Nanofiber scaffolds for cell culture

Abstract

The present invention relates to a method for preparing nanofibrous scaffolds and their use for promoting cell culture. In particular, the nanofibrous scaffolds contain functionalized cellulose nanofibers that significantly improve expansion capacity and protein production. The method includes a step of splitting initial cellulose nanofibers by dispersion.

Description

[Technical Field]

[0001] The present invention relates to a method for preparing a nanofibrous scaffold and its use for promoting cell culture. In particular, the nanofibrous scaffold comprises functionalized cellulose nanofibers that significantly improve expansion capacity and protein production. [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 in various fields. Most complex biological products with specific glycosylation patterns can be produced by recombinant DNA technology in mammalian cell culture and include enzymes, synthetic hormones, and monoclonal antibodies. However, biopharmaceutical manufacturing is challenging because the 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] 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 flat surface, greatly increasing the capacity to accommodate more cells in a limited volume. The most commonly used commercially available microcarriers are solid spherical particles that retain cells on their surface. This approach has increased production yields because cell growth is highly correlated with the surface area available for cell attachment.

[0006] Suspension-based bioreactors are preferred in many practical applications because they allow for the addition of more microcarriers and medium during the culture process, providing process flexibility and control. In suspension-based bioreactors, microcarriers are freely suspended within the bioreactor, but provide the support matrix necessary for adherent cells to attach and grow. Important parameters for microcarriers include the available surface area they provide and the dead volume they occupy.

[0007] Typically, suspension-based bioreactors are equipped with agitation means to ensure that nutrients and gases, such as oxygen, are efficiently distributed within the bioreactor. However, agitation comes with drawbacks: high agitation rates can cause harmful collisions between microcarriers or against the bioreactor, while low agitation rates can prevent the necessary distribution of nutrients and gases, both of which can lead to suboptimal cell growth. Therefore, hydrodynamic forces in closed-system bioreactors play an important role in ensuring that they do not adversely affect cell growth or risk damaging cells attached to microcarriers.

[0008] Therefore, scaling up cell growth for biopharmaceutical production remains challenging, as large quantities of high-quality cells are required at cost-effective scales if commercialization is to be viable. There is now a common understanding that not only the available surface area but also the local spatial environment experienced by cells during culture is critical to the size 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 and cell differentiation is poor.

[0009] Simply increasing the concentration of microcarriers in a bioreactor to increase the available surface area and attachment points is not a viable solution because it results in more collisions between solid spherical microcarriers, gradually increasing stress and damage to cells. Furthermore, high concentrations of microcarriers have been shown in some instances to increase the production of cellular metabolic products, resulting in faster consumption of culture medium, as well as a harmful growth environment and cytotoxicity. Therefore, increasing the concentration of microcarriers does not necessarily lead to enhanced cell growth rates.

[0010] As biomanufacturing of biopharmaceuticals rapidly evolves, scaling production is expected to be a bottleneck in the biopharmaceutical revolution over the next few years. However, existing microcarrier designs limit available surface area and do not present the in vivo-like environment required for many mammalian cells to grow efficiently. Effective microcarriers are at the forefront of solving this problem and will help accelerate the commercialization of biopharmaceuticals.

[0011] Thus, there is an unmet need for the provision of new and improved microcarriers that offer high quality and scalable options for cell culture.

[0012] It would therefore be advantageous to provide a sustainable microcarrier material that provides an increased surface area for cells in culture while simultaneously replicating the extracellular environment found within the human body.

[0013] In particular, it would be advantageous to provide a simple and scalable method for producing improved microcarrier materials that can be utilized in a variety of ways, from stock cultures to bioreactor production. Summary of the Invention

[0014] The microcarriers presented herein are based on a processed form of cellulose nanofibers that provide a matrix with increased surface area and low dead volume for cell growth compared to commercially available competing solutions. 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.

[0015] It is therefore an object of the present invention to provide microcarriers that have the ability to increase cell growth with minimal volume usage.

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

[0017] Thus, one aspect of the present invention is 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) functionalizing the processed cellulose nanofiber material by adding a reagent containing a functional moiety; (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] Yet another aspect of the present invention relates to a nanofibrous cellulose scaffold comprising a cellulose nanofiber material having an average cellulose nanofiber length of less than about 250 μm, wherein the cellulose nanofiber material is functionalized with a functional moiety.

[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] 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.

[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; (iv) optionally extracting the expanded cell population from the cell culture device. [Brief explanation of the drawings]

[0024] [Figure 1](A) Microscopic image of a quaternary ammonium functionalized nanofibrous cellulose scaffold (Cellevate QA). (B) Falcon tube containing nanofibrous cellulose scaffold in dry powder form. (C) Scanning electron microscope (SEM) image of the nanofibrous cellulose scaffold demonstrating the spatial arrangement of individual nanofibers. (D) HEK293 cells cultured on the nanofibrous cellulose scaffold. (E) HEK293 cells cultured on Cytodex-1 beads. [Figure 2] Figure 1 shows cell growth of (A) HEK293 cells and (B) ARN8 cells on different microcarriers. Cell numbers were quantified by lactate dehydrogenase activity. Cells were quantified on the day of seeding (day 0) and on subsequent days to determine their growth potential on different microcarriers. The amount of microcarriers was adjusted to present the same available surface area to the cells, regardless of microcarrier type. [Figure 3] Luciferase production of (A) HEK293 cells and (B) ARN8 cells grown on different microcarriers is shown. The amount of microcarriers was adjusted to present the same available surface area to the cells regardless of the microcarrier type. [Figure 4] Luciferase production in HEK293 cells is shown when taking into account the volume occupied by different microcarriers (i.e., dead volume). (A) Microcarriers were decanted, and the gray lines indicate the approximate volumes of the microcarriers (from left to right: Cellevate CMC ("52"), Cellevate QA ("53"), and Cytodex-1). Equal volumes of microcarriers were used. (B) Luciferase production after 24 hours (black bars) and 48 hours (gray bars) of cells grown on different microcarriers (equal dead volume). [Figure 5]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 6] Microscopic images of cellulose nanofibers split by blending (A-E) or dispersing (F-J) for different times are shown. [Figure 7] 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 8] (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 9] 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 10] 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 11]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 12] 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 13] 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 14] Figure 1 shows how cells behave on nanofibrous cellulose scaffolds. (A) Cell growth after 72 h as a function of the degree of functionalization, given as a titer (mmol / Cl). Quantification over time of (B) concentration and (C) viability of HEK293 cells cultured on nanofibrous cellulose scaffolds prepared by dispersing cellulose nanofibers for 1, 5, 15, or 60 min. Data are presented as averages, and each data point represents technical replicates (n=3). 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] distributed 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. The disperser comprises one or more heads that constitute a means for dividing the cellulose nanofiber material. The heads may be in the form of disk blades. The disperser heads may comprise blades with a propeller design. The disperser generates turbulence and vortices, ensuring uniform division of the cellulose nanofiber material into smaller pieces, i.e., cellulose nanofibers with shorter lengths compared to 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] 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 in vivo cell function.

[0035] 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.

[0036] 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.

[0037] 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.

[0038] Adherent cultures are distinguished from suspension cultures in which cells grow freely floating in suspension.

[0039] Average diameter (of cellulose nanofibers) As used herein, the term "average diameter" refers to the average diameter of cellulose nanofibers in a 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 nanofibers in a sample, for example, by using image analysis software such as ImageJ.

[0040] The average diameter of the cellulose nanofibers can be adjusted during the process of preparing the initial cellulose nanofiber material, which can be achieved, for example, by varying the parameters of the electrospinning process.

[0041] 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.

[0042] Average length (of cellulose nanofibers) In this context, the term "average length" refers to the average length of cellulose nanofibers in a nanofibrous cellulose scaffold. The average length may be determined as the volume-weighted average (D[4,3]) measured by light scattering, for example, on a Malvern Mastersizer S, where D[4,3] is also known as the de Bourquer mean.

[0043] 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

[0044] 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.

