Nanofibrous scaffolds for cell culture

Abstract

The present invention relates to a method for preparing a nanofibrous scaffold, and to the use of the nanofibrous scaffold for promoting cell culture. In particular, the nanofibrous scaffold comprises functionalized cellulose nanofibers that significantly increase the amplification ability and the amount of protein production. The method comprises the step of splitting the initial cellulose nanofibers by dispersion.

Description

Technical Field

[0001] The present invention relates to a method for preparing a nanofibrous scaffold and the use of the nanofibrous scaffold for promoting cell culture. Specifically, the nanofibrous scaffold comprises functionalized cellulose nanofibers that significantly increase the expansion capacity and protein production. Background Art

[0002] Biological products (biologics) such as therapeutic proteins, vaccines, or cell and gene therapy products represent a new medical paradigm. These therapies can bring significant therapeutic benefits to patients and have revolutionized the treatment of many diseases in multiple fields. The most complex biologics with specific glycosylation patterns can be produced in mammalian cell culture by recombinant DNA technology and include enzymes, synthetic hormones, and monoclonal antibodies. However, the manufacture of biologics is challenging because the production of the final product relies on sensitive living host cells, which require more attention than, for example, the chemical synthesis of small molecule drugs.

[0003] The biologics manufacturing process can be divided into upstream and downstream processes. Upstream processes are defined as the entire process from early cell isolation and culture, through cell banking and cell culture expansion, to final harvest—in other words, termination of culture and collection of viable cell batches for purification.

[0004] Therefore, although cell culture is key to the upstream process, cell immunogenicity, adverse events, and efficacy can be affected by even the slightest variation in the manufacturing process. As a result, scaling up from research to clinical quantities is difficult, and many promising therapies fail to reach the market. Furthermore, the most significant disadvantage is low product yield, followed by the costs associated with extensive cell line development and limited cell production viability. The manufacturing process is extremely expensive, and not all patients have access to biologics.

[0005] To aid upstream processes, microcarriers are often used to support the proliferation of adherent cell populations in bioreactors. Microcarriers are support matrices that allow cells to be cultured in three dimensions rather than on a traditional flat surface, significantly increasing the ability to accommodate more cells within a limited volume. The most commonly used commercially available microcarriers are solid, spherical particles that retain cells on their surface. Because cell growth is largely dependent on the surface area available for cell attachment, this approach can accelerate throughput.

[0006] Suspension-based bioreactors are preferred for many practical applications because they allow for the addition of more microcarriers and culture medium during the culture process, which provides process flexibility and controllability. In suspension-based bioreactors, microcarriers float freely within the bioreactor but provide the necessary support matrix for adherent attachment and proliferation. Important parameters of microcarriers include the available surface area they provide and the dead volume they occupy.

[0007] Typically, agitation is installed in suspension-based bioreactors to ensure efficient distribution of nutrients and gases such as oxygen throughout the bioreactor. However, agitation carries the disadvantage that high agitation speeds can cause harmful collisions between microcarriers or into the bioreactor, while low agitation speeds can prevent the necessary distribution of nutrients and gases, both of which lead to suboptimal cell growth. Therefore, fluid dynamics in closed-system bioreactors play an important role in avoiding adverse effects on cell proliferation or the risk of damaging cells adhered to the microcarriers.

[0008] Therefore, scaling up cell growth for biologics production remains nontrivial, as large quantities of high-quality cells are required if any commercialization is to be viable. Currently, it is widely accepted that not only the available surface area but also the local spatial environment experienced by cells during culture is important for the quantity and quality of the final cell product. If cells are not exposed to sufficient attachment sites and a microenvironment that mimics their natural environment, cell signaling will be hampered and cell differentiation will be poor.

[0009] Simply increasing the concentration of microcarriers in a bioreactor to increase available surface area and attachment sites is not a viable solution because it results in more collisions between the solid spherical microcarriers and gradually increases stress and damage to the cells. In addition, in some cases, high concentrations of microcarriers have been shown to increase the production of cellular metabolites, leading to accelerated culture medium consumption and harmful growth environments and cytotoxicity. Therefore, increasing microcarriers does not necessarily translate into enhanced cell expansion rates.

[0010] With the rapid advancement of biomanufacturing technologies for biologics, it is increasingly recognized that large-scale production will be a bottleneck in the biologics revolution over the next few years. However, the design of existing microcarriers limits the available surface area and fails to provide the in vivo-like environment required for the efficient proliferation of many mammalian cells. Effective microcarriers are at the forefront of addressing this issue and helping to accelerate the commercialization of biologics.

[0011] Therefore, there is an unmet need to provide new and improved microcarriers that offer high quality and scalable options for cell culture.

[0012] Therefore, it would be advantageous to provide sustainable microcarrier materials that provide increased surface area for cells in culture while replicating the extracellular environment found in the human body.

[0013] In particular, it would be advantageous to provide a simple and scalable method to generate improved microcarrier materials that can be used in all processes from stock culture to bioreactor production. Summary of the Invention

[0014] The microcarriers presented herein are based on cellulose nanofibers that, in a processed form, provide a matrix with increased surface area and low dead volume for cell growth compared to competing solutions on the market. The cellulose nanofibers provide a scaffold matrix whose physical properties mimic the collagen and elastic fiber structure that make up the human extracellular matrix. As a result, cells grown on the microcarriers presented herein have physiological properties similar to those of the natural tissue from which they are derived, leading to improved intercellular communication and, in turn, restoration or maintenance of function in the body.

[0015] Therefore, an object of the present invention relates to providing a microcarrier having the ability to increase cell growth with minimal volume usage.

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

[0017] Therefore, aspects of the present invention relate to a method for preparing a nanofibrous cellulose scaffold, the method comprising the steps of:

[0018] (i) providing an initial cellulose nanofiber material,

[0019] (ii) dividing the initial cellulose nanofibrous material into processed cellulose nanofibrous materials,

[0020] (iii) functionalizing the processed cellulose nanofiber material by adding a reagent comprising a functional moiety, and

[0021] (iv) drying the processed cellulose nanofiber material,

[0022] Thereby, the nanofibrous cellulose scaffold is provided.

[0023] Another aspect of the present invention relates to a nanofibrous cellulose scaffold obtainable by the method as described herein.

[0024] Yet another aspect of the present invention relates to a nanofiber-like cellulose scaffold comprising a cellulose nanofiber material having an average length of cellulose nanofibers less than about 250 μm, and wherein the cellulose nanofiber material is functionalized with a functional moiety.

[0025] A further aspect of the invention relates to a microcarrier comprising a nanofibrillar cellulose scaffold as described herein.

[0026] Still further aspects of the present invention relate to the use of the nanofibrous cellulose scaffold as described herein as a microcarrier for cell culture.

[0027] An even further aspect of the invention relates to a cell culture device comprising a container loaded with a nanofibrous cellulose scaffold or microcarrier as described herein.

[0028] Another aspect of the present invention relates to a method for cell culture, comprising the following steps:

[0029] (i) providing a cell culture device as described herein,

[0030] (ii) adding a composition comprising a cell population to the cell culture device,

[0031] (iii) incubating the cell population to provide a expanded cell population, and

[0032] (iv) Optionally, extracting the expanded cell population from the cell culture device. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 Shown are (A) Microscope image of a nanofibrous cellulose scaffold functionalized with quaternary ammonium (Cellevate QA). (B) Falcon tube containing a nanofibrous cellulose scaffold in dry powder form. (C) Scanning electron microscopy (SEM) image of a nanofibrous cellulose scaffold, demonstrating the spatial arrangement of individual nanofibers. (D) HEK293 cells cultured on a nanofibrous cellulose scaffold. (E) HEK 293 cells cultured on Cytodex-1 beads.

[0034] Figure 2Figure 20 illustrates the cell growth of (A) HEK293 cells and (B) ARN8 cells on different microcarriers. Cell number is quantified by lactate dehydrogenase activity. On the inoculation day (day 0) and in subsequent days, cells are quantified to determine the growth capacity on different microcarriers. Regardless of the type of microcarrier, the amount of microcarrier is adjusted to provide identical available surface area to the cells.

[0035] Figure 3 Shown are the luciferase production of (A) HEK293 cells and (B) ARN8 cells grown on different microcarriers. Regardless of the type of microcarrier, the amount of microcarriers was adjusted to provide the same available surface area to the cells.

[0036] Figure 4 Figure 2 shows the production of luciferase in HEK293 cells when considering the volume occupied by different microcarriers (i.e., dead volume). (A) Microcarriers were decanted, with the grey line indicating the approximate volume 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 cells were grown on different microcarriers (equal dead volumes) for 24 hours (black bars) and 48 hours (grey bars).

[0037] Figure 5 Examples of laser-cut fibers are shown. (A) Laser-cut cellulose material produces entrapped bubbles and floating cellulose fragments. (B) Electrospun cellulose material cut with a laser. Laser-burned cellulose sheet. (C) SEM image of laser-cut cellulose nanofibers. The cellulose nanofibers melt and fuse together.

[0038] Figure 6 Microscope images of cellulose nanofibers separated by blending (AE) or dispersion (FJ) for different times are shown.

[0039] Figure 7 Figure 2 shows the difference between blended and dispersed cellulose nanofibers. (A) Nanofiber length as a function of segmentation mode and segmentation duration (0.5-10 minutes). Nanofiber length was determined from SEM images using ImageJ software. (B) Representative SEM image of cellulose nanofibers after 10 minutes of blending. (C) Representative SEM image of cellulose nanofibers after 10 minutes of dispersion.

[0040] Figure 8Figure 4 shows the determination of cellulose nanofiber diameter. (A-B) SEM micrographs of cellulose nanofibers after dispersion at 18,000 rpm for 1 hour. (C) Histogram depicting the distribution of cellulose nanofiber diameters for five individual samples. Approximately 2,500 individual cellulose nanofibers were measured using ImageJ software.

[0041] Figure 9 Scanning electron microscopy (SEM) images of electrospun nanofibers are shown; (A) PCL, (B) PLA, (C) PLA / PCL, and (D) cellulose. All images were recorded at 600x magnification, with a scale bar of 50 μm.

[0042] Figure 10 Representative photographs (left) and SEM images (right) of different electrospun nanofibers after 5 minutes of mixing in a blender. (AB) PCL, (CD) PLA, (EF) PLA / PCL. Scale bar in SEM images: 200 μm.

[0043] Figure 11 Representative photographs (left) and SEM images (right) of different electrospun nanofibers after 5 minutes of mixing in a disperser. (AB) PCL, (CD) PLA, (EF) PLA / PCL. Scale bar in SEM images: 50 μm.

[0044] Figure 12 Scanning electron microscopy (SEM) images of blended (left) or dispersed (right) cellulose nanofibers are shown. The cellulose nanofibers were mixed for different time periods; 1 minute (AB), 5 minutes (CD), 15 minutes (EF), or 60 minutes (GH).

[0045] Figure 13 Shown are histograms of the size (length) distribution of cellulose nanofibers that have been fragmented by (A) blending or (B) dispersion. The histograms represent samples fragmented for 1 minute, 5 minutes, 15 minutes, or 60 minutes (from left to right). Nanofiber length is shown as the relative frequency relative to the average nanofiber length at a bin size of 200 μm.

[0046] Figure 14 Figure 2 shows the performance of cells on nanofiber cellulose scaffolds. (A) Cell growth as a function of the degree of functionalization after 72 hours, given as titration (mmol / Cl). Time course quantification of (B) concentration and (C) viability of HEK293 cells cultured on nanofiber cellulose scaffolds prepared by dispersing cellulose nanofibers for 1 minute, 5 minutes, 15 minutes, or 60 minutes. Data are presented as mean values, with each data point representing technical replicates (n=3). DETAILED DESCRIPTION

[0047] definition

[0048] Before outlining the present invention in more detail, a set of terms and conventions are first defined:

[0049] Nanofibers

[0050] In the present context, the term "nanofiber" refers to fibers with a diameter in the range of 10-2000 nm. The fibers can be produced from different types of polymers such as cellulose.

[0051] Cellulose nanofiber materials

[0052] In the present context, the term "cellulose nanofiber material" refers to a starting material prepared from cellulose. The cellulose nanofiber material can be prepared by any method suitable for preparing a cellulose sheet, which can then be processed as described herein to provide a nanofiber-like cellulose scaffold. These methods include, but are not limited to, electrospinning, meltblowing, and drawing of cellulose nanofibers.

[0053] Preferably, the cellulose nanofiber material is prepared by electrospinning cellulose nanofibers. Electrospinning can be performed from a cellulose acetate solution.

[0054] container

[0055] In the present context, the term "container" refers to any defined vessel suitable for culturing cells. The container is preferably a conventional culture vessel, including but not limited to bioreactors, cell culture plates, cell culture bottles, spinner flasks, shaker flasks and roller bottles, petri dishes and tubes.

[0056] The nanofibrous cellulose scaffolds as described herein are easily scalable, and thus the container can be any volume suitable for culturing cells.

[0057] dispersion

[0058] In the present context, the term "dispersing" refers to the process of dividing the cellulose nanofiber material by a disperser. Dispersing is preferably performed in a liquid.

