Passaging of cells

Abstract

The present invention relates to a methodology for simple passaging of cells during cell culturing. The method is easily applicable to industrial scale cell culturing and requires no detachment of adherent cells from the support matrix upon passaging.

Description

[0001] P97013PC

[0002] Passaging of cells

[0003] Technical field of the invention

[0004] The present invention relates to a methodology for simple passaging of cells during cell culturing. The method is easily applicable to industrial scale cell culturing and requires no detachment of adherent cells from the support matrix upon passaging.

[0005] Background of the invention

[0006] Adherent cells are vital for the production of biopharmaceuticals, vaccines, and other therapeutic products, including complex proteins and other biologies used in treating various diseases and medical conditions. Accordingly, ensuring a reliable and scalable source of adherent cells is a crucial competitive parameter for businesses supplying the healthcare and research sectors with high quality adherent cells or products thereof.

[0007] Industrial-scale culturing of adherent cells is typically performed using bioreactors equipped with surfaces or microcarriers that provide the necessary support for cell attachment and growth. The cells may be kept in suspension by agitation of the liquid medium resulting in better nutrient and gas exchange, leading to higher cell densities and productivity. The methodology requires precise control of environmental parameters such as temperature, pH, and oxygen levels to ensure optimal cell growth. These requirements are largely met by modern industrial bioreactor systems.

[0008] Despite advancements in cell culturing technology, the process of passaging adherent cells remains a significant rate-limiting step in industrial-scale operations. Passaging involves detaching cells from their growth surface, typically using enzymatic treatments like trypsin or other proteolytic enzymes to degrade attachment points, and transferring them to fresh culture vessels. This process, also known as subculturing, is crucial for creating a new subculture with fresh nutrients and space, allowing continued cell growth and proliferation. However, it is labour-intensive and time-consuming, requiring centrifugation and / or filtration steps in the bioprocess seed train, careful handling to maintain cell viability and prevent contamination, and optimization work for reproducible outcome across different bioreactor volumes. Additionally, the efficiency of passaging can be affected by factors such as the effectiveness of the detachment process, and subsequent steps (e.g. washing and centrifugation) to remove serum or residual enzyme. These challenges make passaging a bottleneck in the production process, highlighting the need for more efficient and automated systems to improve scalability and productivity. P97013PC

[0009] Thus, there is an unmet need for provision of a simple and efficient methodology for culturing adherent cells which mitigates the risks of contamination and reduces production time.

[0010] Hence, it would be advantageous to provide a method for culturing adherent cells with an improved passaging step that reduces risk of contamination, improves cell production and is easily applicable for large scale industrial settings.

[0011] Summary of the invention

[0012] Herein is provided a simple method for culturing adherent cells which obviates the step of detaching the cells from the support matrix upon passaging. This is accomplished by utilising a support matrix comprising cellulose nanofibers which enables direct transfer of attached cells to a new culture vessel without compromising cell viability or subsequent mobility. Importantly, cells passaged according to the present method consistently produces healthy subcultures that proliferate at normal rate. By significantly reducing the complexity of the cell passaging, the present invention unlocks a cell production method that is markedly faster and less prone to contamination than conventional cell production techniques.

[0013] Thus, an object of the present invention relates to the provision of a high-throughput cell production methodology that can be easily scaled for industrial production.

[0014] Another object of the present invention relates to provision of a method for culturing adherent cells which reduces time consumption and risk of contamination.

[0015] Thus, an aspect of the present invention relates to a method for culturing of cells, said method comprising the steps of:

[0016] (i) providing a first container comprising a first cell culture and a support matrix;

[0017] (ii) passaging at least part of said first cell culture to a second container to provide a subculture;

[0018] (iii) adding an additional amount of support matrix to said second container; and

[0019] (iv) culturing said subculture; wherein said support matrix comprises cellulose nanofibers, and wherein said passaging does not comprise a step of detaching cells from the support matrix.

[0020] Another aspect of the present invention relates to a method for culturing of cells, said method comprising the steps of: P97013PC

[0021] (i) seeding an inoculum on a support matrix;

[0022] (ii) culturing said inoculum to provide a first cell culture;

[0023] (iii) passaging at least part of said first cell culture to provide a subculture;

[0024] (iv) adding an additional amount of support matrix to the subculture;

[0025] (v) culturing said subculture; wherein said support matrix comprises cellulose nanofibers, and wherein said passaging does not comprise a step of detaching cells from the support matrix.

[0026] Another aspect of the present invention relates to a nanofibrous cellulose scaffold comprising a cellulose nanofiber material with a mean length of the cellulose nanofibers of less than about 250 pm, and wherein the cellulose nanofiber material is functionalized with a functional moiety.

[0027] Yet another aspect of the present invention relates to a method of preparing a nanofibrous cellulose scaffold, said method comprising the steps of:

[0028] (i) providing an initial cellulose nanofiber material,

[0029] (ii) dividing said initial cellulose nanofiber material into a processed cellulose nanofiber material,

[0030] (iii) functionalizing the processed cellulose nanofiber material by addition of a reagent comprising a functional moiety, and

[0031] (iv) drying the processed cellulose nanofiber material, thereby providing the nanofibrous cellulose scaffold.

[0032] A further aspect of the present invention relates to a nanofibrous cellulose scaffold obtainable from the method of preparing a nanofibrous cellulose scaffold as described herein.

[0033] Brief description of the figures

[0034] Figure 1 shows (A) microscopy image of nanofibrous cellulose scaffold functionalized with quaternary ammonium (Cellevate QA). (B) Centrifuge tube comprising the nanofibrous cellulose scaffold in dry form. (C) Scanning electron microscopy (SEM) image of the nanofibrous cellulose scaffold demonstrating the spatial arrangement of individual nanofibers.

[0035] Figure 2 shows passaging of cells grown on cellulose nanofibers (A-C) or grown on a beadbased microcarrier (D-E). (A) Passaging of the same three-dimensional (3D) spheroid culture on cellulose nanofibers in a 125 mL spinner flask into a new 125 mL spinner flask P97013PC every 72h-96h without cell dissociation, for in total three consecutive 3D passages. The dotted line represents 150.000 cells per mL of culture volume at seeding. (B) Seeding from 2D flask into a 125 mL spinner flask with a working volume of 75mL. After 72h, the 3D spheroid culture on cellulose nanofibers is subsequently passaged into same format of 75mL (pl) and upscaled to 600mL (pl) in a IL spinner flask. The passaged 75mL culture pl was passaged again into the same culture format of 75mL (p2), generating similar growth trend and cell density. (C) Cell growth curves showing the total number of live cells in culture during the passage and upscale of a 75mL 3D spheroid culture on cellulose nanofibers into a 600mL 3D culture. (D) Cell growth curves of HEK293T cells on beadbased microcarriers for 72 hours and the subsequent passage and continued growth for additional 72 hours. (E) Microscope images at 24h, 48h, and 72h timepoints post passaging of HEK293T cells on bead-based microcarriers.

[0036] Figure 3 shows (A) cell growth curves showing seeding in a IL spinner flask followed by spheroid passaging after 72h into a 2.4L Applikon AppliFlex stirred tank single-use (SU)- bioreactor. (B) Cells seeded in a IL spinner flask and kept for 72h before spheroid passage into a 10L Xcellerex XDR stirred tank SU bioreactor.

[0037] Figure 4 shows (A) images of a 3D spheroid culture in a IL spinner flask with a culture volume of IL. The spheroids increase in number, but do not grow noticeably in size during the 120h cell culture. (B) Images taken from a IL spinner flask that was a passage from another IL spinner flask. The spheroids tend to grow in size over the course of 120 h. (C) Images from the XDR10 bioreactor where spheroids from a IL spinner were passaged into the XDR10 bioreactor. The spheroids tend to grow in size after 3D passage.