[0045] surface area In this context, the term "surface area" refers to the Brunauer-Emmett-Teller (BET) surface area. The BET surface area is typically determined by measuring the physical adsorption of a gas such as nitrogen, and a value for the sample is obtained. The BET method can accurately determine the surface area of the nanofiber cellulose scaffold because gas molecules can move within the nanofiber matrix and examine the internal surface as well.

[0046] The surface area is given as the area per unit mass (e.g., cm 2 / g) and can be measured in accordance with ISO9277:2022 - Determination of the specific surface area of solids by gas adsorption - BET method.

[0047] Degree of substitution (DS) In this context, the term "degree of substitution (DS)" refers to the average number of functional moieties attached per basic unit of the condensed polymer cellulose. The basic unit of cellulose is β(1→4)-linked D-glucose and contains three hydroxyl groups that can be substituted. Therefore, the theoretical maximum value of DS is 3.

[0048] The degree of substitution (DS) can be determined using the following equation. DS = (162N / (1400 - CA×N)) Here, 162 is the molecular weight of the anhydroglucose unit (AGU), N is the proportion of nitrogen, and CA is the molecular weight of the cationic reagent.

[0049] The substitution of cellulose nanofibers can also be quantified as the equivalent of charge per basic unit mass of cellulose and is given in the unit meq / g. This value can be determined by zeta potential measurement, pH titration, or electrokinetic chromatography.

[0050] 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.

[0051] 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).

[0052] Microcarriers In the present context, the term "microcarrier" refers to any support matrix on which adherent cells can be grown in adherent culture.

[0053] Suspension-Based Bioreactors In this context, the term "suspension-based bioreactor" refers to a bioreactor in which the microcarriers and the cells attached thereto are freely suspended in the bioreactor. Suspension-based bioreactors allow for the addition of more microcarriers and / or medium during cultivation.

[0054] Thus, the term "suspension-based bioreactor" includes, but is not limited to, stirred tank bioreactors, fluidized bed bioreactors, and airlift bioreactors.

[0055] In contrast, suspension-based bioreactors are distinguished from bioreactors in which the microcarriers are fixed within the bioreactor, for example, packed-bed bioreactors in which the microcarriers are fixed within the bed.

[0056] 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%).

[0057] Nanofibrous cellulose scaffolds Described herein are nanofibrous cellulose scaffolds that can be used as microcarriers for cell culture. Nanofibrous cellulose scaffolds have properties such as a large surface area and low volume, significantly increasing cell growth per unit culture volume. Importantly, nanofibrous cellulose scaffolds can be produced in a simple and cost-effective manner, resulting in a commercially attractive and easily scalable end product.

[0058] Thus, one aspect of the present invention relates to 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) functionalizing the processed cellulose nanofiber material by adding a reagent containing a functional moiety; (iv) drying the processed cellulose nanofiber material; Thereby providing a nanofibrous cellulose scaffold.

[0059] 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.

[0060] 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.

[0061] Functional moieties substituted onto the nanofibrous cellulose scaffold can include, for example, ECM proteins, peptides, and / or charged groups to increase the level of cell attachment to the scaffold, promote cell differentiation, or aid in the release and isolation of cells from the scaffold.

[0062] Thus, the method provides an improved microcarrier material that can be used to increase cell growth and product yield.

[0063] 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 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.

[0064] 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.

[0065] 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.

[0066] Another embodiment of the present invention relates to the method described herein, wherein the dispersing is carried out in a high speed disperser.

[0067] Further embodiments of the present invention relate to the methods described herein, wherein dividing the initial nanofiber material comprises cutting the initial nanofiber material with a disperser.

[0068] 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.

[0069] 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.

[0070] Another embodiment of the present invention relates to a method as described herein, wherein the dispersing 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.

[0071] A further embodiment of the present invention relates to a method as described herein, wherein the dispersing is carried out for a period ranging from 2 minutes to 120 minutes, such as from 2 minutes to 90 minutes, such as from 5 minutes to 60 minutes, for example from 10 minutes to 60 minutes, such as from 15 minutes to 60 minutes.

[0072] A preferred embodiment of the present invention relates to the method described herein, wherein the dispersion is carried out for at least 5 minutes.

[0073] Another preferred embodiment of the present invention relates to the method described herein, wherein the dispersion is carried out for at least 15 minutes.

[0074] A further embodiment of the present invention relates to the method described herein, wherein the dispersion is carried out for at least 60 minutes.

[0075] 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.

[0076] 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.

[0077] Thus, one embodiment of the present invention relates to a method as described herein, wherein the dispersing is carried out at a speed in the range of about 10,000 rpm to about 30,000 rpm, such as about 12,000 rpm to about 25,000 rpm, for example about 15,000 rpm to about 20,000 rpm.

[0078] Another embodiment of the present invention relates to a method as described herein, wherein the dispersing is carried out at at least 15000 rpm, such as at least 20000 rpm, such as at least 25000 rpm.

[0079] During the splitting step, the length of the cellulose nanofibers is reduced. 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.

[0080] Thus, one embodiment of the present invention relates to a method as described herein, wherein the average length of cellulose nanofibers in the processed cellulose nanofiber material is shorter than the average length of cellulose nanofibers in the initial cellulose nanofiber material.

[0081] Another 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 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.

[0082] 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 ranges from about 30 μm to about 250 μm, such as from about 40 μm to about 200 μm, for example from about 50 μm to about 150 μm, preferably from about 60 μm to about 100 μm.

[0083] Yet a further embodiment of the present invention relates to the method 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.

[0084] The splitting step may be carried out under cooling to reduce the ductility of the cellulose nanofibers, making them more brittle and easier to split. Cooling may occur before or during splitting of the initial cellulose nanofiber material. Cooling may 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.

[0085] Thus, one embodiment of the present invention relates to a method as described herein, wherein the initial cellulose nanofiber material is cooled before or during the splitting step.

[0086] 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.

[0087] 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.

[0088] 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.

[0089] A further embodiment of the present invention relates to a method as described herein, wherein the solvent of the liquid sample comprises ethanol.

[0090] Yet another 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 0.1 wt % and about 10 wt %, such as between about 0.5 wt % and about 5 wt %, for example between about 0.75 wt % and about 4 wt %, preferably between about 1 wt % and about 3 wt %, based on the total weight of the liquid sample.

[0091] 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.

[0092] Accordingly, 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.

[0093] Another embodiment of the present invention relates to a method as described herein, wherein the initial cellulose nanofiber material is prepared by electrospinning.

[0094] 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 ranges 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.

[0095] 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.

[0096] The initial cellulose nanofiber material can be prepared from a cellulose acetate solution, for example, by electrospinning the cellulose acetate solution. However, 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.

[0097] Thus, one embodiment of the present invention relates to a method as described herein, wherein the initial cellulose nanofiber material is prepared by electrospinning a cellulose acetate solution into a cellulose acetate sheet.

[0098] Another embodiment of the present invention relates to a method as described herein, wherein the cellulose acetate sheets are regenerated into cellulose sheets by treatment with NaOH.

[0099] Yet a further embodiment of the present invention relates to a method as described herein, wherein the initial cellulose nanofiber material is regenerated in a regeneration solution comprising sodium hydroxide.

[0100] 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, such as 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.

[0101] 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.

[0102] Accordingly, one embodiment of the present invention relates to a method as 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.

[0103] 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.

[0104] 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.

[0105] 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.

[0106] 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.

[0107] Another embodiment of the present invention relates to the method described herein, wherein the natural polymer is selected from the group consisting of collagen, silk fibroin, keratin, gelatin and polysaccharides, and combinations thereof.

[0108] Further embodiments of the present invention relate to the 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.

[0109] 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 resuspending the processed cellulose nanofiber material. Filtration ensures that excess water is removed from the processed cellulose nanofiber material. Washing aids in the removal of any acetate ions still present after previous treatment. When resuspending the processed cellulose nanofiber material, the concentration of the cellulose nanofibers can be adjusted as needed.