[0059] In the present context, a disperser is a high-speed mixing device that can pulverize solids such as cellulose sheets into smaller fragments. The disperser comprises one or more heads that constitute a device for dividing the cellulose nanofiber material. The head can be in the form of disk blades. The head of the disperser can include blades with a propeller design. The disperser generates turbulence and eddies that ensure that the cellulose nanofiber material is evenly divided into smaller fragments, i.e., cellulose nanofibers that are reduced in length compared to the initial cellulose nanofiber material.

[0060] Any type of disperser can be used, including but not limited to laboratory-scale, pilot-scale, and production-scale high-speed dispersers. The disperser can be selected based on the batch size to be divided, and the diameter of the disperser blades can be adjusted accordingly. The disperser blades can be raised and lowered during dispersion to eliminate stratification, and such mechanisms can be automated. For large batches, the disperser can be a floor-standing or tank-mounted model and / or a multi-shaft model.

[0061] Functional part

[0062] In the present context, the term "functional moiety" refers to a chemical or biological group or molecule located on a nanofiber cellulose scaffold that interacts with cells associated with the scaffold. The functional moiety can interact with cells through interactions including, but not limited to, electrostatic interactions, affinity, and hydrophobic / hydrophilic interactions. The functional moiety can promote cell attachment / adhesion to the nanofiber cellulose scaffold, induce cell differentiation and proliferation, and / or help maintain cell function in vivo.

[0063] Chemical moieties 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). For many cells, cell membranes are negatively charged, and for nanofiber-shaped cellulose scaffolds functionalized with positively charged functional moieties such as QA or DEAE, electrostatic interactions are induced.

[0064] The biological moiety can be any type of biomolecule that can ensure cell attachment to the nanofibrillar 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 help cell differentiation through receptor-ligand interactions, antigen-antibody interactions, protein interactions, and / or immunostimulatory signaling.

[0065] Adherent cells

[0066] In the present context, the term "adherent cells" refers to any cells that require a surface or an artificial substrate such as microcarriers to form an adherent cell culture. Preferably, the adherent cells are derived from solid tissue.

[0067] Adherence culture differs from suspension culture in which cells grow freely floating in suspension.

[0068] Average diameter (of cellulose nanofibers)

[0069] In the present context, the term "average diameter" refers to the average diameter of the cellulose nanofibers in the nanofiber-like cellulose scaffold. The average diameter can be determined from an SEM image of the nanofiber-like cellulose scaffold. Preferably, the average diameter is determined from measurements of at least 100 individual nanofibers in a sample, for example, by using image analysis software such as ImageJ.

[0070] The average diameter of the cellulose nanofibers can be adjusted during the preparation of the initial cellulose nanofiber material, for example by changing the parameters of the electrospinning process.

[0071] Preferably, the average diameter of the cellulose nanofibers in the nanofibrillar cellulose scaffold is from about 250 nm to about 750 nm, such as from about 400 nm to about 600 nm.

[0072] Average length (of cellulose nanofibers)

[0073] In the present context, the term "average length" refers to the average length of the cellulose nanofibers in the nanofiber-like cellulose scaffold. The average length can be determined as the volume-weighted average (D[4,3]) measured by light scattering, for example on a Malvern Mastersizer S. D[4,3] is also known as the de Bruker average.

[0074] The average fiber length in a sample can be determined using the following settings on a Malvern Mastersizer S:

[0075] Range lens: 300RF mm

[0076] Indicates: 3OHD

[0077] Analysis model: Polydisperse particles Refractive index: (1.5295, 0.1000)

[0078] Dispersant refractive index: (1.33000)

[0079] Density: 1.5000g / cm 3

[0080] Preferably, the average length of the cellulose nanofibers is from about 30 μm to about 250 μm, such as from about 40 μm to about 200 μm, such as from about 50 μm to about 150 μm, preferably from about 60 μm to about 100 μm.

[0081] Surface area

[0082] In the present context, the term "surface area" refers to the Brunauer-Emmett-Teller (BET) surface area. The BET surface area can be determined by measuring the physical adsorption of a gas, typically nitrogen, to yield a value for the sample. The BET method can accurately determine the surface area of ​​nanofibrous cellulose scaffolds because gas molecules can travel within the nanofibrous matrix and probe the internal surface.

[0083] Surface area is expressed as area per unit mass (e.g. cm 2 / g) and can be measured according to ISO 9277:2022 - Determination of specific surface area of ​​solids by gas adsorption - BET method.

[0084] Degree of substitution (DS)

[0085] In the present context, the term "degree of substitution (DS)" refers to the average number of functional moieties attached per unit of the condensation polymer cellulose. The basic unit of cellulose is β(1→4)-linked D-glucose, which contains three hydroxyl groups that can be substituted. Therefore, the theoretical maximum DS is 3.

[0086] The degree of substitution (DS) can be determined using the following formula:

[0087] DS = (162N / (1400-CAxN))

[0088] where 162 is the molecular weight in anhydrous glucose units (AGU); N is the percentage of nitrogen; and CA is the molecular weight of the cationic agent.

[0089] The substitution of cellulose nanofibers can also be quantified as the charge equivalent per basic unit mass of cellulose and is given in meq / g. This value can be determined by zeta potential measurement, pH titration or electrokinetic chromatography.

[0090] Dead volume

[0091] In the present context, the term "dead volume" refers to the volume occupied by microcarriers when cells are cultured. Ideally, dead volume is minimized to allow more cells to proliferate per unit volume in the vessel used to culture cells.

[0092] Mercerizing

[0093] In the present context, the term "mercerization" refers to a process comprising swelling the cellulose nanofiber material in an aqueous NaOH solution or an ethanol solution to disrupt the internal hydrogen bonds of cellulose and increase the number of available hydroxyl groups (-OH).

[0094] microcarriers

[0095] In the present context, the term "microcarrier" refers to any support matrix on which adherent cells can grow in adherent culture.

[0096] Suspension-based bioreactors

[0097] In the present context, the term "suspension-based bioreactor" refers to a bioreactor in which the microcarriers and cells attached thereto are free floating in the bioreactor. In a suspension-based bioreactor, it is possible to add more microcarriers and / or culture medium during the culture period.

[0098] Thus, the term "suspension-based bioreactor" includes, but is not limited to, stirred tank bioreactors, fluidized bed bioreactors, and airlift bioreactors.

[0099] In contrast, suspension-based bioreactors are distinguished from bioreactors in which the microcarriers are immobilized within the bioreactor, such as packed bed bioreactors in which the microcarriers are immobilized in the bed.

[0100] about

[0101] Whenever the term "about" is used herein in the context of a quantity, e.g., an absolute quantity, such as amount, purity, weight, size, etc., or a relative quantity (e.g., a percentage, equivalent, or ratio), a time frame, and a parameter such as temperature, pressure, etc., it is understood that such variables are approximate and, therefore, may vary by ±10%, e.g., ±5%, and preferably ±2% (e.g., ±1%) relative to the actual quantity specified. This is true even if such a number is first presented as a percentage (e.g., 'about 10%' may mean ±10% about the number 10, i.e., anywhere between 9% and 11%).

[0102] Nanofibrous cellulose scaffolds

[0103] This paper describes a nanofiber-like cellulose scaffold that can be used as a microcarrier for culturing cells. The nanofiber-like cellulose scaffold possesses properties such as a large surface area and a small size, which significantly enhances cell proliferation per unit culture volume. Importantly, the nanofiber-like cellulose scaffold can be produced using a simple and economical method, resulting in a commercially attractive end product that is easily scalable.

[0104] Therefore, aspects of the present invention relate to a method for preparing a nanofibrous cellulose scaffold, the method comprising the steps of:

[0105] (i) providing an initial cellulose nanofiber material,

[0106] (ii) dividing the initial cellulose nanofibrous material into processed cellulose nanofibrous materials,

[0107] (iii) functionalizing the processed cellulose nanofiber material by adding a reagent comprising a functional moiety, and

[0108] (iv) drying the processed cellulose nanofiber material,

[0109] Thereby, the nanofibrous cellulose scaffold is provided.

[0110] The resulting nanofibrous cellulose scaffold mimics the extracellular matrix (ECM) and, due to the uniform distribution of the cellulose nanofibers, has a high surface area. The ECM is crucial for cell survival, proliferation, differentiation, and migration, and microcarriers that mimic the properties of the ECM are therefore considered a step towards in vivo-like cell culture.

[0111] The flexible nature of cellulose nanofibers allows them to form a network of strands that efficiently utilizes occupied space and reduces the dead volume of microcarriers, which cells cannot enter. As a result, more surface area can be packed into a smaller volume, which is beneficial for use in cell culture vessels where only limited volume is available.

[0112] Functional moieties substituted onto the nanofiber cellulose scaffolds may include ECM proteins, peptides, and / or charged groups to, for example, increase the level of cell attachment to the scaffold, promote cell differentiation, or aid in the release and detachment of cells from the scaffold.

[0113] Thus, the present method provides an improved microcarrier material that can be used to enhance cell growth and product yield.

[0114] Fragmentation of the initial cellulose nanofiber material is important as it ensures uniform distribution of the cellulose nanofibers in the final scaffold. Shorter cellulose nanofibers also significantly reduce the risk of nanofiber entanglements and nanofiber cluster formation, which reduces the available surface area exposed to cells and increases the risk of clogging the bioreactor impeller. In addition, larger cellulose nanofiber tangles or clusters can also make the final nanofiber-like cellulose scaffold difficult to handle and may cause tube clogging or blockage during pipetting. As part of the upstream biomanufacturing process, continuous pipetting or aspiration is performed from the bioreactor for cell counting, viability and yield results. Therefore, even a small risk of clogging will make the microcarrier solution unviable.

[0115] It has been found that dispersing the initial cellulose nanofiber material produces a uniform material suitable for use as a microcarrier. Without being bound by theory, it is thought that dispersing the initial cellulose nanofiber material so that it is distributed with a certain spacing allows cells to have multiple attachment points, resulting in improved proliferation.

[0116] Therefore, embodiments of the present invention relate to methods as described herein, wherein dividing the initial cellulose nanofibrous material is achieved by dispersion.

[0117] Another embodiment of the present invention is directed to the method as described herein, wherein the dispersing is performed with a high speed disperser.

[0118] Further embodiments of the present invention relate to a method as described herein, wherein dividing the initial nanofibrous material comprises the step of cutting the initial nanofibrous material with a disperser.

[0119] Increasing the dispersion time reduces the average length of the cellulose nanofibers in the nanofiber-like cellulose scaffold. Specifically, it is advantageous to disperse the material for at least several minutes to reduce nanofiber entanglement and cluster formation. Furthermore, without being bound by theory, it is contemplated that longer cellulose nanofibers are less susceptible to substitution at the hydroxyl groups, thereby resulting in a lower degree of substitution (DS) of the functional moiety.

[0120] Thus, embodiments of the present invention relate to methods as described herein, wherein the dispersing is performed for at least 2 minutes, such as at least 5 minutes, such as at least 10 minutes.

[0121] Another embodiment of the present invention relates to a method as described herein, wherein the dispersion is carried out for at least 2 minutes, such as at least 5 minutes, such as at least 10 minutes, such as at least 15 minutes, such as at least 20 minutes, such as at least 30 minutes, such as at least 40 minutes, such as at least 50 minutes, such as at least 60 minutes, such as at least 90 minutes, such as at least 120 minutes.

[0122] Further embodiments of the present invention relate to a method as described herein, wherein the dispersing is performed for a time period in the range of 2 minutes to 120 minutes, such as 2 minutes to 90 minutes, such as 5 minutes to 60 minutes, such as 10 minutes to 60 minutes, such as 15 minutes to 60 minutes.

[0123] A preferred embodiment of the present invention relates to a method as described herein, wherein the dispersing is performed for at least 5 minutes.

[0124] Another preferred embodiment of the present invention relates to the method as described herein, wherein the dispersing is performed for at least 15 minutes.

[0125] A further preferred embodiment of the present invention relates to a method as described herein, wherein the dispersing is performed for at least 60 minutes.

[0126] It is advantageous to disperse the initial cellulose nanofibrous material for at least 15 minutes, and even at least 60 minutes, as this reduces entanglement and cluster formation of the cellulose nanofibers.

[0127] Dispersion is preferably carried out at a high speed, such as at about 18000 rpm. The speed can be adjusted according to the type of disperser. Any type of disperser can be used, including but not limited to laboratory high-speed dispersers, pilot-scale high-speed dispersers, and production-scale high-speed dispersers. The speed can be adjusted to produce a good vortex in the solution, and can depend on the volume and viscosity of the solution.

[0128] Thus, embodiments of the present invention relate to methods as described herein, wherein the dispersing is performed at a speed in the range of about 10,000 rpm to about 30,000 rpm, such as at least 12,000 rpm to about 25,000 rpm, such as at least 15,000 rpm to about 20,000 rpm.

[0129] Yet another embodiment of the present invention relates to the method as described herein, wherein the dispersing is performed at at least 15000 rpm, such as at least 20000 rpm, such as at least 25000 rpm.

[0130] During the segmentation step, the length of the cellulose nanofibers decreases. It is important that the cellulose nanofibers are not too long, otherwise the nanofibers will tangle and form clusters. Cells cannot penetrate and migrate into these tightly tangled fiber clusters, and thus lose some of the large surface area of ​​the nanofibers.