[0038] Figure 5 shows the size of spheroids over time in different culture formats (shake flask, spinner flask, bioreactor) and volumes. Larger volumes tend to form smaller spheroids.

[0039] Figure 6 shows examples of cellulose nanofibers cut by laser. (A) The cellulose material cut by laser results in pieces of cellulose that trap air bubbles and float. (B) Electrospun cellulose material cut with a laser. The laser burns the cellulose sheets. (C) Scanning electron microscopy (SEM) images of cellulose nanofibers cut by laser. The cellulose nanofibers are melted and fused together.

[0040] Figure 7 shows determination of cellulose nanofiber diameters. (A-B) SEM micrographs of cellulose nanofibers after 1 hour dispersing at 18000 rpm. (C) Histogram depicting the cellulose nanofiber diameter distribution of five separate samples. Approximately 2500 individual cellulose nanofibers were measured using Image! software. P97013PC

[0041] Figure 8 shows scanning electron microscopy (SEM) images of cellulose nanofibers blended (left) or dispersed (right). The cellulose nanofibers were mixed for different periods of time; 1 min (A-B), 5 min (C-D), 15 min (E-F) or 60 min (G-H).

[0042] Figure 9 shows histograms of size (length) distributions of cellulose nanofibers that have been divided by (A) blending or (B) dispersing. The histograms are for samples divided for 1, 5, 15, or 60 min (left to right). Nanofiber lengths are displayed as relative frequency of mean nanofiber lengths in bins of 200 pm.

[0043] Figure 10 shows scanning electron microscopy (SEM) images of electrospun nanofibers; (A) PCL, (B) PLA, (C) PLA / PCL, and (D) cellulose. All images are recorded at 600x magnification, scale bar is 50 pm.

[0044] Figure 11 shows representative photographs (left) and SEM images (right) of different electrospun nanofibers subsequent to 5 minutes of mixing in a blender. (A-B) PCL, (C-D) PLA, (E-F) PLA / PCL. Scale bar of SEM images is 200 pm.

[0045] Figure 12 shows representative photographs (left) and SEM images (right) of different electrospun nanofibers subsequent to 5 minutes of mixing in a disperser. (A-B) PCL, (C- D) PLA, (E-F) PLA / PCL. Scale bar of SEM images is 50 pm.

[0046] Figure 13 shows (A) viable cell density (VCD) and viability for cells cultured at various rotational speeds, and (B) cell growth curves before and post passaging performed in a IL spinner flask at the rotational speed of 120 rpm.

[0047] Detailed description of the invention

[0048] Definitions

[0049] Prior to outlining the present invention in more details, a set of terms and conventions is first defined:

[0050] Inoculum

[0051] In the present context, the term "inoculum" refers to the initial cell population introduced into a culture medium to initiate growth. The inoculum should be carefully prepared and transferred using sterile techniques to ensure that the desired concentration cells and cellulose nanofibers are introduced into the culture medium.

[0052] Support matrix P97013PC

[0053] In the present context, the term "support matrix" refers to a scaffold or material that provides a surface for cells to attach to, grow, and proliferate. The support matrix ideally mimics the natural extracellular matrix (ECM) found in tissues, offering both mechanical support and optionally biochemical signals that influence cell behaviour. These support matrices are crucial for culturing of adherent cells, as they provide the necessary environment for cells to thrive and function properly.

[0054] A support matrix may also be referred to as microcarrier.

[0055] Passaging

[0056] In the present context, the term "passaging" refers to the process of transferring cells from one culture vessel to another to maintain or expand the cell culture. This technique is essential for the routine maintenance of cell lines and helps prevent over-confluence, which can lead to cell stress or death. Passaging ensures that cells have enough space and nutrients to continue growing and proliferating, maintaining healthy and viable cell populations for research and industrial applications.

[0057] During conventional passaging, cells are typically detached from their growth surface, often using enzymatic treatments like trypsin or other proteolytic enzymes, and then reseeded into fresh culture vessels with new growth medium. Cells may also be mechanically detached, e.g. by pipetting, prior to or as part of the passaging step. However, mechanical detachment is suitable only for lab-scale cell culturing and not viable for industrial scale production.

[0058] It is to be understood that the term "detach" or "detachment" may be used interchangeably with terms such as "separate", "remove", "release", "disassociate" or "dissociate".

[0059] Subculture

[0060] In the present context, the term "subculture" refers to a new cell culture that is created by transferring some or all cells from a previous cell culture into fresh growth medium. This process is done to prolong the lifespan of the cells and to increase their number. Subculturing helps maintaining healthy cell populations by providing fresh nutrients and space for continued growth.

[0061] As used herein, a subculture has undergone at least one passage. It is to be understood that a subculture can be passaged to provide a further subculture. P97013PC

[0062] Container

[0063] In the present context, the term "container" refers to a confined volume used to grow and maintain cell cultures under controlled conditions. A container may preferably be a culture vessel, including, but not limited to, flasks, petri dishes, multi-well plates or bioreactors. A container (or culture vessel) allows culturing of cells under conditions suitable for cell growth.

[0064] Spheroid

[0065] In the present context, the term "spheroid" refers to a three-dimensional (3D) structure of aggregated cells that forms when cells self-assemble together with the cellulose nanofibers into a spherical shape.

[0066] In contrast, non-spheroid cell aggregates are irregular clusters without a defined spherical shape. Such disorganized cell aggregates grow uncontrollable and inefficiently in large clusters, with low reproducibility and huge variations in size and morphology.

[0067] The size of the spheroids may be given as a diameter. It is to be understood that the diameter is the mean diameter of the spheroids in the cell culture. The mean diameter of the spheroids may be determined by microscopy and measuring the diameter of a representative number of spheroids (e.g. 100 or 1000 spheroids).

[0068] The diameter of individual spheroids may be determined by image analysis software, such as ImageJ / Fiji.

[0069] Coefficient of variance ( CV)

[0070] In the present context, the term "coefficient of variance (CV)" refers to a statistical measure of relative variability. It may be used to determine how uniformly cells grow in spheroid form, e.g. by determining the CV of the spheroid diameter. The CV is calculated as CV=(standard deviation of diameter / mean diameter)*100%.

[0071] It is to be understood that a CV value of equal to or above 50% is considered a very high variance. A CV value of equal to or above 40% is considered a high variance.

[0072] Preferably, the spheroids grown on the support matrix has less than very high variance, more preferably less than high variance.

[0073] Agitation system P97013PC

[0074] In the present context, the term "agitation system" refers to any mechanism or device used to mix the culture medium and maintain cells in suspension while ensuring proper distribution of nutrients, gases, and temperature.

[0075] The agitation system may involve positioning the culturing vessel on an agitation device or include an agitation element inside the culturing vessel.

[0076] Adherent cells

[0077] In the present context, the term "adherent cells" refers to any cell that requires a surface or artificial substrate, such as a support matrix, to form an adherent cell culture. Preferably, the adherent cell is derived from a solid tissue.

[0078] Adherent cells are sometimes referred to as anchorage-dependent cells.

[0079] Adherent culture is to be distinguished from suspension culture in which single cells are grown freely floating in suspension.

[0080] Nanofiber

[0081] In the present context, the term "nanofiber" refers to fibers with diameters in the range of 10-2000 nm. The fibers may be generated from different types of polymers, such as cellulose.