[0110] Thus, one embodiment of the present invention relates to a method as described herein, wherein the splitting step (ii) is followed by steps comprising filtering, washing and suspending the processed cellulose nanofiber material.

[0111] Another embodiment of the present invention relates to a method as described herein, wherein said filtering comprises sieving the processed cellulose nanofiber material.

[0112] Yet another embodiment of the present invention relates to the method described herein, wherein the suspension comprises water and / or ethanol.

[0113] 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.

[0114] Thus, one embodiment of the present invention relates to the method described herein, further comprising the step of mercerizing said processed cellulose nanofiber material.

[0115] Another embodiment of the present invention relates to the method described herein, wherein the mercerization step immediately precedes or follows the division step (ii).

[0116] Yet another embodiment of the present invention relates to the method described herein, wherein said mercerization step comprises the addition of NaOH.

[0117] A further embodiment of the present invention relates to a method as described herein, wherein the concentration of NaOH ranges from about 0.05 M to about 2 M, such as from about 0.1 M to about 1.5 M, for example, from about 0.25 M to about 0.75 M, preferably from about 0.4 M to about 0.6 M.

[0118] Yet a further embodiment of the present invention relates to the method described herein, wherein the mercerization step is carried out for a period of about 1 hour to about 3 hours, preferably about 2 hours.

[0119] Yet a further embodiment of the present invention relates to the method described herein, wherein the degree of substitution (DS) of the functional moiety ranges from about 0.01 to about 2.

[0120] Another embodiment of the present invention relates to a method as described herein, wherein the degree of substitution (DS) of the functional moiety is in the range of from about 0.01 to about 1.5, such as from about 0.05 to about 1.2, such as from about 0.1 to about 1, such as from about 0.2 to about 0.8, such as from about 0.4 to about 0.6.

[0121] 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.

[0122] Thus, one embodiment of the present invention relates to a method as described herein, wherein the functional moiety is QA and / or DEAE, and the equivalent of charge per elementary 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, such as 1 meq / g to about 2 meq / g, preferably in the range of about 1.25 meq / g to about 1.75 meq / g.

[0123] The functionalization of nanofibrous cellulose scaffolds can be quantified in terms of the ion exchange capacity of the microcarrier. Ion exchange capacity can be defined as the ability of functional moieties attached to cellulose nanofibers to replace ions attached to the structure with ions of opposite charge present in the surrounding solution. Ion exchange capacity is measured in mmol Cl - It is given in units of / g and is determined by titration.

[0124] Thus, one embodiment of the present invention is the method described herein, wherein the ion exchange capacity of the nanofibrous cellulose scaffold is about 0.1 mmol Cl - / g~about 1.5mmol Cl - / g, e.g., about 0.3 mmol Cl - / g to about 1 mmol Cl - per g, and relates to a method.

[0125] Another embodiment of the present invention is the method described herein, wherein the ion exchange capacity of the nanofiber cellulose scaffold is at least about 0.1 mmol Cl - per g, such as at least about 0.2 mmol Cl - per g, preferably at least about 0.3 mmol Cl - per g, and relates to a method.

[0126] A further embodiment of the present invention is the method described herein, wherein the functional moiety is QA and / or DEAE, and the ion exchange capacity of the nanofiber cellulose scaffold is at least about 0.3 mmol Cl - per g, and relates to a method.

[0127] The functional moiety adhered to the cellulose nanofiber may be of either chemical or biological origin. In particular, positively charged groups are advantageous as they induce electrostatic interactions between the nanofiber cellulose scaffold and cells with negatively charged membranes, thereby increasing cell adhesion to the microcarrier. Biological moieties typically include proteins and peptides, which are essential parts of the interaction between cells and the extracellular environment. This interaction can further promote cell adhesion to the nanofiber cellulose scaffold.

[0128] Thus, one embodiment of the present invention is the method described herein, wherein the functional moiety is selected from chemical moieties or biological molecules, and relates to a method.

[0129] Another embodiment of the present invention is the method described herein, wherein the chemical moiety is selected from the group consisting of negatively charged groups, positively charged groups, zwitterionic groups, hydrophobic groups, hydrophilic groups, amphiphilic groups, ligands, and combinations thereof, and relates to a method.

[0130] A further embodiment of the present invention relates to the 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.

[0131] A preferred embodiment of the present invention relates to the method described herein, wherein the chemical moiety is a quaternary ammonium (QA).

[0132] 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.

[0133] Yet further embodiments of the present invention relate to the methods 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.

[0134] 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 followed by lyophilization, or in a single step, such as freeze-drying. A freeze-dryer performs a water removal process that can extend shelf life and / or make the material easier to transport. Freeze-dryers operate by freezing the material and then reducing pressure and applying heat to sublimate the frozen water in the material.

[0135] Thus, one embodiment of the present invention relates to a method as described herein, wherein the drying step (iv) comprises freezing and / or freeze-drying the processed cellulose nanofiber material.

[0136] Another embodiment of the present invention relates to the method 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.

[0137] The dried nanofibrous cellulose scaffold may be further processed to provide a dry powder, which may be achieved by grinding the dried product.

[0138] 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. The microcarrier product can be in the form of a dry powder that is added to a cell culture vessel, such as a bioreactor, to form a support matrix onto which cells can attach and grow.

[0139] Thus, one aspect of the present invention relates to a nanofibrous cellulose scaffold obtainable from the method described herein.

[0140] Yet another aspect of the present 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 functionalized with a functional moiety.

[0141] Nanofibrous cellulose scaffolds have a high surface area. Without being bound by theory, it is believed that the high surface area is caused, inter alia, 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 provide an excellent available surface area that is advantageous for culturing adherent cells.

[0142] One embodiment of the present invention is a nanofibrous cellulose scaffold as described herein, wherein the nanofibrous cellulose scaffold has a fiber density 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 of ​​nanofibrous cellulose scaffolds.

[0143] Another embodiment of the present invention is 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 of ​​nanofibrous cellulose scaffolds.

[0144] Yet another embodiment of the present invention relates to a 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.

[0145] Preferably, the cellulose nanofiber scaffold is provided as a dry material for ease of use and handling for end users. The dry material can be packaged in a container suitable for direct addition to cell culture vessels. Microcarriers in dry powder form can also be reconstituted / rehydrated in an aqueous solution such as PBS and / or culture medium before addition to cell culture vessels.

[0146] 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 material.

[0147] Another embodiment of the present invention relates to a nanofibrous cellulose scaffold as described herein, wherein the nanofibrous cellulose scaffold is provided as a freeze-dried material.

[0148] 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 that 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. Microcarriers are a key product that continues to make biomanufacturing more cost-effective.

[0149] The nanofibrous cellulose scaffolds described herein offer all the necessary properties for an efficient microcarrier: high surface area, low dead volume, and customization. Importantly, when used in large quantities, the nanofibrous cellulose scaffolds can be easily scaled up for industrial applications without compromising the cost and applicability of microcarriers.

[0150] Accordingly, one aspect of the present invention relates to a microcarrier comprising the nanofibrous cellulose scaffold described herein.

[0151] Another aspect of the present invention relates to the use of the nanofibrous cellulose scaffolds described herein as microcarriers for cell culture.

[0152] 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.

[0153] 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.

[0154] 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.

[0155] 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 manufacturing. 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. Nanofibrous cellulose scaffolds are suitable for use at any point in this process and are particularly advantageous at production scales where other microcarriers may be too expensive or otherwise unsuitable for use.

[0156] Thus, one embodiment of the present invention relates to a cell-cultivation device as described herein, wherein the vessel is a bioreactor, preferably a suspension-based bioreactor.

[0157] Nanofibrous cellulose scaffolds are suitable for use from laboratory scale (1 ml-5 L), through pilot scale / product development (1 L-100 L), to production scale (100 L-1000 L). If desired, this material can also be used as a microcarrier in even larger bioreactors.