[0131] Therefore, embodiments of the present invention relate to methods as described herein, wherein the average length of the cellulose nanofibers in the processed cellulose nanofiber material is shorter than the average length of the cellulose nanofibers in the initial cellulose nanofiber material.

[0132] 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, such as less than about 150 μm, such as less than about 120 μm, such as less than about 100 μm, such as less than about 80 μm.

[0133] Further embodiments of the present invention relate to a method as described herein, wherein the average length of the cellulose nanofibers in the processed cellulose nanofiber material is in the range of about 30 μm to about 250 μm, such as about 40 μm to about 200 μm, such as about 50 μm to about 150 μm, preferably about 60 μm to about 100 μm.

[0134] Still further embodiments of the present invention are directed to a method as described herein, wherein the average length of the cellulose nanofibers in the processed cellulose nanofiber material is in the range of about 70 μm to 120 μm.

[0135] The splitting step can be performed in a cooled state to reduce the ductility of the cellulose nanofibers, making them more brittle and easier to split. Cooling can be performed before or during the splitting of the initial cellulose nanofiber material. Cooling can include cooling the container containing the initial cellulose nanofiber material 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 placing the initial cellulose nanofiber material in a refrigerator or freezer before the splitting step.

[0136] Therefore, embodiments of the present invention relate to methods as described herein, wherein the initial cellulose nanofiber material is cooled before or during the dividing step.

[0137] If the cellulose sheet is prepared by electrospinning, it may generate high static electricity and be difficult to handle. Therefore, the initial cellulose nanofiber material can be conveniently provided as a liquid sample, which is easy to process, for example by dispersion.

[0138] Thus, embodiments of the present invention relate to methods as described herein, wherein the starting cellulose nanofiber material is provided as a liquid sample.

[0139] Another embodiment of the present invention relates to the method as described herein, wherein the solvent of the liquid sample comprises water and / or ethanol.

[0140] Another embodiment of the present invention is directed to the method as described herein, wherein the solvent of the liquid sample comprises ethanol.

[0141] 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 from about 0.1 wt % to about 10 wt %, such as from about 0.5 wt % to about 5 wt %, such as from about 0.75 wt % to about 4 wt %, preferably from about 1 wt % to about 3 wt %, relative to the total weight of the liquid sample.

[0142] The average diameter of cellulose nanofibers can be guided during the preparation of initial cellulose nanofiber material. This can be achieved by changing the parameters of for example electrostatic spinning or melt-blown process, such as the speed and type of voltage or heat, injection and / or the rotational speed of collector drum applied. The average diameter of cellulose nanofibers can be based on application, for example, the difference of the type of cell to be cultivated changes. It has been found that for many applications, average diameter is about 400nm to about 600nm, and it is favourable as about 500nm.

[0143] Thus, embodiments of the present invention relate to methods as described herein, wherein the initial cellulose nanofiber material is prepared by a process selected from the group consisting of electrospinning, meltblowing, fiber drawing, self-assembly, template synthesis, and thermally induced phase separation.

[0144] Another embodiment of the present invention relates to the method as described herein, wherein the initial cellulose nanofiber material is prepared by electrospinning.

[0145] Further embodiments of the present invention relate to a method as described herein, wherein the average diameter of the cellulose nanofibers in the initial cellulose nanofiber material and / or the processed cellulose nanofiber material is in the range of about 10 nm to about 2000 nm, such as about 50 nm to about 1500 nm, such as about 100 nm to about 1000 nm, preferably about 250 nm to about 750 nm.

[0146] A preferred 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 processed cellulose nanofiber material is in the range of about 400 nm to about 600 nm, preferably about 500 nm.

[0147] Initial cellulose nanofiber material can be prepared from a cellulose acetate solution, for example, by electrospinning the cellulose acetate solution. However, before any further processing, the resulting cellulose acetate sheet is preferably regenerated into a cellulose sheet in a sodium hydroxide bath. This treatment opens up hydroxyl groups, which can then be used to attach functional moieties.

[0148] Thus, embodiments of the present invention relate to methods as described herein, wherein the initial cellulose nanofiber material is prepared by electrospinning a cellulose acetate solution into a cellulose acetate sheet.

[0149] Another embodiment of the present invention is directed to the method as described herein, wherein the cellulose acetate sheet is regenerated into a cellulose sheet by treatment with NaOH.

[0150] Still further embodiments of the present invention are directed to methods as described herein, wherein the initial cellulose nanofiber material is regenerated in a regeneration solution comprising sodium hydroxide.

[0151] 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, such as about 0.4 M to about 0.6 M, preferably about 0.5 M.

[0152] Regeneration of the cellulose sheet is preferably carried out in an ethanol solution comprising sodium hydroxide. It is possible to use different amounts of ethanol, such as 5% vol / vol to 99% vol / vol.

[0153] Therefore, embodiments of the present invention relate to methods as described herein, wherein the regeneration solution is an ethanol solution comprising about 5% vol / vol to about 99% vol / vol ethanol, such as about 10% vol / vol to about 95% vol / vol ethanol, such as about 20% vol / vol to about 90% vol / vol ethanol, such as about 30% vol / vol to about 80% vol / vol ethanol, such as about 40% vol / vol to about 70% vol / vol ethanol.

[0154] 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 ethanol content include elasticity and brittleness. Without being bound by theory, it is contemplated that materials with greater elasticity and lower hardness may be more conducive to cell interaction and proliferation.

[0155] Thus, embodiments of the present invention relate to methods as described herein, wherein the regeneration solution is an ethanol solution comprising at least about 50% vol / vol ethanol, such as at least 60% vol / vol ethanol, such as at least 70% vol / vol ethanol, such as at least 80% vol / vol ethanol, such as at least 90% vol / vol ethanol, preferably about 90% vol / vol ethanol.

[0156] Cellulose nanofiber can be combined with other types of nanofibers to enhance the novel properties of nanofiber-like cellulose scaffolds. This may be useful to specific cell types, wherein adding other types of nanofibers broadens the options for mimicking the local extracellular matrix of the specific tissue of the cell. Other polymers can be natural or synthetic. For natural nanofibers, especially the nanofiber types that are traditionally present in the extracellular environment such as collagen can synergize with cellulose to mimic ECM.

[0157] Thus, embodiments of the present invention relate to methods as described herein, wherein the starting cellulose nanofiber material further comprises one or more nanofibers selected from natural polymers or synthetic polymers.

[0158] Another embodiment of the present invention is directed to the method as described herein, wherein the natural polymer is selected from the group consisting of collagen, fibroin, keratin, gelatin, and polysaccharides, and combinations thereof.

[0159] Further embodiments of the present invention are directed to a method as described herein, wherein the synthetic polymer is selected from the group consisting of polycaprolactone (PCL), polylactic 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.

[0160] After the initial cellulose nanofiber material is divided, the resulting processed material is preferably treated to prepare it for functionalization. Part of the treatment may include filtering, washing, and resuspending the processed cellulose nanofiber material. Filtration ensures that excess water is removed from the processed cellulose nanofiber material. Washing helps remove any acetate ions that remain after the previous treatment. When resuspending the processed cellulose nanofiber material, the concentration of cellulose nanofibers can be adjusted as needed.

[0161] Thus, embodiments of the present invention relate to a method as described herein, wherein the dividing step (ii) is followed by steps comprising filtering, washing and suspending the processed cellulose nanofibrous material.

[0162] Another embodiment of the present invention is directed to the method as described herein, wherein said filtering comprises sieving the processed cellulose nanofiber material.

[0163] Yet another embodiment of the present invention relates to the method as described herein, wherein the suspension comprises water and / or ethanol.

[0164] Prior to functionalization, the cellulose nanofibers are preferably mercerized to improve the substitution of functional moieties on the cellulose nanofibers. Mercerization is a process in which the cellulose nanofibers are swollen by immersion in a concentrated aqueous NaOH solution, washed with water and dried. During the mercerization process, the crystal structure of the cellulose nanofibers is transformed from cellulose I to cellulose II. Under the action of a concentrated alkaline solution, the cellulose undergoes chemical, physicochemical and structural modifications. Cellulose II is formed after washing and neutralization. As the alkali penetrates into the crystal lattice, the internal hydrogen bonds are destroyed and the number of available hydroxyl groups (-OH) in the cellulose nanofibers increases. Therefore, mercerization is considered to improve the degree of substitution (DS). The mercerization step can be performed before or after the segmentation step.

[0165] Therefore, embodiments of the present invention relate to a method as described herein, further comprising the step of mercerizing the processed cellulose nanofiber material.

[0166] Another embodiment of the present invention relates to a method as described herein, wherein the mercerizing step is immediately before or after the dividing step (ii).

[0167] Yet another embodiment of the present invention is directed to the method as described herein, wherein the mercerizing step comprises adding NaOH.

[0168] Further embodiments of the invention relate to a method as described herein, wherein the concentration of NaOH is in the range of about 0.05M to about 2M, such as about 0.1M to about 1.5M, such as about 0.25M to about 0.75M, preferably about 0.4M to about 0.6M.

[0169] Still further embodiments of the present invention relate to a method as described herein, wherein the mercerizing step is performed for a period of about 1 hour to about 3 hours, preferably about 2 hours.

[0170] Even further embodiments of the present invention relate to the method as described herein, wherein the degree of substitution of the functional moiety (DS) is in the range of about 0.01 to about 2.

[0171] Another embodiment of the present invention relates to the method as described herein, wherein the degree of substitution of the functional moiety (DS) is in the range of about 0.01 to about 1.5, such as about 0.05 to about 1.2, such as about 0.1 to about 1, such as about 0.2 to about 0.8, such as about 0.4 to about 0.6.

[0172] The amount of substitution can also be quantified as charge equivalents per basic unit mass of cellulose (meq / g).For positively charged functional moieties such as QA and DEAE, a range of 0.5 meq / g to 3.5 meq / g was found to be advantageous.

[0173] Thus, embodiments of the present invention relate to methods as described herein, wherein the functional moiety is QA and / or DEAE, and wherein the charge equivalent per basic unit mass of cellulose on the nanofibrous cellulose scaffold is in the range of about 0.5 meq / g to about 3.5 meq / g, 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.

[0174] The functionalization of the nanofiber-like cellulose scaffold can also be quantified based on the ion exchange capacity of the microcarrier. The ion exchange capacity can be defined as the ability of the functional moieties coupled to the cellulose nanofibers to replace ions attached to their structure with ions of opposite charge in the surrounding solution. The ion exchange capacity is expressed as mmolCl - The unit is / g and is determined by titration.

[0175] Thus, embodiments of the present invention relate to methods as described herein, wherein the ion exchange capacity of the nanofibrous cellulose scaffold is about 0.1 mmol Cl - / g to about 1.5mmol Cl - / g, such as about 0.3mmol Cl - / g to about 1 mmol Cl - / g range.

[0176] Another embodiment of the present invention is directed to the method as described herein, wherein the nanofibrous cellulose scaffold has an ion exchange capacity of at least about 0.1 mmol Cl - / g, such as at least about 0.2 mmol Cl - / g, preferably at least about 0.3 mmolCl - / g.

[0177] Further embodiments of the present invention relate to the methods as described herein, wherein the functional moiety is QA and / or DEAE, and wherein the ion exchange capacity of the nanofiber cellulose scaffold is at least about 0.3 mmol Cl - / g.

[0178] The functional moieties attached to the cellulose nanofibers can be of chemical or biological origin. Specifically, positively charged groups are advantageous because they induce electrostatic interactions between the nanofiber-shaped cellulose scaffold and cells with negatively charged membranes, thereby increasing cell attachment to the microcarriers. Biological moieties include proteins and peptides, which are typically components of the interaction between cells and the extracellular environment. This interaction can further promote cell attachment to the nanofiber-shaped cellulose scaffold.

[0179] Thus, embodiments of the present invention relate to the methods described herein, wherein the functional moiety is selected from a chemical moiety or a biomolecule.

[0180] Another embodiment of the present invention is directed to the method as described herein, wherein the chemical moiety is selected from the group consisting of a negatively charged group, a positively charged group, a zwitterionic group, a hydrophobic group, a hydrophilic group, an amphiphilic group, a ligand, and combinations thereof.

[0181] Additional embodiments of the present invention relate to the method as 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, epoxides, and combinations thereof.

[0182] A preferred embodiment of the present invention relates to a method as described herein, wherein the chemical moiety is a quaternary ammonium (QA).

[0183] It will be appreciated that conventional chemical methods can be used to attach chemical moieties to the cellulose backbone. 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 for attachment of QA, DEAE, and CM, respectively.

[0184] Even further embodiments of the present invention relate to the method as described herein, wherein the biomolecule is selected from the group consisting of proteins, peptides, antibodies, amino acids, polypeptides, glycoproteins, lipoproteins and antigens, and combinations thereof.

[0185] After functionalizing the processed cellulose nanofiber scaffold, the material is dried to obtain its final nanofiber cellulose scaffold form. Drying can be performed in two steps, such as freezing followed by freeze-drying, or in a single step, such as freeze-drying. The dehydration process in a freeze dryer can extend shelf life and / or make the material easier to transport. A freeze dryer works by freezing the material, then reducing the pressure and applying heat to sublime the frozen water in the material.