[0082] Cellulose nanofiber material

[0083] In the present context, the term "cellulose nanofiber material" refers to an initial material prepared from cellulose. The cellulose nanofiber material may be prepared by any method suitable for preparing sheets of cellulose that can subsequently be processed as described herein to provide the nanofibrous cellulose scaffold. These methods include, but are not limited to, electrospinning, meltblowing, and drawing of cellulose nanofibers.

[0084] Preferably, the cellulose nanofiber material is prepared by electrospinning of cellulose nanofibers. Electrospinning may be performed from a solution of cellulose acetate.

[0085] Processed cellulose nanofiber material

[0086] In the present context, the term "processed cellulose nanofiber material" refers to a cellulose nanofiber material that has been treated to reduce the length of the cellulose nanofibers.

[0087] Dispersing P97013PC

[0088] In the present context, the term "dispersing" refers to the process of dividing the cellulose nanofiber material by means of a disperser. Dispersing is preferably performed in a liquid.

[0089] In the present context, a disperser is a high-speed mixing device that can comminute a solid, such as a cellulose sheet, into smaller fragments. The disperser comprises one or more heads which constitute the means of dividing the cellulose nanofiber material. The head may be in the form of a disc blade. The head of the disperser may comprise a blade with a propeller design. The disperser creates a turbulent flow and a vortex which ensures homogenous dividing of the cellulose nanofiber material into smaller fragments, i.e. cellulose nanofibers of reduced length compared to the initial cellulose nanofiber material.

[0090] Any type of disperser may be used, including, but not limited to, laboratory high-speed disperser, pilot-scale high-speed dispersers, and production-scale high-speed dispersers. The disperser can be chosen according to the batch size to be divided, and the diameter of the disperser blades are adjusted accordingly. The disperser blade may be raised and lowered during dispersing to eliminate stratification, such mechanism may be automatic. For large batches the disperser may be a floor-mounted or tank-mounted model and / or be a multishaft model.

[0091] Functional moiety

[0092] In the present context, the term "functional moiety" refers to a chemical or biological group or molecule positioned on the nanofibrous cellulose scaffold, and which interacts with the cells associated with the scaffold. A functional moiety may interact with the cells via interactions including, but not limited to, electrostatic interactions, affinity, and hydrophobicity / hydrophilicity. The functional moiety may promote attachment / adhesion of cells to the nanofibrous cellulose scaffold, induce cell differentiation and / or proliferation, and / or assist with maintenance of in vivo cellular functions.

[0093] Chemical moieties may have one or more positive or negative charges to induce electrostatic interaction with the charged cell membranes. Examples hereof include, but are not limited to, quaternary ammonium (QA), carboxymethyl (CM), and diethylaminoethyl (DEAE). For many cells, the cell membrane would be negatively charged, and electrostatic interactions would be induced for nanofibrous cellulose scaffolds functionalized with positively charged functional moieties, such as QA or DEAE.

[0094] Biological moieties may be any type of biological molecule that can secure cell attachment to the nanofibrous cellulose scaffold, including but not limited to, lipid anchors, celladhesion molecules (CAMs), antigens, receptors, and antibodies. CAMs include integrins, P97013PC immunoglobulins, cadherins, and selectins. Biological moieties may also assist cellular differentiation through receptor-ligand interactions, antigen-antibody interactions, protein interactions, and / or immune stimulatory signalling.

[0095] Mean diameter (of cellulose nano fiber)

[0096] In the present context, the term "mean diameter" refers to the average diameter of cellulose nanofibers in the nanofibrous cellulose scaffold. The mean diameter may be determined from SEM images of the nanofibrous cellulose scaffold. Preferably, the mean diameter is determined from measurement of at least 100 individual nanofibers within the sample, e.g. by use of image analysis software, such as Image!.

[0097] The mean diameter of the cellulose nanofibers can be adjusted in the process of preparing the initial cellulose nanofiber material. This may be achieved by varying the parameters of e.g. the electrospinning process.

[0098] Preferably, the mean diameter of the cellulose nanofibers in the nanofibrous cellulose scaffold is from about 250 nm to about 750 nm, such as about 400 nm to about 600 nm.

[0099] Mean length (of cellulose nanofiber)

[0100] In the present context, the term "mean length" refers to the average length of cellulose nanofibers in the nanofibrous cellulose scaffold. The mean length may be determined as the volume weighted mean value (D[4,3]) measured by light scattering, e.g. on a Malvern Mastersizer S. D[4,3] is also known as the De Brouckere mean value.

[0101] The mean fiber length in a sample may be determined using the following settings on a Malvern Mastersizer S:

[0102] Range lens: 300RF mm

[0103] Presentation: 3OHD

[0104] Analysis model: Polydisperse

[0105] Particle refractive index: (1.5295, 0.1000)

[0106] Dispersant refractive index: (1.33000)

[0107] Density: 1.5000 g / cm3

[0108] Preferably, the mean length of the cellulose nanofibers is from about 30 pm to about 250 pm, such as about 40 pm to about 200 pm, such as about 50 pm to about 150 pm, preferably about 60 pm to about 100 pm.

[0109] Surface area P97013PC

[0110] In the present context, the term "surface area" refers to the Brunauer-Emmett-Teller (BET) surface area. The BET surface area may be determined by measurement of the physiosorption of a gas, usually nitrogen, to give a value of the sample. The BET method can accurately determine the surface area of the nanofibrous cellulose scaffold since the gas molecules can travel within the nanofibrous matrix to probe also interior surfaces.

[0111] The surface area is given as area per unit mass (e.g. cm2 / g) and can be measured according to ISO 9277:2022 - Determination of the specific surface area of solids by gas adsorption — BET method.

[0112] Degree of substitution (DS)

[0113] In the present context, the term "degree of substitution (DS)" refers to the average number of functional moieties attached per base unit of the condensation polymer cellulose. The base unit of cellulose is |3(1— >4) linked D-glucose, which comprise three hydroxyl groups (-OH) that may be subjected to substitution. Accordingly, the theoretical maximum value of DS is 3.

[0114] Degree of substitution (DS) may be determined using the following formula:

[0115] DS = (162N / (1400-CAxN)) where 162 is the molecular weight of the anhydrous glucose unit (AGU); N is the percentage of nitrogen; CA is the molecular weight of the cationic reagent.

[0116] Substitution of the cellulose nanofibers can also be quantified as equivalent of charge per base unit mass of cellulose, and is given in units of meq / g. This value can be determined by potentiometric titration, zeta potential measurements, pH titration or electrokinetic chromatography.

[0117] Mercerization

[0118] In the present context, the term "mercerization" refers to a process comprising swelling of the cellulose nanofiber material in an aqueous or ethanolic NaOH solution to break internal hydrogen bonds of cellulose and increase the number of available hydroxyl groups (-OH).

[0119] Suspension based bioreactor

[0120] In the present context, the term "suspension based bioreactor" refers to a bioreactor wherein the support matrix and cells adhered thereto are freely floating as 3D cultures in the bioreactor. In a suspension based bioreactor, it is possible to add more support matrix and / or medium during culturing. P97013PC

[0121] Thus, the term "suspension based bioreactor" includes, but is not limited to, stirred tank bioreactors, fluidized bed bioreactors, wave and rocking bioreactors, and airlift bioreactors.

[0122] In contrast, a suspension based bioreactor is to be distinguished from a bioreactor wherein the support matrix is fixed within the bioreactor, such as a packed bed bioreactor wherein the support matrix is immobilised in a bed.