[0158] Therefore, one embodiment of the present invention relates to a cell culture device as described herein, wherein the volume of the container is in the range of about 1 mL to about 1000 L, for example, about 1 L to about 1000 L, for example, about 100 L to about 1000 L.

[0159] Another embodiment of the present invention relates to a cell culture device as described herein, 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, such as at least 1000 L.

[0160] 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.

[0161] Another embodiment of the present invention relates to a cell culture device as described herein, comprising a medium.

[0162] A further embodiment of the present invention relates to a cell-cultivation device as described herein, wherein the solvent is a cell culture medium.

[0163] 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 step 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.

[0164] Accordingly, one aspect of the present 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; (iv) optionally extracting the expanded cell population from the cell culture device.

[0165] One embodiment of the present invention relates to a method of culturing the cells described herein, wherein the composition comprises a solvent.

[0166] Another embodiment of the invention relates to a method of culturing the cells described herein, wherein the solvent comprises cell culture medium.

[0167] 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.

[0168] Thus, one embodiment of the present invention relates to a method of culturing cells as described herein, wherein the cell population comprises adherent cells.

[0169] Another 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.

[0170] Further embodiments of the present invention relate to methods 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.

[0171] 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.

[0172] 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.

[0173] 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.

[0174] Further embodiments of the present invention relate to methods of culturing the cells described herein, wherein the cell population comprises stem cells.

[0175] Another embodiment of the present invention relates to a method of culturing the cells described herein, wherein the cell population comprises the HEK293 cell line.

[0176] Methods for culturing cells can also be readily used for cellular agriculture products, as well as new methods for producing existing agricultural products such as milk and (cultured) meat from cells. Cellular agriculture is considered a means to achieve animal-free agriculture. It is contemplated herein that nanofibrous cellulose scaffolds can be used in cellular agriculture, as cellulose is a biocompatible polymer.

[0177] The method of culturing cells can be carried out with or without agitation of the cell culture in the vessel. If present, agitation is preferably achieved by stirring the medium in which the cells are grown. Agitation promotes better transport of nutrients and oxygen to the cells.

[0178] Thus, one embodiment of the present invention relates to a method of culturing the cells described herein, wherein the incubation is carried out without agitation.

[0179] Another embodiment of the present invention relates to a method of culturing the cells described herein, wherein the incubation is carried out under agitation.

[0180] Further embodiments of the present invention relate to methods of culturing cells as described herein, wherein the agitation is selected from the group consisting of stirring, shaking, rocking, shaking, and whisking, and is preferably stirring.

[0181] 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.

[0182] 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 themselves 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.

[0183] 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 materials; (iii) functionalizing the processed cellulose nanofiber material by adding a reagent containing a functional moiety; (iv) drying the processed cellulose nanofiber material; Thereby, providing a nanofibrous cellulose scaffold. X2. The method according to item X1, wherein the division of the initial cellulose nanofiber material is achieved by dispersion. X3. The method according to item X2, wherein the dispersion is carried out using a high-speed disperser. X4. The method according to item X2 or X3, wherein the dispersion is carried out for at least 2 minutes, such as at least 5 minutes, for example at least 10 minutes. X5. The method according to any one of items X2 to X4, 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. X6. The method according to any one of items X1 to X5, 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. X7. The method according to any one of items X1 to X6, wherein the average length of the cellulose nanofibers in the processed cellulose nanofiber material is less than 250 μm, such as 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. X8. The method according to any one of Items X1 to X7, 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. X9. The method of any one of items X1 to X8, wherein the initial cellulose nanofiber material is provided as a liquid sample. X10. The method according to item X9, wherein the solvent of the liquid sample comprises water and / or ethanol. X11. The method according to any one of items X1 to X10, wherein the concentration of cellulose nanofibers in the processed cellulose nanofiber material is about 0.1 wt% to about 10 wt%, for example, about 0.5 wt% to about 5 wt%, for example, about 0.75 wt% to about 4 wt%, preferably about 1 wt% to about 3 wt%, based on the total weight of the liquid sample. X12. The method according to any one of Items X1 to X11, 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. X13. The method of any one of items X1 to X12, wherein the initial cellulose nanofiber material further comprises one or more nanofibers selected from natural or synthetic polymers. X14. The method according to item X13, wherein the natural polymer is selected from the group consisting of collagen, silk fibroin, keratin, gelatin and polysaccharides, and combinations thereof. X15. The method according to item X13 or X14, 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. X16. The method of any one of items X1 to X15, 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. X17. The method according to any one of items X1 to X16, further comprising mercerizing the processed cellulose nanofiber material. X18. The method according to item X17, wherein the mercerization step is immediately before or after the division step (ii). X19. The method according to item X17 or X18, wherein the mercerization step comprises the addition of NaOH. X20. The method according to item X19, 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. X21. The method according to any one of items X17 to X20, wherein the mercerization step is carried out for a period of about 1 hour to about 3 hours, preferably about 2 hours. X22. The method according to any one of items X1 to 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 X1 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 X1 to X27, wherein the splitting step (ii) is followed by steps including filtering, washing and suspending the processed cellulose nanofiber material. X29. The method according to item X28, wherein the filtering comprises sieving the processed cellulose nanofiber material. X30. The method according to item X28 or X29, wherein the suspension comprises water and / or ethanol. X31. The method according to any one of items X1 to X30, wherein the drying step (iv) comprises freezing and / or lyophilization of the processed cellulose nanofiber material. X32. The method according to any one of items X1 to X31, wherein the drying step (iv) comprises freezing the processed cellulose nanofiber material, followed by freeze-drying the processed cellulose nanofiber material after freezing. Z1. A nanofibrous cellulose scaffold obtainable by the method according to any one of claims X1 to X32. Y1. 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 functionalized with a functional moiety. Y2. The nanofibrous cellulose scaffold according to item Y1, wherein the average length of the cellulose nanofibers 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. Y3. The nanofibrous cellulose scaffold according to item Y1 or Y2, wherein the average diameter of the cellulose nanofibers 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. Y4. The nanofibrous cellulose scaffold according to any one of items Y1 to Y3, wherein the processed cellulose nanofiber material is electrospun, meltblown or drawn, preferably electrospun. Y5. The nanofibrous cellulose scaffold according to any one of paragraphs Y1 to Y4, wherein the functional moiety is selected from a chemical moiety or a biological molecule. Y6. The nanofibrous cellulose scaffold according to item Y5, 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. Y7. The nanofibrous cellulose scaffold according to item Y5 or Y6, 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. Y8. The nanofibrous cellulose scaffold of any one of paragraphs Y5-Y7, wherein the chemical moiety is a quaternary ammonium (QA). Y9. The nanofibrous cellulose scaffold according to any one of items Y5 to Y8, wherein the biological molecule is selected from the group consisting of proteins, peptides, antibodies, amino acids, polypeptides, glycoproteins, lipoproteins, and antigens, and combinations thereof. Y10. The nanofibrous cellulose scaffold according to any one of paragraphs Y1 to Y9, wherein the degree of substitution (DS) of the functional moiety ranges from about 0.01 to about 2. Y11. The nanofibrous cellulose scaffold of any one of paragraphs Y1 to Y10, wherein the nanofibrous cellulose scaffold is provided as a dry material. Y12. The nanofibrous cellulose scaffold of any one of paragraphs Y1 to Y11, wherein the nanofibrous cellulose scaffold is provided as a freeze-dried material. Y13. The nanofibrous cellulose scaffold according to any one of paragraphs Y1 to Y12, wherein the nanofibrous cellulose scaffold further comprises one or more nanofibers selected from natural or synthetic polymers. Y14. The nanofibrous cellulose scaffold according to item Y13, wherein the natural polymer is selected from the group consisting of collagen, silk fibroin, keratin, gelatin and polysaccharides, and combinations thereof. Y15. The nanofibrous cellulose scaffold according to any one of items Y13 or Y14, 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. Y16. 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 / g of a BET surface area of ​​the nanofibrous cellulose scaffold of any one of items Y1 to Y15. A1. A microcarrier comprising the nanofibrous cellulose scaffold described in any one of paragraphs Y1-Y16 or Z1. U1. Use of a nanofibrous cellulose scaffold according to any one of paragraphs Y1 to Y16 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 Y16 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, a roller bottle, a Petri dish, and a tube, preferably a bioreactor. V3. The cell-cultivation device according to paragraph V1 or V2, wherein the vessel is a bioreactor, preferably a suspension-based bioreactor. V4. The cell culture device according to any one of items V1 to V3, 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. V5. The cell culture device according to any one of items V1 to V4, wherein the cell culture device includes a solvent. V6. The cell culture device of item V5, 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 V6; (ii) adding a composition comprising a cell population to the cell culture device; (iii) incubating the cell population to provide an expanded cell population; (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-T7, wherein the incubation is carried out without stirring. T9. The method of any one of paragraphs T1-T8, wherein the incubation is carried out under agitation. T10. The method according to item T9, wherein the agitation is selected from the group consisting of stirring, shaking, oscillating, shaking, and whisking, preferably stirring.