[0186] Thus, embodiments of the present invention relate to methods as described herein, wherein the drying step (iv) comprises freezing and / or freeze-drying the processed cellulose nanofiber material.

[0187] Another embodiment of the present invention relates to the method as described herein, wherein the drying step (iv) comprises freezing the processed cellulose nanofibrous material and then freeze-drying the frozen processed cellulose nanofibrous material.

[0188] The dried nanofibrous cellulose scaffold can be further processed to provide a dry powder. This can be achieved by grinding the dried product.

[0189] The methods described herein provide a nanofiber-like cellulose scaffold with a large surface area and low dead volume that can be advantageously used as a microcarrier for culturing cells. 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 to which cells can attach and proliferate.

[0190] Thus, aspects of the present invention relate to a nanofibrous cellulose scaffold obtainable by a method as described herein.

[0191] Another aspect of the present invention relates to a nanofiber-like cellulose scaffold comprising a processed cellulose nanofiber material having an average length of cellulose nanofibers less than about 250 μm, and wherein the processed cellulose nanofiber material is functionalized with a functional moiety.

[0192] Nanofibrous cellulose scaffolds have a high surface area. Without being bound by theory, it is believed that the high surface area is caused in particular by the uniform distribution of processed cellulose nanofibers in the material, which ensures that large entanglements and clusters of nanofibers are avoided. Therefore, the nanofibrous cellulose scaffold provides an excellent available surface area that is conducive to the cultivation of adherent cells.

[0193] Embodiments of the present invention relate to nanofibrous cellulose scaffolds as described herein, wherein the nanofibrous cellulose scaffold has a BET surface area of ​​at least about 40,000 cm 2 / g, such as at least about 50,000 cm 2 / g, such as at least about 55000cm 2 / g, such as at least about 60,000 cm 2 / g, such as at least about 70,000 cm 2 / g, such as at least about 80,000 cm 2 / g.

[0194] Another embodiment of the present invention is directed to a nanofibrous cellulose scaffold as described herein, wherein the nanofibrous cellulose scaffold has a BET surface area of ​​at least about 25,000 cm 2 / g, such as at least about 30,000 cm 2 / g, such as at least about 40,000 cm 2 / g, such as at least about 50,000 cm 2 / g, such as at least about 60,000 cm 2 / g, such as at least about 70,000 cm 2 / g.

[0195] Yet another embodiment of the present invention is directed to the nanofibrous cellulose scaffold as described herein, wherein the BET surface area is measured according to ISO 9277:2022 - Determination of specific surface area of ​​solids by gas adsorption - BET method.

[0196] Preferably, the cellulose nanofiber scaffold is provided as a dry material, which is convenient and easy to handle for the end user. The dry material can be contained in a container suitable for direct addition to a cell culture vessel. Microcarriers in dry powder form can also be reconstituted / hydrated in an aqueous solution such as PBS and / or culture medium and then added to the cell culture vessel.

[0197] Thus, embodiments of the present invention relate to nanofibrous cellulose scaffolds as described herein, wherein the nanofibrous cellulose scaffold is provided as a dry material.

[0198] Another embodiment of the present invention is directed to a nanofiber-cellulose scaffold as described herein, wherein the nanofiber-cellulose scaffold is provided as a freeze-dried material.

[0199] Although bacterial, yeast, and insect cell expression systems are capable of overexpressing recombinant proteins, mammalian cell culture remains the cornerstone of biomanufacturing of biologics due to its ability to propagate human viruses, express monoclonal antibodies, and incorporate post-translational modifications such as glycosylation, which are crucial for the production of effective biologics. The most commonly used mammalian cell line is the human embryonic kidney (HEK) 293 cell, which has been engineered to produce high levels of therapeutic proteins and antibodies. However, HEK 293 cells and other adherent mammalian cells are highly dependent on a suitable support matrix for cell proliferation and viability. Therefore, microcarriers are an important product in the continued push towards more cost-effective biomanufacturing.

[0200] The nanofibrous cellulose scaffolds described here possess all the properties required for efficient microcarriers (high surface area, low dead volume, and customizability). Importantly, the nanofibrous cellulose scaffolds can be easily scaled up for industrial use without compromising large-scale use due to cost and microcarrier suitability.

[0201] Thus, aspects of the present invention relate to a microcarrier comprising a nanofibrillar cellulose scaffold as described herein.

[0202] Another aspect of the present invention relates to the use of the nanofibrous cellulose scaffold as described herein as a microcarrier for cell culture.

[0203] A further aspect of the present invention relates to a cell culture device comprising a container loaded with the nanofibrous cellulose scaffold or microcarrier as described herein.

[0204] The container of the cell culture apparatus is not limited to any particular container, as long as it is a vessel suitable for culturing cells.

[0205] Thus, embodiments of the present invention relate to a cell culture device as described herein, wherein the container is selected from the group consisting of: a bioreactor, a cell culture plate, a cell culture bottle, a spinner flask, a shaker flask and a roller bottle, a petri dish and a tube, preferably a bioreactor.

[0206] Bioreactors are particularly important for large-scale industrial production. Compared to laboratory or pilot research equipment, they can accommodate larger volumes and be used for upstream processes to expand and scale up cell cultures for production. A typical process involves initial expansion of cells in smaller vessels, followed by continuous expansion into larger culture vessels. When the culture volume and density are optimal, the cells are transferred to production bioreactors, which provide a controlled microenvironment and nutrient delivery to regulate cell growth and differentiation, thereby improving standardization and reproducibility. Nanofibrous cellulose scaffolds are suitable for any point in the process and are particularly advantageous at production scale, where other microcarriers may be too expensive or unsuitable for use.

[0207] Thus, embodiments of the present invention relate to a cell culture device as described herein, wherein the container is a bioreactor, preferably a suspension-based bioreactor.

[0208] The nanofiber-like cellulose scaffold is suitable for all processes from laboratory scale (1 ml to 5 L), through pilot scale / product development (1 L to 100 L), and to production scale (100 L to 1000 L). If necessary, the material can also be used as microcarriers in even larger bioreactors.

[0209] Thus, embodiments of the present invention relate 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, such as about 1 L to about 1000 L, such as about 100 L to about 1000 L.

[0210] Another embodiment of the present invention relates to a cell culture device as described herein, wherein the volume of the container is at least 10 L, such as at least 50 L, such as at least 100 L, such as at least 200 L, such as at least 500 L, such as at least 1000 L.

[0211] The container is not limited to any particular material, but can be made of, for example, stainless steel or disposable materials, the latter providing flexibility and reducing downtime caused by the need to clean and sterilize the culture device.

[0212] Another embodiment of the present invention is directed to a cell culture device as described herein, wherein the cell culture device comprises a solvent.

[0213] Further embodiments of the present invention relate to a cell culture device as described herein, wherein the solvent is a cell culture medium.

[0214] Nanofiber-shaped cellulose scaffold can be used as microcarrier in the traditional method for cell culture.Nanofiber-shaped cellulose scaffold can be provided as a part for cell culture device, and described cell culture device comprises container, and described container is loaded with microcarrier.Considering that nanofiber-shaped cellulose scaffold is applicable to cultivating any cell line, described any cell line can benefit from the interaction with supporting matrix during incubation.It is understandable that nanofiber-shaped cellulose scaffold can be used for any traditional expansion step for cell culture, namely first microcarrier and cell are added in small container (such as flask), then cell colony is transferred to large container, finally cell culture is transferred on bioreactor.

[0215] Therefore, aspects of the present invention relate to a method for culturing cells, comprising the steps of:

[0216] (i) providing a cell culture device as described herein,

[0217] (ii) adding a composition comprising a cell population to the cell culture device,

[0218] (iii) incubating the cell population to provide a expanded cell population, and

[0219] (iv) Optionally, extracting the expanded cell population from the cell culture device.

[0220] Embodiments of the invention relate to methods for cell culture as described herein, wherein the composition comprises a solvent.

[0221] Further embodiments of the invention relate to methods for cell culture as described herein, wherein the solvent comprises cell culture medium.

[0222] Methods for cell culture are particularly advantageous for culturing adherent cells that grow while attached to the culture vessel. Ideally, cultured cells should be cultured in a manner that reflects the conditions they would experience in a living organism. Adherent cells are cells that adhere to tissue under natural conditions.

[0223] Accordingly, embodiments of the present invention relate to methods for cell culture as described herein, wherein the cell population comprises adherent cells.

[0224] Another embodiment of the present invention relates to a method for cell culture as 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.

[0225] Additional embodiments of the invention relate to methods for cell culture 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, heart cells, ovarian cells, hybridoma cells, and immortalized cells.

[0226] Still further embodiments of the invention relate to methods for cell culture as described herein, wherein the cell population comprises a cell type selected from the group consisting of: myocytes, myoblasts, and adipocytes.

[0227] For biomanufacturing purposes, some cell lines are preferred due to properties such as ease of handling, ability to propagate human viruses, or implementation of favorable glycosylation patterns, etc. An example of this is HEK293 cells, which can be used for packaging and amplification of recombinant adenoviruses.

[0228] Accordingly, embodiments of the present invention relate to methods for cell culture as 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.

[0229] Further embodiments of the invention relate to methods for cell culture as described herein, wherein the cell population comprises stem cells.

[0230] Another embodiment of the present invention relates to a method for cell culture as described herein, wherein the cell population comprises the HEK 293 cell line.

[0231] The methods used for cell culture can also be readily applied to cellular agriculture products, as well as new methods for producing existing agricultural products like milk and (cultured) meat from cells. Cellular agriculture is considered a way to achieve animal-free agriculture. Since cellulose is a biocompatible polymer, this paper considers nanofiber-like cellulose scaffolds for cellular agriculture.

[0232] The method for culturing cells can be carried out with or without agitation of the cell culture within the container. Agitation, if present, is preferably achieved by stirring the culture medium in which the cells are grown. Agitation promotes better delivery of nutrients and oxygen to the cells.

[0233] Therefore, embodiments of the present invention relate to methods for cell culture as described herein, wherein the incubation is performed in the absence of agitation.

[0234] Another embodiment of the present invention relates to the method for cell culture as described herein, wherein the incubation is performed with agitation.

[0235] Further embodiments of the invention relate to a method for cell culture as described herein, wherein agitation is selected from the group consisting of stirring, shaking, rocking, waving and bubbling, preferably stirring.

[0236] The listing or discussion of an apparently prior-published document in this specification should not necessarily be taken as an admission that the document is part of the state of the art or is common general knowledge.

[0237] Unless the context indicates otherwise, preferences, options and embodiments of a given aspect, feature or parameter of the invention should be considered to have been disclosed in conjunction with any and all preferences, options and embodiments of all other aspects, features and parameters of the invention. This is particularly true for the description of the method for preparing the nanofibrous cellulose scaffold and all its features, which can itself easily become part of the nanofibrous cellulose scaffold, or use or method of cell culture using the same. Embodiments and features of the invention are also summarized in the following clauses.

[0238] Terms

[0239] X1. A method for preparing a nanofibrous cellulose scaffold, the method comprising the following steps:

[0240] (i) providing an initial cellulose nanofiber material,

[0241] (ii) dividing the initial cellulose nanofibrous material into processed cellulose nanofibrous materials,

[0242] (iii) functionalizing the processed cellulose nanofiber material by adding a reagent comprising a functional moiety, and

[0243] (iv) drying the processed cellulose nanofiber material,

[0244] Thereby, the nanofibrous cellulose scaffold is provided.

[0245] X2. The method according to clause X1, wherein dividing the initial cellulose nanofibrous material is achieved by dispersion.

[0246] X3. The method according to item X2, wherein the dispersion is carried out using a high-speed disperser.

[0247] X4. The method of any of clauses X2 or X3, wherein the dispersing is performed for at least 2 minutes, such as at least 5 minutes, such as at least 10 minutes.

[0248] X5. A method according to any one of clauses X2 to X4, wherein the dispersing is carried out at a speed in the range of about 10,000 rpm to about 30,000 rpm, such as at least 12,000 rpm to about 25,000 rpm, such as at least 15,000 rpm to about 20,000 rpm.

[0249] X6. The method according to any of the preceding clauses, 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.

[0250] X7. A method according to any of the preceding clauses, 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, such as less than about 150 μm, such as less than about 120 μm, such as less than about 100 μm, such as less than about 80 μm.

[0251] X8. A method according to any of the preceding clauses, wherein the average length of the cellulose nanofibers in the processed cellulose nanofiber material is in the range of about 30 μm to about 250 μm, such as about 40 μm to about 200 μm, such as about 50 μm to about 150 μm, preferably about 60 μm to about 100 μm.

[0252] X9. A method according to any of the preceding clauses, wherein the initial cellulose nanofiber material is provided as a liquid sample.

[0253] X10. A method according to item X9, wherein the solvent of the liquid sample comprises water and / or ethanol.

[0254] X11. A method according to any of the preceding clauses, wherein the concentration of cellulose nanofibers in the processed cellulose nanofiber material is about 0.1 wt% to about 10 wt%, such as about 0.5 wt% to about 5 wt%, such as about 0.75 wt% to about 4 wt%, preferably about 1 wt% to about 3 wt%, relative to the total weight of the liquid sample.