[0123] About

[0124] Wherever the term "about" is employed herein in the context of amounts, for example absolute amounts, such as numbers, purities, weights, concentrations, sizes, etc., or relative amounts (e.g. percentages, equivalents or ratios), timeframes, and parameters such as temperatures, pressure, etc., it will be appreciated that such variables are approximate and as such may vary by ±10%, for example ± 5% and preferably ± 2% (e.g. ± 1%) from the actual numbers specified. This is the case even if such numbers are presented as percentages in the first place (for example 'about 10%' may mean ±10% about the number 10, which is anything between 9% and 11%).

[0125] Non-enzymatic passaging of cells

[0126] It is problematic to passage adherent cells to a new culture vessel without detachment from the support matrix. There are several reasons for this. Adherent cells rely on attachment to a surface for their growth and proliferation, but if they remain attached to the original microcarrier, they cannot spread and attach to the new surfaces available in the new vessel. The lack of spread can lead to uneven distribution in the new culture vessel, resulting in suboptimal growth conditions due to overcrowding in some areas and underpopulation in others. Especially cells confined in overcrowded areas will experience limited access to fresh nutrients and space, resulting in nutrient depletion and waste accumulation ultimately inhibiting cell growth and viability. Therefore, proper detachment of cells is essential in conventional cell production processes to ensure that cells can reattach and proliferate effectively in the new environment, maintaining healthy and productive cultures.

[0127] Unfortunately, the detachment requirement comes with some major process economy drawbacks. The most common detachment procedure is enzymatic treatment of the cell culture by a protease enzyme that breaks down the proteins responsible for cell adhesion, e.g. trypsin. P97013PC

[0128] In brief the trypsinization process is as follows. First, cells are washed with a calcium- and magnesium-free buffer, such as phosphate-buffered saline (PBS). This step helps to remove any residual serum that could inhibit the action of trypsin. Next, a solution of trypsin, often combined with EDTA (a chelating agent that binds calcium and magnesium ions), is then added to the cells. The culture vessel is then incubated to allow trypsin to cleave the adhesion proteins, causing the cells to detach from the support matrix (or microcarrier). If upon observation (e.g. by microscopy) it is confirmed that cells are floating freely, then trypsin inhibitors are added to stop the trypsinization process. Finally, the residual enzyme may be removed from the detached cells (e.g. by washing and centrifugation).

[0129] Accordingly, one of the most obvious disadvantages with this procedure is the labour- intensive steps that slow cell production down.

[0130] Moreover, the trypsinization process present some risks. First, the process must be conducted under sterile conditions to avoid contamination, which could compromise the entire culture. Contamination can lead to huge profit loss due to entire batches of cells having to be discarded. Next, over-trypsinization, where cells are exposed to trypsin for too long, can damage cell membranes and reduce cell viability, making it crucial to optimise and monitor the process closely and neutralize trypsin promptly. Sensitive cell types are particularly vulnerable to damage from trypsin, potentially affecting their growth and function. Incomplete detachment can occur, necessitating additional trypsinization steps that may further stress the cells and be time-consuming. Finally, post-trypsinization, cells may clump together, complicating the creation of a uniform cell suspension.

[0131] Thus, the detachment process involves many quality assurance steps that are timeconsuming and potentially costly if not handled appropriately.

[0132] An alternative to enzymatic detachment of cells is mechanical detachment of cells. Mechanical detachment refers to physically separating adherent cells from the support matrix or microcarrier, e.g. by scraping, pipetting or vibration. This is typically done when cells are sensitive to enzymes or when maintaining certain surface proteins is important. Methods for mechanical detachment of cells attached to a microcarrier includes agitation (shear force) often combined with washing, and pipetting to dislodge cells. Common for mechanical detachment is that it yields lower recovery and higher cell damage than enzymatic methods. Importantly, mechanical detachment is very labour-intensive and not suitable for industrial scale production. P97013PC

[0133] Herein are described a method of culturing cells that does not require detachment of cells adhered to the support matrix (or microcarrier) upon passaging to create a new subculture. This is a significant advantage because it reduces production time and mitigates some of the contamination risks that manual handling of the cells entails. The method includes utilization of a support matrix comprising cellulose nanofibers that can be easily transferred to another culture vessel. The support matrix allows passaged cells to colonize / grow on the new support matrix added in the next culture vessel.

[0134] Accordingly, an aspect of the present invention relates to a method for culturing of cells, said method comprising the steps of:

[0135] (i) providing a first container comprising a first cell culture and a support matrix;

[0136] (ii) passaging at least part of said first cell culture to a second container to provide a subculture;

[0137] (iii) adding an additional amount of support matrix to said second container; and

[0138] (iv) culturing said subculture; wherein said support matrix comprises cellulose nanofibers, and wherein said passaging does not comprise a step of detaching cells from the support matrix.

[0139] It is to be understood that the first cell culture provided with the support matrix comprises cells that are adhered or attached to the cellulose nanofibers of the support matrix. These may be cells that naturally adhere to the support matrix via a combination of biochemical and physical interactions involving cell surface receptors and extracellular matrix (ECM) components.

[0140] An embodiment of the present invention relates to the method as described herein, wherein said at least part of the first cell culture is passaged together with at least part of the support matrix to said second container.

[0141] Another embodiment of the present invention relates to the method as described herein, wherein said at least part of the first cell culture is passaged to said second container without detaching cells from the support matrix.

[0142] A further embodiment of the present invention relates to the method as described herein, wherein said step of providing a first container comprising a first cell culture and a support matrix comprises seeding an inoculum on a support matrix followed by culturing of said inoculum. P97013PC

[0143] Another embodiment of the present invention relates to the method as described herein, wherein said method comprising the steps of:

[0144] (ia) seeding an inoculum on a support matrix;

[0145] (ib) culturing said inoculum to provide a first cell culture;

[0146] (ii) passaging at least part of said first cell culture to provide a subculture;

[0147] (iii) adding an additional amount of support matrix to the subculture;

[0148] (iv) culturing said subculture; wherein said support matrix comprises cellulose nanofibers, and wherein said passaging does not comprise a step of detaching cells from the support matrix.

[0149] Another aspect of the present invention relates to a method for culturing of cells, said method comprising the steps of:

[0150] (i) seeding an inoculum on a support matrix;

[0151] (ii) culturing said inoculum to provide a first cell culture;

[0152] (iii) passaging at least part of said first cell culture to provide a subculture;

[0153] (iv) adding an additional amount of support matrix to the subculture;

[0154] (v) culturing said subculture; wherein said support matrix comprises cellulose nanofibers, and wherein said passaging does not comprise a step of detaching cells from the support matrix.

[0155] The cells are passaged before confluency and when cell density elevates to a degree where it starts to impede cell proliferation. Thus, the cells should ideally still be in the logarithmic (log) growth phase when passaging is performed. In this way it can be avoided that nutrient depletion or toxic accumulation of waste products influence cell proliferation and passaged cells are not stressed to a degree that it will impact their propensity to proliferate in the new culture. Following passaging, the new culture may be referred to as a subculture. Such subcultures can exist for several generations, i.e. undergoing several passages typically performed as part of the seed-train in industrial scale-up processes.

[0156] In some variants of the method, cells may be cultured in containers of increasing size upon each passage, or at least for the first few passages until a volume of container suitable for industrial production is reached. The containers are preferably culture vessels that are designed for accommodating cell cultures. Containers utilised after the first passage may be referred to as subculturing containers. P97013PC

[0157] Thus, an embodiment of the present invention relates to the method as described herein, wherein the support matrix of step (i) is provided in a first container.

[0158] Another embodiment of the present invention relates to the method as described herein, wherein the at least part of said first cell culture is passaged to a second container.