[0184] The invention will now be described in further detail in the following non-limiting examples. [Example]

[0185] 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 and degree of substitution, were characterized.

[0186] 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.

[0187] 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.

[0188] 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.

[0189] 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 step. 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%.

[0190] This non-functionalized sample yields a nanofibrous cellulose scaffold called "Cellevate No-Func."

[0191] Functionalization of engineered cellulose nanofiber materials The processed cellulose nanofiber material was functionalized with different chemical moieties according to the following process.

[0192] 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.

[0193] 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. The reaction mixture was then 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% suspension again for further experiments.

[0194] This functionalization protocol resulted in a nanofibrous cellulose scaffold called "Cellevate QA."

[0195] 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.

[0196] 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.

[0197] This functionalization protocol resulted in a nanofibrous cellulose scaffold called "Cellevate CMC."

[0198] Carboxymethyldiethylaminoethyl (CM-DEAE): 8.7 g of diethylaminoethyl chloride hydrochloride (DAECH) was dissolved in 100 ml of water. 1 g of regenerated cellulose nanofiber material was then added to the solution with stirring. The reaction was carried out at 40°C for 15 minutes.

[0199] The regenerated cellulose nanofiber material was then transferred to a 0.5 M NaOH solution and the reaction was completed at 80° C. for 10 minutes. Filtration and washing were carried out in the same manner as for QA functionalization.

[0200] The DEAE-functionalized cellulose nanofiber material was then subjected to the above-mentioned CM protocol, resulting in a DEAE-CM-functionalized nanofiber cellulose scaffold, designated “Cellevate DEAE+CM.”

[0201] Drying The functionalized or non-functionalized cellulose nanofiber material was transferred to a -85°C freezer and left overnight. The frozen cellulose nanofiber material was then transferred to a freeze dryer and processed for 48 hours to obtain a dry product. The dried product was then pulverized to obtain a dry powder of nanofiber cellulose scaffolds.

[0202] 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.

[0203] 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.

[0204] Surface area measurement The Brunauer-Emmett-Teller (BET) model is 0.162 nm2 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).

[0205] 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.

[0206] The degree of substitution (DS) of cellulose functionalized with QA (i.e., Cellevate QA) was calculated using the following formula: DS=(162N / (1400-CAx×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.

[0207] result Nanofibrous cellulose scaffolds with different types of functional moieties were prepared, and the dry powder products (Figure 1B) were visualized by optical microscopy (Figure 1A) and SEM (Figure 1C). The resulting nanofibrous cellulose materials are highly homogeneous, in the sense that individual nanofibers are clearly visible without any significant degree of entanglement or clustering. The homogeneous distribution of nanofibers in the material ensures optimal exposure of surface area for cell adhesion and interaction.

[0208] The elemental composition and degree of substitution (DS) of a nanofibrous cellulose scaffold functionalized with QA (Cellevate QA) 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 the negatively charged cell membrane.

[0209] Table 1 lists the prepared nanofibrous cellulose scaffold samples as well as a commercially available microcarrier, Cytodex-1 (Cytiva), as a benchmark. Cytodex-1 is a spherical dextran particle functionalized with diethylaminoethyl (DEAE).

[0210] The surface area of ​​a nanofibrous cellulose scaffold can be approximated by theoretical calculation using the following formula: Theoretical surface area per weight (cm 2 / g)=2 / (r×δ) where m is the mass of the nanofibrous cellulose material, r is the radius of the cellulose nanofiber, and δ is the density of the cellulose sheet.

[0211] The nanofibrous cellulose scaffold offers an increased surface area compared to standard commercially available microcarriers. The accuracy of the theoretical calculations is supported by measurements of the BET surface area of ​​a 0.5% cellulose sample, which is 58,000 cm. 2 / g gave a BET surface area. [Table 1] Table 1. Summary of samples compared herein. The surface area of ​​the Cytodex-1 microcarriers was obtained from the product datasheet, and the surface area of ​​the nanofibrous cellulose scaffolds is a theoretical calculation. Fiber length and diameter were obtained as described in Example 4.

[0212] conclusion This example demonstrates that several different variants of nanofibrous cellulose scaffolds can be produced in a simple manner that is readily scalable for industrial applications. The nanofibrous cellulose scaffolds have a large surface area accessible to cells and do not contain collapsed or tangled nanofibers that prevent cells from accessing the surface.

[0213] Example 2: Cell growth on nanofibrous cellulose scaffolds In this example, the ability of cells to grow on nanofibrous cellulose scaffolds was evaluated. To explore the properties of cellulose nanofibers over the available surface area, the amount of microcarrier added was adjusted to present cells with an equal amount of surface area, allowing for a direct comparison between nanofibrous cellulose scaffolds and the commercially available standard microcarrier, Cytodex-1.

[0214] method Cell expansion HEK293AD and ARN8 cells were cultured at 10,000 cells / cm in 30 ml of Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS). 2 (Total cell count 1.75x10 6 pcs) density, 175cm 2 The cells were seeded into Nunc flasks. After 3 days, the cell culture medium was removed. The cell monolayer was then washed once with PBS buffer. To detach the cells from the flask, 1 ml of trypsin-EDTA (0.25%) was added to the flask and incubated at 37°C for at least 1 minute. Once the cells had detached from the flask, 9 ml of DMEM containing 10% FCS was added to stop trypsin activity. The cells were suspended and homogenized by pipetting, and then counted on a hemocytometer.

[0215] Preparation of microcarriers Nanofibrous cellulose scaffolds were prepared as described in Example 1. The following samples were evaluated: Cellevate No-Func, Cellevate CMC, Cellevate QA, and Cellevate DEAE+CMC.

[0216] In this experiment, Cytodex-1 and nanofiber cellulose scaffold microcarriers were prepared with approximately the same available surface area. Because the surface area per volume varies between carriers, different amounts of microcarriers were used.

[0217] Cytodex-1 microcarriers were prepared in PBS according to the manufacturer's recommendations. 1.33 g of Cytodex-1 dry beads were diluted with 40 ml of PBS and then autoclaved. The autoclaved beads had a diameter of 190 μm and a mass of 3000 cm. 2 / g of surface area. The total surface area per volume is 100 cm 2 / ml (1.33g x 3000cm 2 / g / 40ml=100cm 2 / ml).

[0218] Nanofibrous cellulose scaffold microcarriers were prepared by diluting 0.5 g of dry microcarriers in 50 ml of PBS. The fibers had a diameter of 500 nm and a length of 72 μm, with a length of 50,000 cm. 2 / g surface area (53333 cm for ease of approximation) 2 / g) and has a total surface area per volume of 500 cm 2 / ml(0.5g×50000cm 2 / g / 50ml=500cm 2 / ml).

[0219] To present the same amount of surface area to the cells, 500 μl of Cytodex-1 solution (500 μl × 100 cm) was used. 2 / ml=50cm 2 ) and 100 μl of nanofiber cellulose scaffold solution (100 μl × 500 cm2 / ml=50cm 2 ) was used.

