to handle due to their light weight and fragility, leading to a tendency to fracture. Samples prepared from 1% and 2% cellulose concentrations exhibited a denser structure that appeared to primarily support cell growth on the exterior surface, as infiltration into the scaffold was hindered by the small pore size. This observation was supported by cell staining (Figure 15, bottom), which confirmed the observation that limited cell infiltration occurred for the 1% and 2% samples.

[0389] For nanofibrous cellulose scaffolds with varying amounts of crosslinker, scaffolds prepared from 0.06% and 0.125% cellulose concentrations allowed higher cell growth than scaffolds prepared from 0.25% cellulose concentration (Figure 16). This confirmed previous findings (Figure 15, center). Without being bound by theory, this difference may be due to the higher porosity of scaffolds prepared from lower cellulose concentrations. The data also showed that 94.9 x 10 -3 It has been shown that a crosslinker to cellulose ratio of less than mol% / mol% results in enhanced cell growth, for example when the scaffold is highly porous.

[0390] conclusion This example demonstrates that nanofibrous cellulose scaffolds can be prepared from various cellulose concentrations. The cellulose concentration changes the porosity of the scaffold, and cellulose concentrations in the range of 0.03% to 0.125% are preferred due to their ability to support cell proliferation by promoting increased cell penetration into the pores. Furthermore, 94.9 x 10 -3It is advantageous to crosslink the cellulose nanofibers at a ratio of crosslinker to cellulose of less than mol % / mol %.

[0391] Example 7: Mechanical stability of nanofibrous cellulose scaffolds In this example, the mechanical stability of nanofibrous cellulose scaffolds was investigated with the aim of assessing their suitability in industrial setups involving shear stress due to agitation.

[0392] method Nanofibrous cellulose scaffold discs were produced in a larger format, 57 mm in diameter, to fit in the bioreactor compared to the scaffolds produced in Example 6 (smaller diameter of 12 mm). Stability was tested based on the ability of the discs to be removed from the mold without breaking and their ability to withstand the shear stresses applied when agitated in a mock-up reactor (Figure 18A).

[0393] Nanofibrous cellulose scaffold discs with different porosities were prepared by crosslinking functionalized cellulose with various concentrations of QA (0.25%, 0.125%, 0.06%, and 0.03%) as described in Example 6. Each concentration of functionalized cellulose was crosslinked with 0.2 μl of crosslinker (19.0 × 10) per mg of cellulose. -3 % / mol%) was added. Scaffolds of different thicknesses were obtained by filling the molds with different volumes (3, 10, 17, and 25 ml) of the cellulose suspension. The molds had an inner diameter of 57 mm, a maximum filling 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 then freeze-dried. Finally, the scaffolds were cured on aluminum trays in an oven at 120 °C for 3 hours.

[0394] The mechanical stability of each scaffold was tested in a mock-up bioreactor. Each scaffold was placed between two perforated support disks attached to a stirrer (Figure 18A, left). The stirrer was attached to a Nano Star 7.5 digital stirrer (IKA) and immersed in a beaker containing 500 ml of PBS to mimic the rotational motion of a stirred bioreactor (Figure 18A, center). The stirring speed was set to 50 rpm, and stability over time was monitored. Figure 18A (right) shows a scaffold (sample IIB) that was destroyed by the shear force applied during rotation.

[0395] result Using this methodology, a wide range of nanofibrous cellulose scaffolds were prepared, varying in thickness and density / porosity (Figures 17(I)-(IV)). From visual inspection, it was apparent that scaffolds prepared from the highest cellulose concentrations, e.g., 0.25% (Figure 17(IV)) and 0.125% (Figure 17(III)), produced the most coherent discs. A summary of the measured thicknesses of the scaffolds is shown in Table 5. [Table 5] Table 5. Samples prepared for testing mechanical stability.