[0255] X12. A method according to any of the preceding clauses, 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, such as about 50 nm to about 1500 nm, such as about 100 nm to about 1000 nm, preferably about 250 nm to about 750 nm.

[0256] X13. A method according to any of the preceding clauses, wherein the initial cellulose nanofiber material further comprises one or more nanofibers selected from natural polymers or synthetic polymers.

[0257] X14. The method according to clause X13, wherein the natural polymer is selected from the group consisting of collagen, fibroin, keratin, gelatin and polysaccharides, and combinations thereof.

[0258] X15. A method according to any one of clauses X13 or X14, wherein the synthetic polymer is selected from the group consisting of: polycaprolactone (PCL), polylactic 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.

[0259] X16. A method according to any of the preceding clauses, wherein the initial cellulose nanofiber material is prepared by a process selected from the group consisting of: electrospinning, melt blowing, fiber drawing, self-assembly, template synthesis and thermally induced phase separation.

[0260] X17. The method according to any of the preceding clauses, further comprising the step of mercerizing the processed cellulose nanofiber material.

[0261] X18. A method according to clause X17, wherein the mercerizing step occurs immediately before or after the dividing step (ii).

[0262] X19. A method according to any of clauses X17 or X18, wherein the mercerizing step comprises adding NaOH.

[0263] X20. A method according to item X19, wherein the concentration of NaOH is in the range of about 0.05M to about 2M, such as about 0.1M to about 1.5M, such as about 0.25M to about 0.75M, preferably about 0.4M to about 0.6M.

[0264] X21. A method according to any one of clauses X17 to X20, wherein the mercerizing step is carried out for a period of about 1 hour to about 3 hours, preferably about 2 hours.

[0265] X22. A method according to any of the preceding clauses, wherein the functional moiety is selected from a chemical moiety or a biological molecule.

[0266] X23. The method of clause 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.

[0267] X24. A method according to any of clauses 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.

[0268] X25. A method according to any one of clauses X22 to X24, wherein the chemical moiety is a quaternary ammonium (QA).

[0269] X26. A method according to any one of clauses X22 to X25, wherein the biomolecule is selected from the group consisting of proteins, peptides, antibodies, amino acids, polypeptides, glycoproteins, lipoproteins and antigens, and combinations thereof.

[0270] X27. A method according to any of the preceding clauses, wherein the degree of substitution of the functional moiety (DS) is in the range of about 0.01 to about 2.

[0271] X28. A method according to any of the preceding clauses, wherein the dividing step (ii) is followed by a step comprising filtering, washing and suspending the processed cellulose nanofibrous material.

[0272] X29. The method of clause X28, wherein the filtering comprises screening the processed cellulose nanofiber material.

[0273] X30. A method according to any of clauses X28 or X29, wherein the suspension comprises water and / or ethanol.

[0274] X31. A method according to any of the preceding clauses, wherein the drying step (iv) comprises freezing and / or freeze-drying the processed cellulose nanofiber material.

[0275] X32. A method according to any of the preceding clauses, wherein the drying step (iv) comprises freezing the processed cellulose nanofiber material and then freeze-drying the frozen processed cellulose nanofiber material.

[0276] Z1. A nanofibrous cellulose scaffold obtainable from a method according to any one of clauses X1 to X32.

[0277] Y1. A nanofiber-like cellulose scaffold comprising a processed cellulose nanofiber material having cellulose nanofibers having an average length of less than about 250 μm, and wherein the processed cellulose nanofiber material is functionalized with a functional moiety.

[0278] Y2. A nanofiber-like 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, such as about 40 μm to about 200 μm, such as about 50 μm to about 150 μm, preferably about 60 μm to about 100 μm.

[0279] Y3. A nanofiber-like cellulose scaffold according to any one of clauses Y1 or Y2, wherein the average diameter of the cellulose nanofibers ranges from about 10 nm to about 2000 nm, such as from about 50 nm to about 1500 nm, such as from about 100 nm to about 1000 nm, preferably from about 250 nm to about 750 nm.

[0280] Y4. The nanofiber-like cellulose scaffold according to any one of clauses Y1 to Y3, wherein the processed cellulose nanofiber material is electrospun, meltblown or drawn, preferably electrospun.

[0281] Y5. The nanofibrillar cellulose scaffold of any one of clauses Y1 to Y4, wherein the functional moiety is selected from a chemical moiety or a biomolecule.

[0282] Y6. The nanofiber-like cellulose scaffold of clause 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.

[0283] Y7. A nanofibrous cellulose scaffold according to any one of clauses 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.

[0284] Y8. The nanofibrous cellulose scaffold of any one of clauses Y5 to Y7, wherein the chemical moiety is a quaternary ammonium (QA).

[0285] Y9. The nanofibrillar cellulose scaffold of any one of clauses Y5 to Y8, wherein the biomolecule is selected from the group consisting of proteins, peptides, antibodies, amino acids, polypeptides, glycoproteins, lipoproteins and antigens, and combinations thereof.

[0286] Y10. The nanofiber-like cellulose scaffold of any one of clauses Y1 to Y9, wherein the degree of substitution of the functional moiety (DS) is in the range of about 0.01 to about 2.

[0287] Y11. The nanofibrous cellulose scaffold of any one of clauses Y1 to Y10, wherein the nanofibrous cellulose scaffold is provided as a dry material.

[0288] Y12. The nanofibrous cellulose scaffold of any one of clauses Y1 to Y11, wherein the nanofibrous cellulose scaffold is provided as a freeze-dried material.

[0289] Y13. The nanofiber-like cellulose scaffold of any one of clauses Y1 to Y12, wherein the nanofiber-like cellulose scaffold further comprises one or more nanofibers selected from natural polymers or synthetic polymers.

[0290] Y14. The nanofiber-like cellulose scaffold of clause Y13, wherein the natural polymer is selected from the group consisting of collagen, fibroin, keratin, gelatin, and polysaccharides, and combinations thereof.

[0291] Y15. A nanofibrous cellulose scaffold according to any one of clauses Y13 or Y14, wherein the synthetic polymer is selected from the group consisting of: polycaprolactone (PCL), polylactic 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.

[0292] Y16. The nanofibrous cellulose scaffold of any one of clauses Y1 to Y15, wherein the nanofibrous cellulose scaffold has a BET surface area of ​​at least about 40,000 cm 2 / g, such as at least about 50,000 cm 2 / g, such as at least about 55000cm 2 / g, such as at least about 60,000 cm 2 / g, such as at least about 70,000 cm 2 / g, such as at least about 80,000 cm 2 / g.

[0293] A1. A microcarrier comprising a nanofibrillar cellulose scaffold according to any one of clauses Y1 to Y16 or Z1.

[0294] U1. Use of a nanofibrous cellulose scaffold according to any one of clauses Y1 to Y16 or Z1 as a microcarrier for cell culture.

[0295] V1. A cell culture device comprising a container loaded with the nanofibrous cellulose scaffold according to any one of clauses Y1 to Y16 or Z1 or the microcarrier according to clause A1.

[0296] V2. The cell culture apparatus according to clause V1, wherein the container is selected from the group consisting of: a bioreactor, a cell culture plate, a cell culture bottle, a spinner flask, a shaker flask and a roller bottle, a petri dish and a tube, preferably a bioreactor.

[0297] V3. A cell culture device according to any of clauses V1 or V2, wherein the container is a bioreactor, preferably a suspension-based bioreactor.

[0298] V4. A cell culture device according to any one of clauses V1 to V3, wherein the volume of the container is at least 10 L, such as at least 50 L, such as at least 100 L, such as at least 200 L, such as at least 500 L, such as at least 1000 L.

[0299] V5. The cell culture device of any one of clauses V1 to V4, wherein the cell culture device comprises a solvent.

[0300] V6. A cell culture device according to clause V5, wherein the solvent is cell culture medium.

[0301] T1. A method for cell culture, comprising the following steps:

[0302] (i) providing a cell culture device according to any one of clauses V1 to V6,

[0303] (ii) adding a composition comprising a cell population to the cell culture device,

[0304] (iii) incubating the cell population to provide a expanded cell population, and

[0305] (iv) Optionally, extracting the expanded cell population from the cell culture device.

[0306] T2. The method of item T1, wherein the composition comprises a solvent.

[0307] T3. The method of item T2, wherein the solvent comprises cell culture medium.

[0308] T4. The method of any one of clauses T1 to T3, wherein the cell population comprises adherent cells.

[0309] T5. The method according to any one of clauses T1 to T4, 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.

[0310] T6. A method according to any one of clauses T1 to 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, heart cells, ovarian cells, hybridoma cells and immortalized cells.

[0311] T7. A method according to any one of clauses 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.

[0312] T8. The method of any one of clauses T1 to T7, wherein the incubation is performed without agitation.

[0313] T9. The method of any one of items T1 to T8, wherein the incubation is carried out with agitation.

[0314] T10. The method of clause T9, wherein agitation is selected from the group consisting of stirring, shaking, shaking, waving and foaming, preferably stirring.

[0315] The present invention will now be described in further detail in the following non-limiting examples.

[0316] Examples

[0317] Example 1: Preparation of nanofibrous cellulose scaffolds

[0318] In this example, a non-limiting demonstration of how a nanofibrillar cellulose scaffold can be prepared is given. The core properties of the material such as surface area and degree of substitution were characterized.

[0319] method

[0320] Preparation of initial cellulose nanofiber materials

[0321] 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 overnight at room temperature using a magnetic stirrer.

[0322] A 19% cellulose acetate solution was electrospun to produce a cellulose acetate nanofiber sheet. The cellulose nanofibers were electrospun (Fluidnatek LE50) at a drum speed of 200 rpm and a flow rate of 10 ml / hour at 18 kV+ (emitter) and 10 kV- (collector). The cellulose acetate nanofibers were collected on an aluminum substrate at a temperature of 23°C and a relative humidity of 63%. After the electrospinning process was completed, the cellulose acetate sheet containing the cellulose nanofibers was removed from the drum.

[0323] A cellulose acetate sheet was immersed in a 0.5 M NaOH solution in ethanol to regenerate it into cellulose. The cellulose acetate sheet was left in the solution for 6 hours, then transferred to a sieve and washed with a large amount of distilled water. The washed sheet was placed in an oven at 80°C for 12 hours to provide a dry cellulose sheet. The cellulose sheet was weighed.

[0324] Processing of initial cellulose nanofiber materials

[0325] Use scissors to cut the dry cellulose sheet into coarse fragments of about 2x2 cm square. The size of the fragments does not have to be accurate, but larger fragments should be avoided because they may hinder the segmentation step. The coarse fragments of cellulose are completely immersed in water and dispersed at 18,000rpm for 60 minutes (intermittent shutdowns to cool the disperser) using a high-speed disperser (IKA T25 Digital Ultra Turrax). The processed cellulose nanofiber material is transferred to a sieve, washed with water to remove any residual acetate ions, and drained to remove excess water. The cellulose nanofiber material is transferred to a flask, and fresh water is added to provide a cellulose concentration of 2wt%.

[0326] This unfunctionalized sample produced a nanofibrous cellulose scaffold referred to as "Cellevate Unfunctionalized."

[0327] Functionalization of processed cellulose nanofiber materials

[0328] The processed cellulose nanofiber materials were functionalized with different chemical compounds according to the following procedure.

[0329] Quaternary ammonium (QA):

[0330] 0.4 g of NaOH was dissolved in 20 ml of water. 0.4 g of regenerated cellulose nanofiber material was then added to the solution. The mercerization process was continued at room temperature for 2 hours.

[0331] 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 filtered, cooled to room temperature, and repeatedly washed with water to remove any unreacted CHPTAC or NaOH. Finally, the filtered and functionalized cellulose nanofiber material was mixed with water to prepare a 2% suspension for further experiments.

[0332] This functionalization protocol resulted in a nanofibrous cellulose scaffold dubbed "Cellevate QA."

[0333] Carboxymethyl (CM):

[0334] 0.6 g of NaOH was dissolved in 25 ml of ethanol. 0.5 g of regenerated cellulose nanofiber material was then added to the solution. The mercerization process was continued at room temperature for 1 hour.

[0335] 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 at 60° C. for 2 hours. Filtration and washing were performed as for the QA functionalization.

[0336] This functionalization scheme resulted in a nanofibrous cellulose scaffold termed "Cellevate CMC."

[0337] Carboxymethyldiethylaminoethyl (CM-DEAE):

[0338] 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 under stirring. The reaction was carried out at 40° C. for 15 minutes.

[0339] Subsequently, the regenerated cellulose nanofiber material was transferred to a 0.5 M NaOH solution at 80° C. for 10 minutes to complete the reaction. Filtration and washing were performed as for QA functionalization.

[0340] The DEAE-functionalized cellulose nanofiber material was then subjected to the CM protocol described above to obtain a DEAE-CM functionalized nanofibrous cellulose scaffold referred to as "Cellevate DEAE+CM."

[0341] dry

[0342] The functionalized or unfunctionalized cellulose nanofiber material was transferred to a -85°C freezer overnight. The frozen cellulose nanofiber material was then transferred to a freeze dryer and processed for 48 hours to obtain a dry product. The dry product was ground to provide a dry powder of the nanofiber-like cellulose scaffold.