[0159] The second container may be referred to as a subculturing container in that it is a vessel utilised to further culture a subset of the first cell culture.

[0160] Thus, an embodiment of the present invention relates to the method as described herein, wherein the at least part of said first cell culture is passaged to a subculturing container.

[0161] Yet another embodiment of the present invention relates to the method as described herein, wherein the first container and the subculturing container are culturing vessels.

[0162] A further embodiment of the present invention relates to the method as described herein, wherein the subculturing container comprises a larger volume than the first container.

[0163] A still further embodiment of the present invention relates to the method as described herein, wherein each passage of the cells is to a container comprising a larger volume than the previous container.

[0164] An even further embodiment of the present invention relates to the method as described herein, wherein each passage of the subculture is to a new subculturing container comprising a larger volume than the previous subculturing container.

[0165] Scaling up the production of adherent cells poses several challenges, one of which is the passaging of cells at larger volumes. Furthermore, securing an even distribution of cells in the culture vessel and that all cells receive an adequate supply of nutrients and oxygen becomes more difficult as the culture volume increases. The method described herein relies on a simple passaging step that is less laborious than conventional techniques. Moreover, it is demonstrated that passaged cells distribute sufficiently in the subculturing container to provide exponential cell growth. Without being bound by theory, it is contemplated that cells passaged with the support matrix are capable of efficiently colonizing and proliferating on the additional amount of support matrix made available in the subculturing container to effectively kick off the next logarithmic growth phase. Thus, the present method can advantageously be used for cell production at the larger culture volumes required for industrial cell production. P97013PC

[0166] Accordingly, an embodiment of the present invention relates to the method as described herein, wherein the volume of the container(s) is in the range of about 15 mL to about 10000 L, such as about 50 mL to about 1000 L, such as about 100 mL to about 750 L, such as about 250 mL to about 500 L, such as about 500 mL to about 250 L, such as about 1 L to about 100 L, such as about 5 L to about 25 L.

[0167] Another embodiment of the present invention relates to the method as described herein, wherein the volume of the first container is in the range of about 50 mL to about 25 L, such as about 75 mL to about 10 L, such as about 100 mL to about 5 L, such as about 150 mL to about 1 L, such as about 200 mL to about 750 mL, such as about 250 mL to about 500 mL.

[0168] A further embodiment of the present invention relates to the method as described herein, wherein the volume of the subculturing container(s) is in the range of about 250 mL to about 10000 L, such as about 500 mL to about 1000 L, such as about 1 L to about 500 L, such as about 5 L to about 250 L, such as about 10 L to about 100 L.

[0169] An even further embodiment of the present invention relates to the method as described herein, wherein the volume of the second container(s) is at least 500 mL, such as at least 1 L, such as at least 2 L, such as at least 5 L, such as at least 10 L, such as at least 25 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, such as at least 10000L.

[0170] A still further embodiment of the present invention relates to the method as described herein, wherein the method is for large scale industrial production.

[0171] Bioreactors are of particular importance to large scale industrial production. They may hold larger volumes than laboratory- or pilot study equipment and are used in upstream processes to expand and scale cell culture for production. Accordingly, containers of the method, including the first container and second container (or subculturing container(s)), are preferably bioreactors.

[0172] Therefore, an embodiment of the present invention relates to the method as described herein, wherein the first container and / or second container are bioreactors.

[0173] Another embodiment of the present invention relates to the method as described herein, wherein the bioreactors are suspension based bioreactors. P97013PC

[0174] A further embodiment of the present invention relates to the method as described herein, wherein the bioreactors are selected from the group consisting of stirred tank bioreactors, fluidized bed bioreactors, wave and rocking bioreactors, and airlift bioreactors.

[0175] The adherent cells are cultured as a three-dimensional (3D) culture, i.e. the cells adhere to the support matrix found in solution within the culture vessel and not as a monolayer in a 2D culture or on a bead-based microcarrier. The adherent cells may grow as spheroids when attached to the support matrix. Some challenges present themselves when producing spheroids at industrial scale. One being to maintain a uniform size of spheroids across large batches. Ideally, spheroids do not grow too large as they may then suffer from limited nutrient and oxygen diffusion to the core, leading to necrotic centers and reduced cell viability. A complexity is therefore to ensuring consistent and reproducible conditions for spheroid formation.

[0176] Herein is has been found that the method consistently produces spheroids of relevant sizes, with larger volumes inducing growth of spheroids of smaller sizes which do not suffer from local nutrient depletion.

[0177] Accordingly, an embodiment of the present invention relates to the method as described herein, wherein the cells grow as three-dimensional (3D) spheroids.

[0178] Another embodiment of the present invention relates to the method as described herein, wherein the diameter of the spheroids is in the range of about 25 pm to about 500 pm, such as about 30 pm to about 400 pm, such as about 40 pm to about 300 pm, such as about 50 pm to about 250 pm, such as about 75 pm to about 200 pm.

[0179] A further embodiment of the present invention relates to the method as described herein, wherein the diameter of the spheroids is in the range of about 20 pm to about 100 pm, such as about 30 pm to about 90 pm, such as about 40 pm to about 80 pm, at the time of the first passaging.

[0180] It is to be understood that the diameter of the spheroids refers to the mean diameter of all the spheroids in the cell culture, and may be determined by measuring the diameter, e.g. by microscopy, of a representative number of spheroids. Thus, an embodiment of the present invention relates to the method as described herein, wherein the diameter of the spheroids is the mean diameter of the spheroids in the cell culture. Another embodiment of the present invention relates to the method as described herein, wherein P97013PC diameter of the spheroids is measured by microscopy. The uniformity of the spheroids may be represented by the coefficient of variance (CV) of the spheroid diameters.

[0181] Process control parameters, such as the agitation of the culture, may be adjusted to promote the growth of spheroids of a certain size. Accordingly, agitation within the culture vessel may advantageously be optimised by precise regulation of a means for adjusting agitation ( / .e. an agitation system), such as impellers, baffles, orbital shakers, bubble columns, magnetic stirrers, rocking board and / or perfusion systems. Impellers are mechanical devices mounted on a shaft inside the culture vessel. They rotate to create fluid motion, ensuring even distribution of cells and nutrients. The rotational speed can be selected based on the requirements and / or shear-sensitivity of the cells and taking into account the volume of the culture vessel. Herein it has been found that higher rotational speed tends to induce formation of smaller spheroids.

[0182] Accordingly, an embodiment of the present invention relates to the method as described herein, wherein the container(s) are connected to or comprise an agitation system.

[0183] Another embodiment of the present invention relates to the method as described herein, wherein the agitation system is selected from the group consisting of impellers, baffles, blades, orbital shakers, bubble columns, magnetic stirrers, rocking board and perfusion systems.

[0184] Yet another embodiment of the present invention relates to the method as described herein, wherein the container(s) comprise an agitation element.

[0185] Still another embodiment of the present invention relates to the method as described herein, wherein the agitation system comprises an agitation element.

[0186] A further embodiment of the present invention relates to the method as described herein, wherein the agitation element is selected from the group consisting of impellers, paddles, blades and magnetic stirring bars.

[0187] A preferred embodiment of the present invention relates to the method as described herein, wherein the container(s) comprise impellers.

[0188] Importantly, cells cultured on the support matrix described herein withstand the larger shear stress induced at higher rotational speed (agitation speed) and continue to display high viability and proliferation. Being able to culture cells at high rotational speed P97013PC enhances nutrient and oxygen distribution throughout the culturing container and limits gradients of pH, dissolved oxygen and metabolites. Especially in larger culture vessels or high-density cultures (i.e. industrial scale production) enhanced mass transfer is advantageous to optimise gas exchange and waste removal.