[0220] To account for the unused space occupied by the interior of the microcarriers, the theoretical "dead volume" of each microcarrier was calculated (Table 2).

[0221] Cytodex-1 beads have a radius of 95 μm, therefore the surface area of ​​a single bead is A=4π×r 2 = 4π × (0.095 mm) 2 =0.113mm 2 is.

[0222] The surface area of ​​the beads is 3000 cm 2 / g, so 2654867 beads / g (300000mm 2 / g / 0.113mm 2 Cytodex-1 microcarriers were prepared at 3,530,973 beads in 40 ml, or 88,274 beads / ml (100 cm 2 / ml solution).

[0223] The volume of the Cytodex-1 beads (spheres) is as follows: V=4 / 3×r 3 = 4 / 3 × π × (0.095 mm) 3 =0.00359mm 3

[0224] Therefore, 100 cm 2 The dead volume of Cytodex-1 beads (88274 beads) is 317 μl / ml of medium (88274 beads / ml x 0.00359 μl / bead = 317 μl / ml).

[0225] The diameter of the cellulose nanofiber is 500 nm, and the length is 72 μm. The surface area of ​​a single fiber (cylinder) is A=2π×r 2 +2π×r×h=2π×(0.00025mm)2 +2π×(0.00025mm)×(0.072mm), which is 0.000113mm 2 becomes.

[0226] The surface area of ​​the fiber is 50,000 cm 2 / g, so 4.4 × 10 10 Fiber / g(5,000,000mm 2 / g / 0.000113mm 2 Nanofiber cellulose scaffold microcarriers were prepared at 2.2 × 10 per 50 ml. 10 fibers, thus 4.4 × 10 8 Fibers / ml (500cm 2 / ml solution).

[0227] The volume of a single fiber (cylinder) is V=π×h×r 2 =π×(0.072mm)×(250x10 -6 mm) 2 =1.4×10 -8 mm 3 is.

[0228] Therefore, 500 cm 2 of fiber (4.4x10 8 The dead volume of the fiber was 6.16 μl / ml of medium (4.4 x 10 8 Fiber / ml x 1.4 x 10 -8 This results in a 100cm fiber of 1.23 μl / ml of medium. 2 gives a dead volume of [Table 2] Table 2. Summary of physical properties of nanofibrous cellulose scaffolds and Cytodex-1.

[0229] Seeding of cells on microcarriers The cells were placed in a 50cm 210,000 cells / cm on each microcarrier (100 μl of nanofiber cellulose scaffold microcarrier solution or 500 μl of Cytodex-1 solution). 2 , 20,000 cells / cm 2 , 40,000 cells / cm 2 , 60,000 cells / cm 2 or 120,000 cells / cm 2 in a final volume of 1 ml of cell culture medium or 1.5 ml of Cytodex-1 (taking into account the dead volume of Cytodex-1).

[0230] The cells were incubated overnight (maximum 18 hours) in an incubator (without shaking) at 37°C and 5% CO. The Falcon tube caps were not completely closed to allow gas exchange.

[0231] The next morning, 9 ml of cell culture medium (DMEM, 10% FCS) was added to the cells and disrupted by pipetting up and down 10 times with a 10 ml pipette. The cells (in 10 ml of medium) were then transferred to a 10 cm uncoated Petri dish. The highest density was achieved by culturing 50 cm 2 The number of cells was 60,000 cells / ml in 10 ml of medium (120,000 cells / cm 2 The Petri dishes were then placed in an incubator at 37°C, 5% CO2 on an orbital shaker at 60 rpm.

[0232] Passaging procedures and cell culture For long-term culture (more than 3 days), cells were passaged. After 48 hours, the cells were transferred to a 125 ml Corning flask after disrupting the fiber aggregates by pipetting up and down 10 times with a 10 ml pipette. Fresh medium (DMEM, 10% FCS) was added to the cells up to 30 ml. Nanofiber cellulose scaffolds or Cytodex-1 were added to the flask (100 cm). 2 , equivalent to 200 μl of nanofibrous cellulose scaffold microcarriers or 1 ml of Cytodex-1). For short-term cell cultures and experiments (less than 3 days), cells were not passaged.

[0233] The cells were incubated at 37°C and 8% CO on an orbital shaker at 90 rpm. After 48 hours, an additional 200 cm 2 of microcarriers was added along with 20 ml of fresh medium to reach 50 ml.

[0234] Cell viability Cells were grown and passaged on nanofiber cellulose scaffold microcarriers or Cytodex-1 as described above. At the indicated times, the cells / fibers were pipetted 10 times with a 10 ml pipette to break up cell / fiber aggregates and obtain a homogenous suspension of cells / fibers in cell culture medium. A 500 μl suspension of cell / fiber medium was then mixed with 500 μl of 2X passive lysis buffer (Promega). The volume was adjusted to minimize sampling effects and maximize accuracy of the readings. The lysate was then centrifuged at 14,000 rpm for 15 min at 4°C. 10 μl of the supernatant lysate was then diluted with 90 μl of 1X passive lysis buffer (diluted 1:10, after which 5 μl of the diluted lysate was added to 25 μl of lysis buffer). Lactate dehydrogenase (LDH) activity was then analyzed according to the manufacturer's recommendations. LDH activity is proportional to the number of cells, represented on the y-axis.

[0235] result Cells initially attached to the nanofibrous cellulose scaffolds better than to standard commercial microcarriers (Figure 1D-E). Both cell lines (HEK293AD and ARN8) grew rapidly and to high density on the nanofibrous cellulose scaffolds over the relevant culture time window, especially on the scaffolds with functionalized moieties (Figure 2A-B). All nanofibrous cellulose scaffolds performed better than the standard commercial benchmark.

[0236] Cells could be easily and cost-effectively passaged onto nanofibrous cellulose scaffolds, and the fact that cell passaging did not require trypsinization is particularly beneficial as it significantly reduces the cost, labor, and risk of damaging or contaminating the cells.

[0237] Encouragingly, nanofibrous cellulose scaffolds allowed cells to grow at a density of 1 × 10 cells per ml of medium. 6 6 x 10 cells per ml of medium compared to conventional adherent cultures grown with 6 Cells (50 cm in 1 ml of medium) 2 at 120,000 cells / cm 2 ) and allowed to grow for at least 48 hours.

[0238] conclusion Nanofibrous cellulose scaffolds theoretically offer significantly more surface area per volume (approximately 250 times more) than commercially available standard microcarriers (Table 2). Thus, in a finite volume (e.g., a bioreactor), the amount of available surface area that can be added is much greater for nanofibrous cellulose scaffolds compared to commercially available standard Cytodex-1.

[0239] In this example, this difference was "neutralized" so that the same amount of surface area was available to the cells by adjusting the amount of microcarriers added. Nevertheless, cells grew best and at a higher density on the nanofibrous cellulose scaffold, demonstrating that cells prefer the natural-like environment presented by cellulose nanofibers.

[0240] Example 3: Transient cell transfection and protein production on nanofibrous cellulose scaffolds In this example, transient transfection and subsequent protein production of cells grown on nanofibrous cellulose scaffolds was evaluated. The experiment was performed in two steps: first, where the surface area provided by the microcarriers was kept constant (as in Example 2), and second, where the volume of medium was kept constant (thus allowing for more nanofibrous cellulose scaffolds within the culture volume). The nanofibrous cellulose scaffolds were benchmarked against a commercially available standard, Cytodex-1.

[0241] method Nanofibrous cellulose scaffolds were prepared as described in Example 1. The following samples were evaluated: Cellevate No-Func, Cellevate CMC, Cellevate QA, and Cellevate DEAE+CMC.

[0242] Transfection was performed using polyethyleneimine (PEI). Renilla luciferase production was measured daily. Luciferase activity is a marker of transfection and protein production. To determine the homogeneity of luciferase production, photographs of cells cultured on nanofibers were taken using fluorescence microscopy. The amount of Renilla luciferase protein was determined using the Bright-Glo Luciferase Assay (Promega).