[0343] microscope

[0344] A sample of the nanofiber-like cellulose scaffold (diluted to 0.2 wt% cellulose) was added to a glass slide. Images of the network of cellulose nanofibers in the sample were captured using an optical microscope (Leica) at 40x magnification.

[0345] Scanning electron microscopy

[0346] The morphology of the nanofibrous cellulose scaffolds was investigated using scanning electron microscopy (SEM). The samples were dried and sputter-coated with gold prior to analysis. Micrographs were acquired on a Hitachi SU3500 in secondary electron (SE) imaging mode at an accelerating voltage of 5 kV and a working distance of 7 mm at various magnifications.

[0347] Surface area measurement

[0348] The Brunauer-Emmett-Teller (BET) model was used, with a molecular cross-sectional area of ​​0.162 nm. 2 The specific surface area was measured based on the presence of nitrogen molecules. The dried samples were degassed under vacuum for 6 hours before measuring the BET surface area. Nitrogen adsorption and desorption isotherms were collected at 77.3 K on an ASAP 2020M analyzer (Micromeritics).

[0349] Elemental analysis and degree of substitution

[0350] Elemental analysis of the nanofibrous cellulose scaffolds was performed using 5 mg of sample on an elemental analyzer FlashEA 1112 (ThermoFischer Scientific).The samples were thoroughly dried prior to analysis to remove any adsorbed moisture.

[0351] The degree of substitution (DS) of QA-functionalized cellulose (i.e., Cellevate QA) was calculated using the following formula:

[0352] DS = (162N / (1400-CAxN))

[0353] Where 162 is the molecular weight of anhydroglucose unit (AGU); N is the percentage of nitrogen; CA is the molecular weight of the cationic agent. For the nanofibrous cellulose scaffold of the present invention, the cationic agent is CHPTAC with a molecular weight of 188.1.

[0354] result

[0355] Nanofibrous cellulose scaffolds with different types of functional moieties were prepared and characterized by optical microscopy ( Figure 1 A) and SEM( Figure 1 C) dry the powder product ( Figure 1 B) Visualization. The resulting nanofiber-like cellulose scaffold material is highly uniform, as individual nanofibers are clearly visible without any significant entanglement or clustering. The uniform distribution of nanofibers throughout the material ensures optimal exposure of the surface area for cell attachment and interaction.

[0356] The elemental composition and degree of substitution (DS) of nanofibrous cellulose scaffolds 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%. Based on the nitrogen content, the DS was calculated to be 0.18. Because positive charges promote electrostatic interactions with negatively charged cell membranes, the amount of QA substituted may affect cell growth.

[0357] The prepared nanofibrous cellulose scaffold samples are listed in Table 1 along with the benchmark commercial microcarrier Cytodex-1 (Cytiva), which are spherical dextran particles functionalized with diethylaminoethyl (DEAE).

[0358] The surface area of ​​the nanofibrous cellulose scaffold can be approximated in theoretical calculations using the following equation:

[0359] Theoretical surface area per unit weight (cm 2 / g)=2 / (r xδ)

[0360] 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.

[0361] The nanofibrous cellulose scaffold provides an increased surface area compared to market standard microcarriers. The accuracy of the theoretical calculations is supported by the measurement of the BET surface area of ​​a 0.5% cellulose sample, which gives a value of 58,000 cm 2 / g of BET surface area.

[0362]

[0363]

[0364] Table 1. Summary of samples compared herein. The surface area of ​​the Cytodex-1 microcarriers was taken from the product datasheet, while the surface area of ​​the nanofiber cellulose scaffolds was theoretically calculated. Fiber length and diameter were obtained as described in Example 4.

[0365] in conclusion

[0366] This example shows that it is possible to produce several different variations of nanofiber-like cellulose scaffolds in a simple manner that is easily scalable for industrial use. The nanofiber-like cellulose scaffolds have a large surface area available to cells and do not contain nanofibers that are folded into clusters or tangles in a way that prevents cells from accessing the surface.

[0367] Example 2: Cell proliferation on nanofibrous cellulose scaffolds

[0368] In this example, the ability of cells to grow on nanofibrous cellulose scaffolds was evaluated. To explore the properties of cellulose nanofibers beyond the available surface area, the amount of added microcarriers was adjusted to provide an equal amount of surface area for the cells, thereby allowing a direct comparison between the nanofibrous cellulose scaffolds and the market standard microcarrier, Cytodex-1.

[0369] method

[0370] Cell expansion

[0371] HEK293AD and ARN8 cells were plated at 10,000 cells / cm 2 (The total is 1.75 x 10 6 The density of cells was seeded at 175 cm 2 30 ml of Dulbecco's modified eagle medium (DMEM) supplemented with 10% fetal calf serum (FCS) was placed in a Nunc flask. After three 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. After the cells were detached from the flask, 9 ml of DMEM 10% FCS was added to stop the trypsin activity. The cells were suspended and homogenized by pipetting and then counted using a hemocytometer.

[0372] Preparation of microcarriers

[0373] Nanofibrous cellulose scaffolds were prepared as described in Example 1. The following samples were evaluated: Cellevate unfunctionalized, Cellevate CMC, Cellevate QA, and Cellevate DEAE+CMC.

[0374] For this experiment, Cytodex-1 and nanofibrous cellulose scaffold microcarriers were prepared with the same approximate available surface area. Because the surface area per unit volume differed between the carriers, varying amounts of microcarriers were used.

[0375] Cytodex-1 microcarriers were prepared in PBS according to the manufacturer's recommendations. 1.33 g of Cytodex-1 dry beads were prepared by diluting in 40 ml of PBS and then autoclaving. The autoclaved beads had a diameter of 190 μm and a surface area of ​​3000 cm 2 / g. The total surface area per unit volume is 100cm 2 / ml. (1.33g x 3000cm 2 / g / 40ml=100cm 2 / ml).

[0376] 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 surface area of ​​50,000 cm 2 / g (for ease of approximation, from 53333cm 2 / g (rounded up). The total surface area per unit volume is 500cm 2 / ml(0.5gx 50000cm 2 / g / 50ml=500cm 2 / ml).

[0377] To provide the same amount of surface area to the cells, 500 μl of Cytodex-1 solution (500 μl x 100 cm 2 / ml=50cm 2 ) and using 100 μl of nanofibrous cellulose scaffold solution (100 μl x 500 cm 2 / ml=50cm 2 ).

[0378] To account for the unused space occupied by the interior of the microcarriers, a theoretical "dead volume" was calculated for each microcarrier (Table 2).

[0379] The radius of a Cytodex-1 bead is 95 μm; therefore, the surface area of ​​a single bead is:

[0380] A=4πx r 2 =4πx(0.095mm) 2 =0.113mm 2 .

[0381] Since the surface area of ​​the beads is 3000 cm 2 / g, so there are 2654867 beads / g (300000mm 2 / g / 0.113mm 2 / beads). Cytodex-1 microcarriers were prepared with 3530973 beads in 40 ml, thus 88274 beads / ml (yielding 100 cm 2 / ml of solution).

[0382] The volume of Cytodex-1 beads (spheres) is:

[0383] V = 4 / 3 x r 3 =4 / 3xπx(0.095mm) 3 =0.00359mm 3

[0384] Therefore, 100cm 2 The dead volume of Cytodex-1 beads (88274 beads) is 317 μl / ml of culture medium (88274 beads / ml x 0.00359 μl / bead = 317 μl / ml).

[0385] The diameter of the cellulose nanofiber is 500 nm, and the length of the cellulose nanofiber is 72 μm. The surface area of ​​a single fiber (cylinder) is:

[0386] A=2πx r 2 +2πx rxh=2πx(0.00025mm) 2 +2πx(0.00025mm)x(0.072mm), which =

[0387] 0.000113mm 2 .

[0388] Since the surface area of ​​the fiber is 50,000 cm 2 / g, so there are 4.4x 10 10 Root fiber / g(5000000mm 2 / g / 0.000113mm 2 / fiber). Nanofibrous cellulose scaffold microcarriers were prepared at 2.2 x 10 10 Root fiber preparation, therefore 4.4 x 10 8 Root fiber / ml (produces 500cm2 / ml of solution).

[0389] The volume of a single fiber (cylinder) is:

[0390] V=πx hxr 2 =πx(0.072mm)x(250x10 -6 mm) 2 =1.4x10 -8 mm 3 .

[0391] Therefore, 500cm 2 of fiber (4.4 x 10 8 Root fibers) with a dead volume of 6.16 μl / ml of culture medium (4.4 x 10 8 Fibers / ml x 1.4 x 10 -8 μl / fiber). This gives 100 cm 2 The dead volume of the culture medium was 1.23 μl / ml.

[0392]

[0393] Table 2. Summary of the physical properties of nanofiber-like cellulose scaffolds and Cytodex-1.

[0394] Seeding cells on microcarriers

[0395] In 1 ml of cell culture medium or 1.5 ml of Cytodex-1 final volume (taking into account the dead volume of Cytodex-1), plate at 10,000 cells / cm 2 , 20,000 cells / cm 2 , 40,000 cells / cm 2 , 60,000 cells / cm 2 or 120,000 cells / cm 2 The cells were seeded at a density of 50 cm 2 Each microcarrier (100 μl of nanofibrous cellulose scaffold microcarrier solution or 500 μl of Cytodex-1 solution) was loaded with 100 μl of nanofibrous cellulose scaffold microcarrier solution.

[0396] The cells were incubated overnight (maximum 18 hours) in an incubator at 37° C., 5% CO 2 (without shaking). The cap of the Falcon tube was not completely closed to allow for gas exchange.

[0397] The next morning, 9 ml of cell culture medium (DMEM, 10% FCS) was added to the cells and pipetted up and down ten times with a 10 ml pipette to break up the cell aggregates. The cells (in 10 ml of culture medium) were then transferred to a 10 cm uncoated Petri dish. 2 The highest density in 10 ml of culture medium was 600,000 cells / ml (120,000 cells / cm 2 The petri dish was then placed in an incubator at 37°C, 5% CO2 on an orbital shaker at 60 rpm.

[0398] Passaging procedures and cell culture

[0399] For long-term culture (> 3 days), cells were passaged. After 48 hours, the cells were pipetted up and down ten times with a 10 ml pipette to break up the cell aggregates, and then the cells were transferred to a 125 ml Corning flask. Fresh culture 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 , which is equivalent to 200 μl of nanofibrous cellulose scaffold microcarriers or 1 ml of Cytodex-1). For short-term cell culture and experiments (<3 days), cells were not passaged.

[0400] The cells were incubated at 37°C, 8% CO2 on an orbital shaker at 90 rpm. After 48 hours, 20 ml of fresh culture medium was added to an additional 200 cm 2 Add microcarriers to make up to 50 ml.

[0401] Cell viability

[0402] As described above, cells are grown and passaged on nanofiber cellulose scaffold microcarriers or Cytodex-1. At the appointed time, 10ml pipettes are pipetted ten times by cells / fiber to break up cell / fiber aggregation, and obtain the uniform suspension of cells / fiber in cell culture medium. Then the suspension of the cell / fiber culture medium of 500 μ l is mixed with 2X passive lysis buffer (Promega) of 500 μ l. The purpose of large volume is to minimize the sampling effect, and the accuracy of reading is maximized. Then the lysate is centrifuged at 14000rpm for 15 minutes at 4 DEG C. Then the supernatant lysate of 10 μ l is diluted in the passive lysis buffer 1X of 90 μ l (dilution ratio is 1: 10. Then the diluted lysate of 5 μ l is added in the lysis buffer of 25 μ l). Then lactate dehydrogenase (LDH) activity is analyzed according to the advice of the manufacturer. LDH activity is proportional to the cell number represented on the y axis.

[0403] result

[0404] Cells initially attach better to the nanofibrous cellulose scaffolds than to market standard microcarriers ( Figure 1 DE). Within the relevant culture time window, two cell lines (HEK293AD and ARN8) proliferated rapidly and at high density on the nanofiber cellulose scaffolds, and specifically on the scaffolds with functional moieties ( Figure 2 AB). All nanofibrous cellulose scaffolds outperformed the market standard benchmark.

[0405] It is possible to easily and cost-effectively passage cells onto nanofibrillar cellulose scaffolds. In particular, the fact that cells are passaged without the need for trypsinization is beneficial as it significantly reduces cost, labor and the risk of damaging or contaminating the cells.

[0406] Encouragingly, the nanofibrous cellulose scaffold allowed cells to grow at a rate of 6 x 10 6 The density of cells (in 1 ml of culture medium, 50 cm 2 120,000 cells / cm 2 ) for at least 48 hours, whereas classical adherent cultures grew 1 x 10 6 cells.

[0407] in conclusion

[0408] The nanofiber cellulose scaffold theoretically provides a significantly greater surface area per unit volume than the market standard microcarrier (approximately 250 times greater) (Table 2). Therefore, compared to the market standard Cytodex-1, the amount of available surface area that can be increased in a limited volume (such as a bioreactor) is much greater.

[0409] In this example, this difference was "neutralized," and the same amount of surface area was made available to the cells by adjusting the amount of microcarriers added. Even so, the cells grew best and at a higher density on the nanofibrous cellulose scaffolds, suggesting that the cells prefer the native-like environment presented by the cellulose nanofibers.