[0189] In conventional industrial scale cell culturing, rotational speed must be balanced against several risks. Higher rotational speed causes increased shear stress in the culture vessel which may lead to cell detachment, cell rounding (loss of morphology), and cell death. This can be particularly problematic when cells are grown as monolayers, such as on beads or other microcarriers that do not facilitate growth as spheroids. Moreover, collisions between beads at high rotational speed can cause physical damage to cells. Thus, for some cell lines, e.g. mammalian and insect cells, it is normal practice to add one or more protectants to the bioreactor to increase resistance against shear stress. These protectants may be, but are not limited to, surfactants, such as non-ionic surfactants like poloxamer 188, protein-based protectants, such as serum proteins or gelatin, anti-foam agents, such as polypropylene glycol, and encapsulation materials, such as alginate beads. Such protectants are regularly used with mammalian cells, such as HEK293, CHO, BHK and Vero, and insect cells, such as Sf9 and Af21.

[0190] Herein, it is not only demonstrated that cells cultured on the support matrix comprising cellulose nanofibers thrive at high rotational speed, but also that that they grow in controllable small-sized spheroids for which nutrient supply is improved compared to larger spheroids or uncontrollable non-spheroid cell aggregates. This is achieved without addition of any protectants against shear stress.

[0191] A preferred embodiment of the present invention relates to the method as described herein, wherein the rotational speed of the agitation system in the container(s) is in the range of about 100 rpm to about 400 rpm, such as about 110 rpm to about 300 rpm, such as about 120 rpm to about 200 rpm.

[0192] Another preferred embodiment of the present invention relates to the method as described herein, wherein the rotational speed of the agitation system in the container(s) is in the range of about 100 rpm to about 250 rpm, more preferably about 120 rpm to about 200 rpm.

[0193] Yet another embodiment of the present invention relates to the method as described herein, wherein the rotational speed of the agitation system in the container(s) is at least P97013PC

[0194] 100 rpm, such at least 120 rpm, such as at least 150 rpm, such as at least 180 rpm, such as at least 200 rpm.

[0195] Thus, an embodiment of the present invention relates to the method as described herein, wherein the rotational speed of the impeller in the container(s) is in the range of about 5 rpm to about 1000 rpm, such as about 10 rpm to about 750 rpm, such as about 20 rpm to about 500 rpm, such as about 25 rpm to about 250 rpm, such as about 50 rpm to about 200 rpm, such as 75 rpm to about 150 rpm.

[0196] Still another embodiment of the present invention relates to the method as described herein, wherein the rotational speed of the agitation system in the first container is in the range of about 25 rpm to about 400 rpm, such as about 50 rpm to about 300 rpm, such as 100 rpm to about 200 rpm.

[0197] Another embodiment of the present invention relates to the method as described herein, wherein the rotational speed of the impeller in the first container is in the range of about 50 rpm to about 1000 rpm, such as about 100 rpm to about 750 rpm, such as 250 rpm to about 500 rpm.

[0198] A further embodiment of the present invention relates to the method as described herein, wherein the rotational speed of the agitation system in the subculturing container(s) is in the range of about 50 rpm to about 500 rpm, such as about 75 rpm to about 400 rpm, such as 100 rpm to about 300 rpm, such as about 120 rpm to about 200 rpm.

[0199] Yet another embodiment of the present invention relates to the method as described herein, wherein the rotational speed of the impeller in the subculturing container(s) is in the range of about 5 rpm to about 100 rpm, such as about 10 rpm to about 75 rpm, such as 20 rpm to about 50 rpm.

[0200] A still further embodiment of the present invention relates to the method as described herein, wherein no protectants against shear stress are added to the container(s) during cell culturing.

[0201] Another embodiment of the present invention relates to the method as described herein, wherein said protectants against shear stress are selected from the group consisting of surfactants, protein-based protectants, anti-foam agents, and encapsulation materials. P97013PC

[0202] A further embodiment of the present invention relates to the method as described herein, wherein said protectants against shear stress are non-ionic surfactants.

[0203] The method described herein can be utilised for consecutive passages of the cells with addition of additional support matrix for the passaged cells to colonize and proliferate on each time. For industrial production of cells, it is valuable to consistently and seamlessly being able to passage cells since some types of cells (e.g. immortalised cells lines) may be passaged up to 200 times, depending on the type of cell line and its intended use. The passage number refers to the number of times a cell culture has been harvested and reseeded into new culture vessels. Having a simple process for passaging the cells is advantageous because each passage can introduce genetic changes if cells are stressed, and minimum handling of the cells reduce the risk of the produced cells deviating from the intended cell product.

[0204] Therefore, an embodiment of the present invention relates to the method as described herein, wherein the method is repeated for consecutive passages of the cells.

[0205] Another embodiment of the present invention relates to the method as described herein, wherein the cells are passaged at least one time, such as at least two times, such as at least three times, such as at least four times, such as at least five times.

[0206] Yet another embodiment of the present invention relates to the method as described herein, wherein steps (iii)-(v) are repeated, such that the subculture is passaged to a further subculturing container and cultured to provide a further subculture.

[0207] A further embodiment of the present invention relates to the method as described herein, wherein steps (iii)-(v) are repeated at least one time, such as at least two times, such as at least three times, such as at least four times, such as at least five times.

[0208] It is to be understood that each passage of the cells entails transfer of a portion or major part of the cell culture, including the support matrix upon which they are attached, from one container to a new container. The passaged cells may be initially from the first cell culture or later from subsequent subcultures. After the first passage of cells from the first cell culture (cultured from the first inoculum with the support matrix), the new cell culture is regarded as a subculture. Subsequent passages of cells will be from a subculture to a new subculture. The containers holding these cell cultures may therefore all be recognised as subculturing containers. As described herein, the volume of the subculturing containers may change (e.g. increase) during the upstream process, and accordingly, so may the P97013PC volume of the new subculture (also referred to as "volume of new culture" or "new working culture volume"). It is to be understood that the volume of "new culture" refers to the total culture volume held within the subculturing container, i.e. the volume of the passaged cells and additional amount of support matrix together with the volume of new cell culture medium. Typically, the volume of new culture is a bit less than the volume of the subculturing container.

[0209] The number of cells transferred for each passage may be adjusted to suit the volume of new culture. The cells are cultured under conditions that allows proliferation following a classical logarithmic growth phase. This includes immersion in a suitable cell culture medium, and careful control of parameters, such as temperature, pH, nutrient supply, oxygen levels, humidity, sterility, and any other special conditions required for specific cell types, such as growth factors and hormones. For each passage, the cells are transferred into fresh medium to provide a new source of nutrients to the passaged cells. Additional support matrix may also be provided for the cells to colonize and proliferate on.

[0210] Thus, an embodiment of the present invention relates to the method as described herein, wherein each passage of the cells involves transfer of the cell culture to a new container.

[0211] Another embodiment of the present invention relates to the method as described herein, wherein each passage comprises transfer of a number of cells in the range of about 5*104to about 3*106cells per mL of new culture, 7.5*104cells per mL of new culture to about l*106cells per mL of new culture, such as about l*105to about 5*105cells per mL of new culture, such as about 1.25*105to about 2.5*105cells per mL of new culture, preferably about 1.5*105cells per mL of new culture.

[0212] A further embodiment of the present invention relates to the method as described herein, wherein cells are cultured under conditions suitable for cell growth.

[0213] A still further embodiment of the present invention relates to the method as described herein, wherein the container(s) comprises cell culture medium.