[0396] Scaffolds prepared from cellulose concentrations below 0.125% tended to collapse upon freeze-drying, resulting in reduced thickness compared to denser scaffolds. The thinnest scaffolds, made from suspensions with volumes of only 3 ml, were impossible to remove from the mold without breaking. With higher suspension volumes, the resulting thicker scaffolds were easier to handle.

[0397] Tests in mock-up bioreactors revealed that only scaffolds prepared from cellulose concentrations of 0.013% and (17 ml (portion), 25 ml) and 0.25% (10 ml, 17 ml, and 25 ml) were able to withstand the shear stress of the mock-up bioreactor.

[0398] conclusion This example demonstrates that it is possible to produce nanofibrous cellulose scaffolds with sufficient mechanical strength to resist fracture when used as microcarriers in a stirred bioreactor. Scaffolds are preferably prepared from a cellulose concentration of at least 0.125% and can advantageously have thicknesses ranging from 6 mm to 10 mm. Higher density scaffolds can be prepared at 3 mm thickness and still maintain structural integrity in a stirred bioreactor.

[0399] Example 8: Cell growth on nanofibrous cellulose scaffolds in a commercial bioreactor In this example, nanofibrous cellulose scaffolds are tested in a commercial bioreactor to evaluate their suitability as microcarriers.

[0400] method Pilot nanofibrous cellulose scaffolds were prepared in the form of discs by crosslinking a 1% suspension of QA-functionalized cellulose by adding 1.0 μl of crosslinker per ml of cellulose suspension, as described in Example 7. 10 ml of the suspension was added to a mold with inner dimensions of 57 mm and a maximum fill height of 10 mm. The mold was frozen at -80°C and then freeze-dried. Finally, the scaffolds were cured in an oven at 120°C for 3 hours on an aluminum tray. The scaffolds were approximately 3 mm thick, and each disc weighed 100 mg.

[0401] Approximately 1m 2 Three disks made from nanofiber cellulose scaffolds, each corresponding to a surface area of ​​100 μg, were placed in a 1 L CellBRX bioreactor. 300 million HEK293 cells were then seeded into the bioreactor and allowed to attach to the scaffolds. Cells were grown for 5 days, with cell number and glucose consumption monitored daily.

[0402] result Cells attached and grew on the nanofiber cellulose scaffold discs in the 1 L CellBRX bioreactor system, as indicated by an increase in cell density over time and a concomitant consumption of glucose (Figure 19).

[0403] conclusion This example demonstrates that nanofibrous cellulose scaffolds are suitable for use in bioreactors in industrial setups for culturing large populations of cells.

Claims

1. 1. A method for preparing a nanofibrous cellulose scaffold, comprising: (i) providing an initial cellulose nanofiber material; (ii) dividing the initial cellulose nanofiber material into processed cellulose nanofiber materials; (iii) cross-linking the processed cellulose nanofiber material; and (iv) drying the processed cellulose nanofiber material; The division is achieved by dispersion, Thereby providing said nanofibrous cellulose scaffold.

2. The method of claim 1 , wherein the dispersion is carried out using a high speed disperser.

3. 3. The method according to claim 1 or 2, wherein the dispersion is carried out for at least 2 minutes, such as at least 5 minutes, for example at least 10 minutes, such as at least 15 minutes, for example at least 20 minutes, such as at least 30 minutes, for example at least 40 minutes, such as at least 50 minutes, for example at least 60 minutes.

4. 4. The method of any one of claims 1 to 3, wherein the average length of the cellulose nanofibers in the processed cellulose nanofiber material is in the range of about 70 μm to about 120 μm.

5. 5. The method of any one of claims 1 to 4, wherein the processed cellulose nanofiber material is crosslinked by the addition of a crosslinking agent 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.

6. 6. The method of claim 5, wherein the crosslinking agent is an epichlorohydrin, such as polyamide epichlorohydrin.