[0243] A fixed amount of microcarrier surface area (50 mm 2 ) Protein production This experiment measured the effect of microcarrier surface area (50 mm) on protein production (luciferase) from HEK293AD and ARN8 cells. 2 The efficiency of different types of microcarriers related to ) was compared.

[0244] Cells (HEK293AD and ARN8) were cultured in a 50cm 2 6x10 per ml onto each microcarrier 6 The cells were transfected with 2.5 μg of Renilla luciferase reporter gene driven by the SV40 constitutive promoter using 7.5 μg of polyethyleneimine (PEI) as the transfection vehicle.

[0245] Cells were grown and passaged on nanofiber cellulose scaffolds or Cytodex-1 as described in Example 2. At the indicated times, the cells / fibers were pipetted 10 times with a 10 ml pipette to break up cell / fiber aggregates and obtain a homogenous suspension of cells / fibers in cell culture medium. A 500 μl suspension of cell / fiber medium was then mixed with 500 μl of 2X passive lysis buffer (Promega). The large volume was intended to minimize sampling effects and maximize accuracy of the readings.

[0246] Luciferase activity was analyzed 24 and 48 hours after transfection using the Bright-Glo luciferase assay (Promega). Cells and microcarriers were homogenized by pipetting, and 50 μl of medium was removed and mixed with 50 μl of Bright-Glo reagent. Luciferase activity was immediately analyzed in a luminometer (Promega) according to the manufacturer's protocol.

[0247] Protein production with fixed-volume microcarriers This experiment compared the efficiency of different types of microcarriers relative to the volume of the microcarriers by measuring protein production (luciferase) from HEK293AD cells in a finite volume of cell culture medium.

[0248] Microcarriers were placed at the same cell density (1 cm 2 20,000 cells per microcarrier) and the same volume of microcarriers (100 mm) in 2 ml of cell culture medium. 3 ) were transiently transfected with 100% ribosomal RNA (RI) to compare luciferase production.

[0249] Cells were transfected with 40,000 molecules of a luciferase reporter gene driven by an SV40 promoter using 7.5 μg of polyethyleneimine (PEI) as the transfection medium. Five hours after transfection, 3 ml of fresh medium was added to a 50 ml Falcon tube (the cap was left loose to allow O2 / CO2 exchange). Luciferase activity was analyzed 24 and 48 hours after transfection using the Bright-Glo luciferase assay (Promega). The cell microcarriers were homogenized by pipetting, and 50 μl of medium was removed and mixed with 50 μl of Bright-Glo reagent. Luciferase activity was immediately analyzed using a luminometer (Promega).

[0250] Importantly, the volume of Cellevate microcarriers was found to increase significantly when wet. 2 Theoretically, the volume per cm of Cytodex-1 microcarriers calculated in Example 2 is 2 This is approximately 250 times smaller than the volume per unit volume. To obtain a realistic value, a series of experiments were performed to estimate the volume of a hydrated nanofibrous cellulose scaffold. It was found to be a good approximation to estimate that the volume occupied by the nanofibrous cellulose scaffold is 5 times smaller than the volume of Cytodex-1 (Figure 4A).

[0251] The wet volume of Cytodex-1 is 100 cm 2 317mm for a surface area of 3 (32cm 2 / 100mm 3 ) If the volume of the wet nanofibrous cellulose scaffold microcarrier is 5 times smaller than Cytodex-1, the volume of the wet nanofibrous cellulose scaffold microcarrier is 160 cm 2 100mm 3 Each microcarrier was placed in a 50 ml Falcon tube at the same cell density, i.e., 20,000 cells / cm in a 100 μl carrier volume.2 to a final volume of 5 ml (the cap was not tightly closed to allow O2 / CO2 exchange).

[0252] result Using the same amount of available surface area, HEK293AD cells produced approximately 70% more luciferase per ml on Cellevate CMC and Cellevate QA than on Cytodex-1 8 days after transfection (Figure 3A). ARN8 cells produced 40-70% more luciferase per ml on nanofiber cellulose scaffold microcarriers (Cellevate No-Func, Cellevate CMC, and Cellevate QA) compared to Cytodex-1 5 days after transfection (Figure 3B).

[0253] By decanting the microcarriers, it was observed that a good approximation to arrive at an equivalent volume of microcarriers was that the volume of nanofibrous cellulose scaffold microcarriers was 5 times smaller than the volume of Cytodex-1 (Figure 4A). Therefore, the volume of nanofibrous cellulose scaffold microcarriers added was adjusted accordingly.

[0254] Evaluation of cell growth upon addition of a fixed volume of microcarriers demonstrated the significant reduction in dead volume of the nanofibrous cellulose scaffolds, thereby advantageously increasing the available surface area within a limited cell culture vessel. Luciferase yields from HEK293AD cells on nanofibrous cellulose scaffold microcarriers (Cellevate CMC and Cellevate QA) were approximately 5.5-6.3-fold higher after 48 hours compared to Cytodex-1 (Figure 4B).

[0255] conclusion This example demonstrates that nanofibrous cellulose scaffold microcarriers enable highly efficient transient transfection of HEK293AD and ARN8 cells, resulting in high-yield production of active recombinant protein. Furthermore, nanofibrous cellulose scaffold microcarriers offer a large surface area per volume, allowing for increased amounts of available surface area to be packed into a finite volume, such as a bioreactor. This example demonstrates that this advantage translates into significantly improved recombinant protein yields.

[0256] Example 4: 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.

[0257] method The preparation of the initial cellulose nanofiber material was carried out as described in Example 1.

[0258] Processing protocol Various methods for partitioning the initial cellulose nanofiber material were evaluated. Therefore, samples were prepared as follows.

[0259] 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.

[0260] 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).

[0261] 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.

[0262] 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).

[0263] Microscopy Samples of the processed cellulose nanofiber material were diluted 10 times with water and applied to a glass slide. Images of the cellulose nanofiber network in the sample were captured with an optical microscope (Leica) using 40x magnification.

[0264] 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.

[0265] Fiber length measurement The fiber length of cellulose nanofibers was determined by SEM (Hitachi SU3500) or light scattering (Malvern Mastersizer S).

[0266] 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.

[0267] 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 method for defining the derived diameter D[m,n], i.e., the mean and other moments of the 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.

[0268] 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.

[0269] 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.

[0270] 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 manner produce fragments large enough to trap air bubbles and float in solution (Figure 5A). Furthermore, laser cutting is not an appropriate technique for splitting cellulose nanofibers because the cellulose nanofibers melt, burn, and stick together (Figure 5B-C).

[0271] Cellulose nanofiber material processed in a blender (Figures 6A-E) produced more densely entangled samples with less homogeneously distributed cellulose nanofibers than cellulose nanofiber material processed in a disperser (Figures 6F-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 (Figures 6F-J). Therefore, dispersing for a minimum of 2 minutes may be advantageous to avoid excessive entanglement and clustering of cellulose nanofibers.

[0272] SEM images of the processed cellulose nanofiber materials confirmed that the samples processed by blending produced longer ( Figure 7A ) and more entangled ( Figure 7B–C ) cellulose nanofibers compared to the processed cellulose nanofiber materials processed by dispersion.

[0273] 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 3. [Table 3] Table 3. Fiber length measurements from a Malvern Mastersizer S.

[0274] SEM measurements were used to determine the diameter of the cellulose nanofibers (Figure 8A-B). Over 2,000 individual cellulose nanofibers were measured using ImageJ software, resulting in an average cellulose nanofiber diameter of 500 nm (Figure 8C).

[0275] 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.

[0276] 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.

[0277] Example 5: 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).

[0278] 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.

[0279] 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.

[0280] 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.

[0281] 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. For each fiber sheet, 1 ml of polymer solution was spun.

[0282] 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.

[0283] 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).

[0284] result Nanofibers could be electrospun from all materials (PCL, PLA, PLA / PCL, and cellulose) to form nanofiber sheets (see Figures 9A-D).