[0214] An even further embodiment of the present invention relates to the method as described herein, wherein the subculturing container(s) comprises fresh cell culture medium.

[0215] The cultured cells to be passaged to the subculturing container may be done so together with the additional amount of support matrix, i.e. in a single step. P97013PC

[0216] Thus, an embodiment of the present invention relates to the method as described herein, wherein the at least part of said first cell culture is passaged to a second container together with the additional amount of support matrix.

[0217] Through careful experimentation, favourable amounts of support matrix (and ratios to passaged cells) have been identified. Using these amounts of passaged cells and additional added support matrix result in a short lag phase (where cells are adjusting to their new environment and preparing for active growth) meaning that cells enter the log growth phase more quickly, leading to faster cell proliferation and potentially higher yields in a shorter time frame. Without being bound by theory, it is contemplated herein that the support matrix comprising cellulose nanofibers enables quick colonization and subsequent establishment of nuclei for spheroid formation.

[0218] Therefore, an embodiment of the present invention relates to the method as described herein, wherein the additional amount of support matrix comprises at least about 0.01 mg support matrix per mL new culture, such as at least 0.02 mg / mL, such as 0.05 mg / mL, such as 0.06 mg / mL, such as 0.075 mg / mL, such as 0.1 mg / mL, such as 0.2 mg / mL.

[0219] Another embodiment of the present invention relates to the method as described herein, wherein the additional amount of support matrix comprises about 0.05 mg to about 2 mg support matrix per l*106passaged cells, such as 0.1 mg to about 1 mg support matrix per l*106passaged cells, 0.2 mg to about 0.5 mg support matrix per l*106passaged cells.

[0220] A further embodiment of the present invention relates to the method as described herein, wherein the ratio between the number of passaged cells of step (iii) and the additional amount of support matrix of step (iv) is in the range of 0.5*106to 20*106cells / mg support matrix for each mL of new culture, 0.75*106to 10*106cells / mg support matrix for each mL of new culture, such as l*106to 3*106cells / mg support matrix for each mL of new culture.

[0221] The cells are preferably passaged when they are still in the log phase. Cells in the log phase are actively dividing and at their healthiest. Passaging them at this stage ensures that the new culture starts with a population of robust, proliferating cells that can recover quickly and resume growth in the new culture vessel. Moreover, delay of passaging may cause overcrowding of cells leading to nutrient depletion, waste accumulation potential cell death. Finally, cells kept too long in culture may enter a stage of senescence in which they stop dividing and are more prone to undergoing unwanted genetic changes. P97013PC

[0222] Accordingly, an embodiment of the present invention relates to the method as described herein, wherein step (iii) is performed when the first cell culture has reached a confluency of at least about 50%, such as at least about 60%, such as at least about 70%, such as at least about 80%, such as at least about 90%, such as at least about 95%, such as at least about 99%, such as about 100%.

[0223] Another embodiment of the present invention relates to the method as described herein, wherein step (iii) is performed when the first cell culture has reached a confluency in the range of about 70%-90%.

[0224] A further embodiment of the present invention relates to the method as described herein, wherein step (iii) is performed when the first cell culture has reached about l*105to about 10*106cells per mL culture, such as about 3*105to about 5*106cells per mL culture, about 5*105to about 2*106cells per mL culture.

[0225] The method described herein is in principle suitable for any type of adherent cell. Accordingly, the method may find utility of a variety of different adherent cells, including, but not limited to, mammalian cells, and insect cells (e.g. Sf9 and Af21). However, the main application of the method is for culturing of mammalian cells.

[0226] Thus, an embodiment of the present invention relates to the method as described herein wherein said first cell culture comprises adherent cells.

[0227] Another embodiment of the present invention relates to the method as described herein, wherein said inoculum comprises adherent cells.

[0228] A further embodiment of the present invention relates to the method as described herein, wherein said first cell culture comprises mammalian cells.

[0229] A still further embodiment of the present invention relates to the method as described herein, wherein said inoculum comprises mammalian cells.

[0230] Yet another embodiment of the present invention relates to the method as described herein, wherein the mammalian cells are selected from the group consisting of HEK293 cells, Vero cells, CHO cells, BHK cells, hybridoma cells, NS0 cells, PER.C6 cells, MDCK cells, stem cells, smooth muscle cells, hepatocytes, endothelial cells, pancreatic cells, immune cells, epidermal cells, muscle cells, adipose cells, and neuron cells. P97013PC

[0231] An even further embodiment of the present invention relates to the method as described herein, wherein the mammalian cells are selected from the group consisting of Vero cells, MDCK cells, HEK293 cells, BHK cells, WI-38 cells, MRC5 cells, and CEF / DF-1 cells.

[0232] Shear stress may affect cells differently during culturing. Some cells are highly resistant to shear stress while others require special strategies for large scale production in stirred bioreactors. Without being bound by theory, it is contemplated herein that the support matrix comprising cellulose nanofibers described herein protects the adhered cells from shear stress through formation of spheroids thereupon which shield the cells from external forces.

[0233] Therefore, the method for culturing of cells described herein may be particular suited for groups of cells. Cells that are fragile and would normally not be effectively cultured in large bioreactors may be cultured using the method described herein. Likewise, more shear resistant cells may be cultured under conditions of higher rotational speed, thereby improving nutrient distribution and cell growth compared to conventional cell culturing techniques depending on different types of microcarriers, such as beads.

[0234] Accordingly, an embodiment of the present invention relates to the method as described herein, wherein the mammalian cells are selected from the group consisting of HEK293 cells, CHO cells, BHK cells, hybridoma cells, NSO cells, PER.C6 cells, and immune cells.

[0235] A preferred embodiment of the present invention relates to the method as described herein, wherein the mammalian cells are selected from the group consisting of HEK293 cells, CHO cells, and BHK cells.

[0236] Another preferred embodiment of the present invention relates to the method as described herein, wherein the mammalian cells are HEK293 cells.

[0237] Accordingly, an embodiment of the present invention relates to the method as described herein, wherein the mammalian cells are selected from the group consisting of, Vero cells, MDCK cells, stem cells, fibroblasts, smooth muscle cells, hepatocytes, endothelial cells, pancreatic cells, epidermal cells, muscle cells, adipose cells, and neuron cells.

[0238] A preferred embodiment of the present invention relates to the method as described herein, wherein the mammalian cells are Vero cells. P97013PC

[0239] The cells can be passaged without detachment from the support matrix. This is a great advantage because many conventional steps of detaching the cells from the support matrix are time-consuming, involves risk of damaging or contaminating the cells, and overall reduce the throughput of cell production. These conventional detachment steps include enzymatic treatment, mainly trypsinization, chemical treatments and any mechanical methods of removing the cells from the support matrix.

[0240] Enzymatic treatments for detachment of cells include, but are not limited to, treatment with trypsin, collagenase, accutase (non-mammalian enzyme) and dispase. Trypsin breaks down proteins that mediate cell adhesion, while accutase and dispase are used for particularly sensitive cells because it is milder than trypsin. Collagenase is mainly relevant for degradation of collagen-based support matrices.

[0241] Chemical treatments for detachment of cells include, but is not limited to, treatment with ethylenediaminetetraacetic acid (EDTA) and treatment with citric saline. Both EDTA and citric saline works as chelator agents that effectively chelate calcium and magnesium ions that are crucial for cell adhesion. By removing these ions, the ionic interactions between the cells and the support matrix are disrupted.

[0242] Mechanical methods for detachment of cells include, but are not limited to, scraping and pipetting. However, none of these methods are suitable for industrial scale production of cells.