7. The ratio of cross-linking agent to processed cellulose nanofiber material was approximately 15 × 10 －３ mol% / mol% to about 80×10 －３ mol% / mol%, for example, about 25×10 －３ mol% / mol% to about 70×10 －３ mol% / mol%, for example, about 30×10 －３ mol% / mol% to about 50×10 －３ mol% / mol%, for example, about 35×10 －３ mol% / mol% to about 40×10 －３ 7. The method of any one of claims 1 to 6, wherein the range of mol% / mol% is:

8. 8. The method of any one of claims 1 to 7, wherein the concentration of cellulose nanofibers in the processed cellulose nanofiber material is from about 0.01 wt% to about 1 wt%, such as from about 0.02 wt% to about 0.5 wt%, for example from about 0.03 wt% to about 0.2 wt%, based on the total weight of the liquid sample.

9. 9. The method of any one of claims 1 to 8, wherein the initial cellulose nanofiber material is prepared by electrospinning, meltblowing, drawing, self-assembly, template synthesis, and thermally induced phase separation.

10. The method of any one of claims 1 to 9, wherein the drying step (iv) comprises freezing and / or lyophilization of the processed cellulose nanofiber material.

11. 11. The method of any one of claims 1 to 10, wherein the drying step (iv) is followed by a step comprising hardening the dried processed cellulose nanofiber material.

12. 12. The method of any one of claims 1 to 11, further comprising the step of functionalizing the processed cellulose nanofiber strands by the addition of a reagent comprising a functional moiety, said further step immediately preceding or following the cross-linking step (iii).

13. A nanofibrous cellulose scaffold obtainable by the method according to any one of claims 1 to 12.

14. 1. A nanofibrous cellulose scaffold comprising a processed cellulose nanofiber material having an average cellulose nanofiber length of less than about 250 μm, wherein the processed cellulose nanofiber material is crosslinked with a crosslinking agent.

15. The ratio of cross-linking agent to processed cellulose nanofiber material was approximately 15 × 10 －３ mol% / mol% to about 80×10 －３ mol% / mol%, for example, about 25×10 －３ mol% / mol% to about 70×10 －３ mol% / mol%, for example, about 30×10 －３ mol% / mol% to about 50×10 －３ mol% / mol%, for example, about 35×10 －３ mol% / mol% to about 40×10 －３ 15. The nanofibrous cellulose scaffold of claim 13 or 14, wherein the cellulose content is in the range of mol% / mol%.

16. The nanofibrous cellulose scaffold of any one of claims 13 to 15, wherein the processed cellulose nanofiber material is functionalized with a functional moiety.

17. The nanofibrous cellulose scaffold of any one of claims 13 to 16, wherein the nanofibrous cellulose scaffold is provided as a dry continuous material.

18. 18. The nanofibrous cellulose scaffold according to any one of claims 13 to 17, wherein the nanofibrous cellulose scaffold is a disc having a thickness in the range of about 3 mm to about 30 mm, such as about 3 mm to about 20 mm, for example about 3 mm to about 15 mm, preferably about 3 mm to about 10 mm.

19. The nanofibrous cellulose scaffold has a thickness of at least about 40,000 cm ２ / g, e.g., at least about 50,000 cm ２ / g, e.g., at least about 55,000 cm ２ / g, e.g., at least about 60,000 cm ２ / g, e.g., at least about 70,000 cm ２ / g, e.g., at least about 80,000 cm ２ The nanofibrous cellulose scaffold according to any one of claims 13 to 18, having a BET surface area of ​​1 / g.

20. A microcarrier comprising the nanofibrous cellulose scaffold of any one of claims 13 to 19.

21. A cell culture device comprising a container loaded with the nanofibrous cellulose scaffold according to any one of claims 13 to 19 or the microcarrier according to claim 20.

22. 1. A method for culturing cells, comprising: (i) providing a cell culture device according to claim 21; (ii) adding a composition comprising a cell population to the cell culture device; (iii) incubating the cell population to provide an expanded cell population; and (iv) optionally extracting the expanded cell population from the cell culture device.

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