[0410] Example 3: Transient cell transfection and protein production on nanofiber cellulose scaffolds

[0411] In this example, transient transfection and subsequent protein production of cells grown on nanofiber cellulose scaffolds were evaluated. The experiment was performed in two steps: a first step in which the surface area provided by the microcarriers was kept constant (as in Example 2); and a second step in which the culture volume was kept constant (thus allowing for more nanofiber cellulose scaffold in the culture volume). The nanofiber cellulose scaffold was benchmarked against the market standard, Cytodex-1.

[0412] method

[0413] Nanofibrous cellulose scaffolds were prepared as described in Example 1. The following samples were evaluated: Cellevate unfunctionalized, Cellevate CMC, Cellevate QA, and Cellevate DEAE+CMC.

[0414] Transfection was performed using polyethyleneimine (PEI). Renilla luciferase production was measured daily. Luciferase activity is a marker for transfection and protein formation. Fluorescence microscopy was used to provide images of cells cultured on nanofibers to determine the uniformity of luciferase production. The amount of Renilla luciferase protein was determined by Bright-Glo luciferase assay (Promega).

[0415] A fixed amount of microcarrier surface area (50 mm 2 ) produces protein

[0416] This study compared the surface area of ​​microcarriers (50 mm) by measuring protein (luciferase) production from HEK293AD and ARN8 cells. 2 ) related to the efficiency of different types of microcarriers.

[0417] Cells (HEK293AD and ARN8) were cultured at 6 x 10 6 Cells were seeded in 50 cm 2 The cells were transfected with 7.5 μg of polyethyleneimine (PEI) and 2.5 μg of Renilla luciferase reporter gene driven by the SV40 constitutive promoter.

[0418] As described in Example 2, cells are grown and passaged on nanofiber cellulose scaffold or Cytodex-1. At the appointed time, 10 ml pipette is pipetted ten times by cell / fiber to break cell / fiber aggregation, and obtains the uniform suspension of cell / fiber in cell culture medium.Then the suspension of the cell / fiber culture medium of 500 μ l is mixed with the 2X passive lysis buffer (Plomag company) of 500 μ l. The purpose of large volume is to minimize the sampling effect, and the accuracy of reading is maximized.

[0419] 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 culture medium was mixed with 50 μl of Bright-Glo reagent and immediately analyzed for luciferase activity on a luminometer (Promega) according to the manufacturer's protocol.

[0420] Protein production using fixed-volume microcarriers

[0421] This experiment compared the efficiency of different types of microcarriers in relation to microcarrier volume by measuring protein (luciferase) production from HEK293AD cells in a limited volume of cell culture medium.

[0422] At the same cell density (per cm 2 The microcarriers were seeded with 20,000 cells to compare the transient transfection of the same volume of microcarriers (100 mm 3 ) after luciferase production.

[0423] Use the polyethyleneimine (PEI) of 7.5 μ g as transfection mode, with 40,000 molecules of the luciferase reporter gene driven by SV40 promoter transfection cell.Five hours after transfection, the fresh culture medium of 3 ml is added in 50 ml Falcon tube (lid is not tightly covered to allow O2 / CO2 exchange).Use Bright-Glo luciferase assay (Puluomai company) to analyze the luciferase activity of 24 hours and 48 hours after transfection.Make cell microcarrier even by pipetting, and get the culture medium of 50 μ l and the Bright-Glo reagent of 50 μ l to mix, on luminometer (Puluomai company), analyze luciferase activity immediately.

[0424] It is important to note that Cellevate microcarriers increase in size significantly when wet. Theoretically, each cm of Cellevate microcarrier fiber 2 The volume of Cytodex-1 microcarriers is calculated as per cm 2 To obtain a realistic value, a series of experiments were performed to estimate the volume of the hydrated nanofibrous cellulose scaffold. It was found that estimating the volume occupied by the nanofibrous cellulose scaffold to be 1 / 5 of the volume occupied by Cytodex-1 was a good approximation ( Figure 4 A).

[0425] For 100cm 2 The surface area of ​​Cytodex-1 is 317 mm 3 (32cm 2 / 100mm 3If the volume of wet nanofibrous cellulose scaffold microcarriers is 1 / 5 of the volume of Cytodex-1, then for 160 cm 2 , the volume of wet nanofibrous cellulose scaffold microcarriers is 100 mm 3 Each microcarrier was seeded with the same cell density: 1 cell per cm in a 50 ml falcon tube. 2 With 20,000 cells, the volume of the carrier was 100 μl for a final volume of 5 ml (the cap was not tightly closed to allow O2 / CO2 exchange).

[0426] result

[0427] When 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 3 A). Five days after transfection, ARN8 cells on nanofiber cellulose scaffold microcarriers (Cellevate unfunctionalized, Cellevate CMC, and Cellevate QA) produced 40-70% more luciferase per ml than ARN8 cells on Cytodex-1 ( Figure 3 B).

[0428] By decanting the microcarriers, it was observed that a good approximation of the microcarrier volume was that the volume of the nanofiber cellulose scaffold microcarriers was 1 / 5 of the volume of the Cytodex-1 ( Figure 4 A). Therefore, the volume of added nanofibrous cellulose scaffold microcarriers was adjusted accordingly.

[0429] Evaluation of cell growth upon addition of a fixed volume of microcarriers showed that the dead volume of the nanofibrous cellulose scaffolds was significantly reduced, and thus the available surface area of ​​the limited cell culture vessel was increased, which is highly advantageous. After 48 hours, the luciferase production of HEK293AD cells on the nanofibrous cellulose scaffold microcarriers (Cellevate CMC and Cellevate QA) was approximately 5.5-6.3 times greater than that of HEK293AD cells on Cytodex-1 ( Figure 4 B).

[0430] in conclusion

[0431] This example demonstrates that nanofiber-based cellulose scaffold microcarriers can efficiently transiently transfect HEK293AD and ARN8 cells, resulting in high yields of active recombinant proteins. Furthermore, the high surface area per unit volume of nanofiber-based cellulose scaffold microcarriers allows for increased surface area to be packed into confined volumes, such as bioreactors. This example demonstrates that this advantage significantly increases recombinant protein yields.

[0432] Example 4: Processing of initial cellulose nanofiber material

[0433] In this example, various methods of fragmenting the initial cellulose nanofiber material were tested and their effects on the nanofiber-like cellulose scaffolds were evaluated.

[0434] method

[0435] The preparation of the initial cellulose nanofiber material was performed as described in Example 1.

[0436] Processing plan

[0437] Different methods of fragmenting the initial cellulose nanofiber material were evaluated. Therefore, samples were prepared according to the following:

[0438] Mechanical cutting (using scissors):

[0439] 2.5 g of the cellulose sheet was cut into 10 x 10 mm pieces using scissors and directly measured as the fragmented cellulose sheet.

[0440] Blending:

[0441] 2.5 g of cellulose sheet was cut into 20x20 mm pieces with scissors and added to a laboratory blender (LB20, Waring Laboratory) along with 250 ml of water. The cellulose nanofiber material was processed at 7000 rpm for different durations (0.5 min, 1 min, 2.5 min, 5 min and 10 min).

[0442] Laser Cutting:

[0443] Cellulose sheets were cut into three different sizes (0.75 x 0.75 mm, 1.5 x 1.5 mm, and 3 x 3 mm) using a laser cutter (Epilog Laser, Zing 24). The cut sheets were measured as is.

[0444] dispersion:

[0445] 2.5 g of cellulose sheet was cut into 20 x 20 mm pieces with scissors and added to an Erlenmeyer flask along with 250 ml of water. The sample was processed at 18,000 rpm for different durations (0.5 min, 1 min, 2.5 min, 5 min, 10 min, 15 min and 60 min) using a disperser (IKA T25 Digital Ultra-Turrax with S25 NB-25G disperser tool).

[0446] microscope

[0447] A sample of the processed cellulose nanofiber material was diluted 10-fold in water and added to a glass slide. An image of the network of cellulose nanofibers in the sample was captured using an optical microscope (Leica) at 40x magnification.

[0448] Scanning electron microscopy (SEM)

[0449] Samples of the processed cellulose nanofiber material were diluted 1,000-10,000 times in water, and a droplet of the sample was applied to the SEM fixture. The sample was dried and sputter-coated with gold, and then images were captured on a Hitachi SU3500. Images were captured at different magnifications.

[0450] Fiber length measurement

[0451] The fiber length of cellulose nanofibers was determined by SEM (Hitachi SU3500) or light scattering (Malvern Mastersizer S).

[0452] SEM images were manually evaluated by visual inspection to ensure that both ends were visible for all measured fibers. Fiber length was determined using ImageJ software. Fiber length was determined from several SEM images to obtain a larger data set.

[0453] Fiber length is also determined using light scattering. Briefly, 1 ml of sample is added to water in a Malvern Mastersizer S and the measurement is performed using the settings described under "Mean Length." Sample is added to the sample container until the haze value is between 15-20%. A fiber length histogram showing the fiber length distribution is generated for each sample. Statistics of the distribution are calculated from the results using the derived diameter D[m,n]—an internationally recognized method for defining mean and other moments of particle size. D(v,0.5), D(v,0.1), and D(v,0.9) are standard "percentile" readings from the analysis. D(v,0.5) is the fiber length below which 50% of the sample falls, and 50% of the sample falls. When applied to particles, this value is also referred to as the mass median diameter (MMD). D(v,0.1) is the fiber length below which 10% of the sample falls. D(v,0.9) gives the fiber length below which 90% of the sample falls. The volume-weighted average fiber length D was also determined [4,3].

[0454] Pipetting / floating test

[0455] Samples of processed cellulose nanofiber material were tested for pipetting ability. 1 ml of sample was aspirated from a sample container and then expelled through a pipette tip into a tube filled with water. The ease of pipetting was assessed, including the potential for clogging of the pipette by the processed cellulose nanofiber material.

[0456] 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 see if the material floated immediately after shaking and 24 hours after shaking.

[0457] result

[0458] Pipetting tests on scissor-cut and laser-cut cellulose sheets showed that the cut fragments were too large to pipette because they would clog the pipette tips. In addition, cellulose nanofiber materials processed in this way produced fragments that were too large to trap bubbles and float in the solution ( Figure 5 A). In addition, cutting with laser causes cellulose nanofibers to melt, burn, and stick together, and is therefore not suitable for splitting cellulose nanofibers ( Figure 5 BC).

[0459] and cellulose nanofiber materials processed by disperser ( Figure 6 FJ) compared to the cellulose nanofiber material processed by the blender ( Figure 6AE) generally produced denser and more tangled samples in which the cellulose nanofibers were not evenly distributed. The cellulose nanofiber material that was dispersed for at least 2.5 minutes appeared to be more evenly distributed throughout the sample and had fewer long cellulose nanofiber strands ( Figure 6 FJ). Therefore, a dispersion of at least, for example, 2 minutes may be advantageous to avoid excessive entanglements and clusters of the cellulose nanofibers.

[0460] The SEM images of the processed cellulose nanofiber materials confirmed that the cellulose nanofibers produced by the samples processed by blending were longer ( Figure 7 A), and the entanglement level is higher ( Figure 7 BC).

[0461] Dispersion time affects the average length of the cellulose nanofibers in the cellulose nanofiber material. The longer the dispersion time, the shorter the fiber length. Shorter fibers may be advantageous because they are not prone to forming clusters and tangles, thereby reducing the amount of inaccessible surface areas and the risk of clogging. A summary of definite average fiber lengths is provided in Table 3.

[0462]

[0463] Table 3. Fiber length measurements from the Malvern Mastersizer S.

[0464] SEM measurements were used to determine the cellulose nanofiber diameter ( Figure 8 AB). More than 2000 individual cellulose nanofibers were measured using ImageJ software, and the average cellulose nanofiber diameter of 500 nm was given ( Figure 8 C).

[0465] Cellulose nanofiber materials processed by dispersion do not trap bubbles or tend to float in solution. Pipetting is least challenging for samples with longer dispersion durations. Therefore, dispersion for at least 10 minutes is preferred to improve flow through the pipette.

[0466] in conclusion

[0467] This example shows that not every method for dividing the initial cellulose nanofiber material is equally effective and suitable for preparing nanofibrous cellulose scaffolds. In particular, dispersion is advantageous because it divides the cellulose nanofiber material uniformly without generating tangles or clumps.

[0468] Example 5: Processing Nanofibers of Materials Other Than Cellulose

[0469] In this example, the processing of various nanofibers other than cellulose was evaluated with the goal of identifying secondary nanofibers suitable for preparing nanofiber-like scaffolds for cell culture growth. The electrospun nanofibers were characterized by scanning electron microscopy (SEM) and by visual inspection (after segmenting the nanofibers).

[0470] method

[0471] electrospinning

[0472] Nanofibers of polycaprolactone (PCL), poly-L-lactic acid (PLA), a mixture of PCL and PLA (PLA / PCL), and cellulose were electrospun. Electrospinning was performed as described in Example 1 on a Fluidnatek LE50 apparatus.

[0473] PCL fibers were obtained by dissolving 8% polycaprolactone pellets (Sigma Aldrich, MW80000) in a 1:1 solution of chloroform:methanol. The distance from the needle to the collector was set at 20 cm, the flow rate was 3 ml / h, and the voltage was 18 kV.