[0285] Sheets made from nanofibers other than cellulose were subjected to two distinct modes of nanofiber partitioning: blending (Figures 10A-F) and dispersion (Figures 11A-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.

[0286] Both the PCL nanofibers (Figures 10A-B and 11A-B) and the PLA / PCL nanofibers (Figures 10E-F and 11E-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.

[0287] Although the PLA nanofibers (Figures 10C-D and 11C-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.

[0288] 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.

[0289] Example 6: 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.

[0290] method QA-functionalized cellulose nanofibers were prepared for further testing. Cellulose acetate sheets were prepared by electrospinning as described in Example 1.

[0291] 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.

[0292] 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 repeated 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 4. The length of individual cellulose nanofibers was estimated from SEM images (four separate 10 μL droplets per time point). In the light scattering experiments, 10,000 fibers were measured per sample.

[0293] 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 residual CHPTAC or NaOH remained. Finally, the filtered fibers were resuspended in water to a 2% solution and lyophilized for further experiments.

[0294] result From the SEM images (Figures 12A-H), it is clear that the disperser quickly provides a homogenous nanofiber population with relatively few long nanofibers and without any significant entanglement. While large clumps of uncut nanofibers are present in the sample after only 1 minute of splitting (Figures 12A-B), the disperser already presents a finer nanofiber population with only a few small clumps of entangled nanofibers after 5 minutes of dispersion (Figure 12D). After 15 and 60 minutes of dispersion, the presence of entangled nanofibers is almost completely eliminated (Figures 12F and 12H). In contrast, the blend sample contains large clumps of entangled fibers even after 60 minutes of blending (Figure 12G).

[0295] The data for each sample is summarized in Figures 13A-B. It is clear that the frequency of long nanofibers is higher in the blend samples compared to the dispersed samples. Furthermore, longer processing times tend to produce fewer long nanofibers.

[0296] This relative trend is supported by measurements of the nanofibers using optical diffraction, and these data are summarized in Table 4. [Table 4] Table 4. 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.

[0297] 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.

[0298] 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.

[0299] Example 7: Degree of functionalization and cell growth on cellulose nanofibers In this example, the effect of nanofiber functionalization and dispersion time on cell growth in the resulting nanofiber scaffolds was investigated.

[0300] method Sample preparation Samples were prepared by weighing 0.05 g of freeze-dried cellulose sample (prepared as described in Example 6) into 50 ml tubes. 25 ml of distilled water was added to each tube, and the fibers were allowed to swell for 2 hours. After swelling, the tubes were centrifuged and the supernatant was discarded.

[0301] To evaluate the effect of the degree of functionalization on cell growth, samples were prepared by adding various amounts of CHPTAC, thereby resulting in different ion exchange capacities.

[0302] To saturate the exchange sites with chloride ions, the samples were incubated with 25 ml of 0.1 M HCl for 1 h with stirring. After incubation, the samples were filtered using a 0.2 μm Millipore filter unit and the supernatant was discarded.

[0303] To remove unbound chloride ions, nanofiber cellulose microcarriers were incubated with 25 ml of 0.1 mM HCl for 10 minutes. The microcarriers were then centrifuged and the supernatant discarded. To replace the bound chloride ions with sulfate ions, the microcarriers were incubated with 40 ml of 10% (w / w) sodium sulfate solution for 2 hours. The microcarriers were then filtered using a 0.2 μm Millipore filter unit, and the filtrate was saved for silver nitrate titration.

[0304] Ion exchange capacity measurement The ionic capacity of each sample was determined by AgNO3 titration using a Mettler-Toledo T5 titrator. 10 ml of each filtrate collected from the last sample preparation step above was diluted with 30 ml of distilled water. The samples were then titrated with 0.01 M AgNO3 to determine the amount of chloride ions bound to the functionalized cellulose fiber. From the equivalence points on each titration curve, the ion exchange capacity (mmol Cl) was calculated using LabX software. - / g) was calculated.

[0305] Cell culture protocol: Cell growth experiments on cellulose fibers were performed in a 12-well plate format using HEK293 cells (ATCC CTRL-3216) at passage 8 (viability 92% on the day of fiber inoculation).

[0306] One milligram of cellulose fiber for each condition was washed three times with DPBS and resuspended in 10 ml of cell culture medium (DMEM with high glucose, GlutaMAX supplement, and pyruvate, 10% heat-inactivated FBS, and 1% penicillin-streptomycin). 300,000 cells per well were seeded into 0.3 ml of medium containing 0.3 ml (equivalent to 0.3 mg) of QA-functionalized cellulose fiber (D1, D5, D15, and D60) prepared as described in Example 6, and the fibers were placed in an incubator (37°C and 5% CO2) overnight at 35 rpm on a 16 mm orbital shaker.

[0307] The following morning, each well was filled with 1.4 ml of medium to bring the final volume to 2 ml, raising the initial cell concentration to 150,000 cells / ml before analysis. The shaker speed was increased to 65 rpm.

[0308] At 24, 48, and 72 hours after seeding, triplicates from separate wells from each time point were collected for further analysis. Cell density and viability were assessed over time using enzymatic dissociation of cells in combination with the microcarrier program on a NucleoCounter (NC-202).

[0309] result Cell growth was enhanced on nanofibrous cellulose scaffolds with higher functionalization (Figure 14A). In particular, the ion exchange capacity of 0.3 mmol Cl - The best cell growth appears to be achieved on cellulose microcarriers exceeding 1000 kJ / g.

[0310] Consistent cell growth was achieved on all cellulose microcarriers. Data show that cells grown on cellulose microcarriers prepared from cellulose nanofibers dispersed for at least 5 minutes proliferated best and exhibited excellent viability (Figures 14B-14C). Cell density reached an 8-fold increase over the initial seeding density over 72 hours of incubation. This was achieved without cell transfer or medium changes.

[0311] conclusion This example demonstrates that cellulose microcarriers can be efficiently functionalized and that minimizing ion exchange capacity is preferable to enhance cell growth. Cellulose microcarriers in which nanofibers were dispersed for more than 1 minute (e.g., 5 minutes) constituted the optimal scaffold for cell growth. Without being bound by theory, it is believed that the reduction in nanofiber clustering and entanglement ensures a larger surface area is available for cells to seed and interact with.

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) functionalizing the processed cellulose nanofiber material by adding a reagent comprising a functional moiety; (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 initial cellulose nanofiber material is prepared by electrospinning, meltblowing, drawing, self-assembly, template synthesis, and thermally induced phase separation.

6. The method of any one of claims 1 to 5, wherein the drying step (iv) comprises freezing and / or freeze-drying the processed cellulose nanofiber material.

7. 7. The method of any one of claims 1 to 6, wherein the functional moiety is a chemical moiety 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.

8. The method of any one of claims 1 to 7, wherein the functional moiety is a quaternary ammonium (QA).

9. The nanofibrous cellulose scaffold has an ion exchange capacity of at least about 0.3 mmol Cl － The method according to any one of claims 1 to 8, wherein the saturation is 0.05 to 0.

15.

10. A nanofibrous cellulose scaffold obtainable by the method according to any one of claims 1 to 9.

11. 1. A nanofibrous cellulose scaffold comprising a cellulose nanofiber material having an average cellulose nanofiber length of less than about 250 μm, wherein the cellulose nanofiber material is functionalized with a functional moiety.

12. 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 12. The nanofibrous cellulose scaffold of claim 11, having a BET surface area of ​​1 / g.

13. A microcarrier comprising the nanofibrous cellulose scaffold of any one of claims 10 to 12.

14. A cell culture device comprising a container loaded with the nanofiber cellulose scaffold according to any one of claims 10 to 12 or the microcarrier according to claim 13.

15. 1. A method for culturing cells, comprising: (i) providing a cell culture device according to claim 14; (ii) adding a composition comprising a cell population to the cell culture device; (iii) incubating the cell population to provide an expanded cell population; (iv) optionally extracting the expanded cell population from the cell culture device.

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