[0474] PLA fibers were obtained by dissolving 8% PLLA pellets (Goodfellow, MFR: 24) in chloroform: methanol, 3: 2. The distance from the needle to the collector was set at 24 cm, the flow rate was 4.5 ml / h, and the voltage was 35 kV.

[0475] PLA / PCL fibers were prepared by dissolving PLA pellets (Gutterford, MFR: 65) and PCL pellets (Sigma-Aldrich, MW 80000) in a 1:2 ratio in an 8% polymer solution of chloroform:methanol (3:2). The needle-to-collector distance was set at 20 cm, the flow rate was 3 ml / h, and the voltage was 18 kV.

[0476] 1 ml of the polymer solution was spun to obtain each fiber sheet.

[0477] Scanning electron microscopy

[0478] Electrospun materials of PCL, PLA, mixed PCL / PLA, and cellulose were imaged on a Hitachi SU3500 as described in Example 1. Images of the nanofibrous materials were obtained for both unfragmented and fragmented nanofibers.

[0479] Processing of PCL, PLA and PCL / PLA nanofiber materials

[0480] The electrospun sheets of PCL, PLA and mixed PLA / PCL were cut into smaller 10x10 mm pieces and mixed for 5 minutes using two different mixing tools. The cut sheets were mixed using a blender at 7000 rpm (LB20E laboratory blender, Waring) or a disperser at 18000 rpm (IKA T25 digital Ultra Turrax).

[0481] result

[0482] Nanofibers can be electrospun from all materials (PCL, PLA, PLA / PCL, and cellulose) and formed into nanofibrous sheets (see Figure 9 AD).

[0483] Sheets made from nanofibers other than cellulose utilize two different modes of segmenting the nanofibers, namely blending ( Figure 10 AF) and dispersion ( Figure 11 AF). Testing has shown that it is not possible to uniformly blend or disperse PCL, PLA, or PLA / PCL nanofibers into a homogeneous mixture of short-strand nanofibers.

[0484] During this process, PCL nanofibers ( Figure 10 AB and Figure 11 AB) and PLA / PCL nanofibers ( Figure 10 EF and Figure 11 EF) both melt or deform. Specifically, the nanofibers melt together to form large fragments of solid polymer, or large entangled nanofiber clusters with semi-molten nanofibers.

[0485] PLA nanofibers ( Figure 10 CD and Figure 11 CD) melts differently than PCL fibers, but all materials can easily get stuck in the mixing tools or tangle on the blades of the blender and stop the process.

[0486] in conclusion

[0487] This example shows that it is not possible to uniformly blend or disperse nanofibers of all materials. Therefore, not all nanofiber materials can be easily converted into shorter strands and then formed into nanofibrous scaffolds as described herein. Therefore, it is preferred to use cellulose nanofibers to prepare nanofibrous scaffolds.

[0488] Example 6: Methods for segmenting initial cellulose nanofibers

[0489] In this example, the effect of the nanofiber segmentation method on the length of cellulose nanofibers was evaluated. Nanofibers were segmented by blending or dispersion, and the length of representative fractions of nanofibers was measured by individual analysis of SEM images or by laser diffraction in solution.

[0490] method

[0491] Preparation of QA functionalized cellulose nanofibers for further testing Cellulose acetate sheets were prepared by electrospinning as described in Example 1.

[0492] Regeneration of cellulose

[0493] Cellulose is obtained by the regeneration (deacetylation) of electrospun cellulose acetate sheets. In short, 25g of cellulose acetate sheets were cut into small pieces (2x2 cm) and added to a beaker with 1.25L of a 95% ethanol solution containing 0.5M NaOH. The cellulose fibers were regenerated at room temperature for 24 hours. After the reaction time, the regenerated cellulose nanofibers were filtered with a Büchner filter, immersed in 500mL dH2O for 1 minute, and filtered again. This process was repeated until the conductivity of the final washing step was 0μS / cm to ensure that there was no residual NaOH or acetate. Finally, the nanofibers were dried in an oven at 60°C overnight.

[0494] Segmentation of cellulose nanofibers

[0495] 1% cellulose suspension is prepared by adding 5g of regenerated cellulose to 500ml of distilled water. The solution is added to a disperser and mixed at different time points at 18,000rpm (IKA T25 digital Ultra Turrax). The 100mL sample of the mark is removed and then mixed for 1 minute (D1), 5 minutes (D5), 15 minutes (D15) and 60 minutes (D60). The procedure is repeated using a blender, but the speed is set to 7,000rpm (LB20E laboratory blender, Waring) (marked samples: B1, B5, B15 and B60). After mixing, the fiber is dried overnight in an oven at 60°C. The size distribution of nanofibers has been analyzed by SEM and dynamic light scattering (DLS). SEM measurements and DLS measurements are carried out as described in Example 4. The length of individual cellulose nanofibers is assessed from SEM images (for each time point, 4 single 10 μL droplets). For light scattering experiments, 10,000 fibers were measured for each sample.

[0496] Functionalization of cellulose nanofibers

[0497] The samples of the regenerated cellulose fibers blended or dispersed at different time points were functionalized with quaternary ammonium (QA). In short, 1 g of the regenerated cellulose at each time point was resuspended in 50 ml of a 1.5 M NaOH solution and then mercerized for 2 hours at room temperature under stirring. After the mercerization step, the temperature was raised to 80° C., and 2.5 ml of CHPTAC was added dropwise. The reaction was continued at 80° C. for 4 hours. After the reaction, the mixture was filtered with a Buchner filter, immersed in 200 mL of dH2O for 1 minute, and filtered again. This process was repeated until the conductivity of the final washing step was 0 μS / cm, to ensure that there was no residual CHPTAC or NaOH. Finally, the filtered fibers were resuspended in water to a 2% solution, and freeze-dried for further experiments.

[0498] result

[0499] From the SEM images ( Figure 12 AH) can be clearly seen that the disperser quickly provides a uniform population of nanofibers with relatively few long nanofibers and without any significant entanglements. Although large pieces of uncut nanofibers are present in the sample after only 1 minute of segmentation ( Figure 12 AB), but after 5 minutes of dispersion, the disperser showed a finer population of nanofibers with only a few smaller entangled nanofiber clumps ( Figure 12 D). After 15 and 60 minutes of dispersion, the presence of entangled nanofibers almost completely disappeared ( Figure 12 F and 12H). In contrast, the blended samples still contained large clumps of entangled fibers even after 60 minutes of blending ( Figure 12 G).

[0500] Figure 13 The data for each sample are summarized in Figures AB. It is clear that the long nanofibers are more frequent in the blended samples compared to the dispersed samples. In addition, there is a trend that longer processing times result in shorter nanofiber lengths.

[0501] Measurements of the nanofibers using light diffraction support this relative trend. These data are summarized in Table 4.

[0502]

[0503] Table 4. Fiber length measurements of cellulose nanofibers prepared by blending (B1, B5, B15, B60) and dispersion (D1, D5, D15, D60). The measurements were performed on a Malvern Mastersizer S.

[0504] In a word, it is expected that the average length of cellulose nanofibers is shortened.Yet it is equally important that the colony of nanofiber does not contain a lot of long nanofibers, because these long nanofibers may serve as the core of larger nanofiber cluster and entanglement.The cluster of these nanofibers or entanglement are undesirable in nanofiber-shaped scaffold, because the cells seeded thereon still can't obtain most of surface area.In addition, it is undesirable that uneven nanofiber colony comprises cluster and entanglement, because the variability between the nanofiber-shaped scaffold of each batch can become inconsistent, and therefore is unreliable concerning cell growth.

[0505] in conclusion

[0506] This example shows that the way in which the cellulose nanofiber material is divided affects the size distribution of the nanofibers, and in particular the fraction of long fibers. Dispersing the nanofibers is preferred because it allows for rapid removal of longer fibers and results in the most uniform population of cellulose nanofibers.

[0507] Example 7: Functionalization degree and cell growth on cellulose nanofibers

[0508] In this example, the effects of nanofiber functionalization and dispersion time on cell growth on nanofibrous scaffolds were investigated.

[0509] method

[0510] Sample preparation

[0511] The samples were prepared by weighing 0.05 g of freeze-dried cellulose sample (prepared as described in Example 6) in a 50 ml tube. 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 discarded.

[0512] To evaluate the effect of the degree of functionalization on cell growth, samples were prepared by adding different amounts of CHPTAC, thereby obtaining different ion exchange capacities.

[0513] To saturate the exchange sites with chloride ions, the sample was incubated with 25 ml of 0.1 M HCl for 1 hour under stirring. After the end of the incubation, the sample was filtered using a 0.2 μm Millipore filter unit and the supernatant was discarded.

[0514] To remove unbound chloride ions, the nanofibrous 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 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.

[0515] Ion exchange capacity measurement

[0516] 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 final sample preparation step was diluted in 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 fibers. The ion exchange capacity (mmol Cl) was calculated using LabX software based on the equivalent points on each titration curve. - / g).

[0517] Cell culture protocol

[0518] Cell growth experiments on cellulose fibers were performed in a 12-well plate format using HEK293 cells (ATCC CTRL-3216) at passage 8 (92% viability on the day of inoculation with fibers).

[0519] 1 mg of cellulose fibers under each condition were washed 3 times with DPBS and resuspended in 10 ml of cell culture medium (DMEM containing high glucose and GlutaMAX supplements and pyruvate, 10% heat-inactivated FBS and 1% penicillin-streptomycin). 300,000 cells per well were seeded in 0.3 ml of culture medium containing 0.3 ml (equivalent to 0.3 mg) of QA functionalized cellulose fibers (D1, D5, D15 and D60) prepared as described in Example 6 and incubated overnight on an orbital shaker (16 mm) at 35 rpm in an incubator (at 37° C., containing 5% CO 2 ).

[0520] The next morning, top up each well with 1.4 ml of medium to a final volume of 2 ml and an initial cell concentration of up to 150,000 cells / ml prior to analysis. Increase the shaker rotation speed to 65 rpm.

[0521] At 24, 48 and 72 hours after seeding, three replicates were taken from individual wells at each time point for further analysis. Enzymatic dissociation of cells was used in conjunction with the microcarrier program of the NucleoCounter (NC-202) to assess changes in cell density and viability over time.

[0522] result

[0523] Cell growth was enhanced on highly functionalized nanofibrous cellulose scaffolds ( Figure 14 A). Specifically, for ion exchange capacities above 0.3 mmol Cl- Optimal cell growth appears to be achieved with cellulose microcarriers containing 100 μg of cellulose.

[0524] Consistent cell growth was achieved on all cellulose microcarriers. The data showed that cells grown on cellulose microcarriers prepared from cellulose nanofibers that had been dispersed for at least 5 minutes proliferated best and displayed high viability ( Figure 14 BC). After 72 hours of incubation, the cell density increased eightfold compared to the initial seeding density. This was achieved without transferring cells or changing the culture medium.

[0525] in conclusion

[0526] This example demonstrates that cellulose microcarriers can be efficiently functionalized, with the lowest possible ion exchange capacity, to promote cell growth. Cellulose microcarriers in which nanofibers were dispersed for more than one minute (e.g., five minutes) formed the best scaffolds for cell growth. Without being bound by theory, it is contemplated that the reduced nanofiber clustering and entanglement ensures a greater surface area for cell seeding and interaction.

Claims

1. A method for preparing a nanofibrous cellulose scaffold, the method comprising the following steps: (i) providing an initial cellulose nanofiber material, (ii) dividing the initial cellulose nanofibrous material into processed cellulose nanofibrous materials, (iii) functionalizing the processed cellulose nanofiber material by adding a reagent comprising a functional moiety, and (iv) drying the processed cellulose nanofiber material, wherein said segmentation is achieved by dispersion, Thereby, the nanofibrous cellulose scaffold is provided.

2. The method according to claim 1, wherein the dispersing is performed using a high-speed disperser.

3. The method according to any one of claims 1 or 2, wherein the dispersing is performed for at least 2 minutes, such as at least 5 minutes, such as at least 10 minutes, such as at least 15 minutes, such as at least 20 minutes, such as at least 30 minutes, such as at least 40 minutes, such as at least 50 minutes, such as at least 60 minutes.

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

5. The method according to any one of the preceding claims, wherein the initial cellulose nanofiber material is prepared by electrospinning, melt blowing, spinning, self-assembly, template synthesis and thermally induced phase separation.

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

7. The method of any one of the preceding claims, 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, epoxides, and combinations thereof.

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

9. The method of any one of the preceding claims, wherein the nanofibrous cellulose scaffold has an ion exchange capacity of at least about 0.3 mmolCl - / g.

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

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

12. The nanofibrous cellulose scaffold of claim 11, wherein the nanofibrous cellulose scaffold has a BET surface area of ​​at least about 40,000 cm 2 / g, such as at least about 50,000 cm 2 / g, such as at least about 55000cm 2 / g, such as at least about 60,000 cm 2 / g, such as at least about 70,000 cm 2 / g, such as at least about 80,000 cm 2 / g.

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

15. A method for cell culture, comprising the following steps: (i) providing the 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 a expanded cell population, and (iv) Optionally, extracting the expanded cell population from the cell culture device.

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