[0243] Accordingly, an embodiment of the present invention relates to the method as described herein, wherein said step of detaching cells from the support matrix is selected from enzymatic treatment, chemical treatment, and mechanical methods.

[0244] Another embodiment of the present invention relates to the method as described herein, wherein said step of detaching cells is enzymatic treatment.

[0245] A further embodiment of the present invention relates to the method as described herein, wherein said step of detaching cells is a mechanical method, such as pipetting or scraping.

[0246] The support matrix comprises cellulose nanofibers that supplies a network with a large surface area for the cells to adhere to. The three-dimensional structure of the support matrix provides an environment resembling the extracellular matrix (ECM) that is optimal for cellular growth. In some variants of the method, the cellulose nanofiber may be processed to a nanofibrous cellulose scaffold. P97013PC

[0247] Thus, an embodiment of the present invention relates to the method as described herein, wherein said support matrix comprises a nanofibrous cellulose scaffold as described herein.

[0248] The preparation of the nanofibrous cellulose scaffold is described in WO 2024 / 133700 A2 and is outlined in the following. It is to be understood that the resulting product from this method may be used as a support matrix in the method for culturing cells described herein. The production of the nanofibrous cellulose scaffold can easily be scaled for industrial use without cost and applicability of the resulting support matrix when used at high volumes being a hindrance.

[0249] Accordingly, an aspect of the present invention relates to a method of preparing a nanofibrous cellulose scaffold, said method comprising the steps of:

[0250] (i) providing an initial cellulose nanofiber material,

[0251] (ii) dividing said initial cellulose nanofiber material into a processed cellulose nanofiber material,

[0252] (iii) functionalizing the processed cellulose nanofiber material by addition of a reagent comprising a functional moiety, and

[0253] (iv) drying the processed cellulose nanofiber material, thereby providing the nanofibrous cellulose scaffold.

[0254] Dividing of the initial cellulose nanofiber material is advantageous as it ensures a homogeneous distribution of the cellulose nanofibers in the final scaffold. The shorter cellulose nanofibers also significantly reduce the risk of entanglement of the nanofibers and formation of clusters of nanofibers, which leads to less available surface area exposed to the cells and increased risk of blocking the impeller of a bioreactor. As part of the upstream biomanufacturing process, sampling (e.g. pipetting or tapping) from the bioreactor is continuously performed in order to perform cell counting, viability and yield results. Therefore, a support matrix with decreased risk of clogging is favoured.

[0255] It has been found that dispersing the initial cellulose nanofiber material results in a homogeneous material that is suitable for use as a support matrix. Without being bound by theory, it is contemplated that dispersing the initial cellulose nanofiber material results in a distribution which are spaced so as to allow multiple attachment points for the cells leading to improved proliferation. P97013PC

[0256] Therefore, an embodiment of the present invention relates to the method of preparing a nanofibrous cellulose scaffold as described herein, wherein dividing said initial cellulose nanofiber material is achieved by dispersing.

[0257] Another embodiment of the present invention relates to the method of preparing a nanofibrous cellulose scaffold as described herein, wherein dispersing is performed with a high-speed disperser.

[0258] A further embodiment of the present invention relates to the method of preparing a nanofibrous cellulose scaffold, wherein dividing said initial nanofiber material comprises a step of cutting the initial nanofiber material with a disperser.

[0259] Increasing the dispersing time reduces the mean length of the cellulose nanofibers in the nanofibrous cellulose scaffold. In particular, it is advantageous to disperse the material for at least a couple of minutes to reduce nanofiber entanglement and cluster formation. Also, without being bound by theory, it is contemplated that longer cellulose nanofibers are not as easily substituted on the hydroxyl (-OH) groups, thereby leading to a lower degree of substitution (DS) of functional moieties.

[0260] Thus, an embodiment of the present invention relates to the method of preparing a nanofibrous cellulose scaffold as described herein, wherein dispersing is performed for at least 2 min, such as at least 5 min, such as at least 10 min, such as at least 15 min, such as at least 20 min, such as at least 30 min, such as at least 40 min, such as at least 50 min, such as at least 60 min, such as at least 90 min, such as at least 120 min.

[0261] Another embodiment of the present invention relates to the method of preparing a nanofibrous cellulose scaffold as described herein, wherein dispersing is performed for a period of time in the range of 2 min to 120 min, such as 2 min to 90 min, such as 5 min to 60 min, such as 10 min to 60 min, such as 15 min to 60 min.

[0262] A preferred embodiment of the present invention relates to the method of preparing a nanofibrous cellulose scaffold as described herein, wherein dispersing is performed for at least 5 min.

[0263] Another preferred embodiment of the present invention relates to the method of preparing a nanofibrous cellulose scaffold as described herein, wherein dispersing is performed for at least 15 min. P97013PC

[0264] A still further preferred embodiment of the present invention relates to the method of preparing a nanofibrous cellulose scaffold as described herein, wherein dispersing is performed for at least 60 min.

[0265] Dispersing of the initial cellulose nanofiber material for at least 15 min, and even at least 60 min, can be advantageous as it reduces entanglement and cluster formation of the cellulose nanofibers.

[0266] Preferably dispersing is performed at high speed, such as at about 18000 rpm. The speed may be adjusted depending on the type of disperser. Any type of disperser may be used, including, but not limited to, laboratory high-speed disperser, pilot-scale high-speed dispersers, and production-scale high-speed dispersers. The speed may be adjusted to produce a good vortex in the solution, and can depend on volume and viscosity of the solution.

[0267] Therefore, an embodiment of the present invention relates to the method of preparing a nanofibrous cellulose scaffold as described herein, wherein dispersing is performed at a speed in the range of about 10000 rpm to about 30000 rpm, such as at about 12000 rpm to about 25000 rpm, such as about 15000 rpm to about 20000 rpm.

[0268] During the dividing step, the cellulose nanofibers are reduced in length. It is advantageous that the cellulose nanofibers are not too long as it can cause entanglement of the nanofibers and cluster formation. Cells are not able to penetrate into these tight clusters of entangled nanofibers and thereby a portion of the large surface area of the nanofibers are lost.

[0269] Accordingly, an embodiment of the present invention relates to the method of preparing a nanofibrous cellulose scaffold as described herein, wherein the mean length of the cellulose nanofibers in said processed cellulose nanofiber material is shorter than the mean length of the cellulose nanofibers in said initial cellulose nanofiber material.

[0270] Another embodiment of the present invention relates to the method of preparing a nanofibrous cellulose scaffold as described herein, wherein the mean length of the cellulose nanofibers in said processed cellulose nanofiber material is in the range of about 30 pm to about 250 pm, such as about 40 pm to about 200 pm, such as about 50 pm to about 175 pm, such as about 60 pm to about 150 pm, preferably about 70 pm to about 120 pm. P97013PC

[0271] Yet another embodiment of the present invention relates to the method of preparing a nanofibrous cellulose scaffold as described herein, wherein the mean length of the cellulose nanofibers in said processed cellulose nanofiber material is from about 30 pm to about 250 pm, such as about 40 pm to about 200 pm, such as about 50 pm to about 150 pm, preferably about 60 pm to about 100 pm.

[0272] A further embodiment of the present invention relates to the method of preparing a nanofibrous cellulose scaffold as described herein, wherein the mean length is the volume weighted mean value (D[4,3]) of the cellulose nanofibers.

[0273] A further embodiment of the present invention relates to the method of preparing a nanofibrous cellulose scaffold as described herein, wherein the mean length is measured by light scattering.

[0274] The dividing step may be performed under cooling to lower the ductility of the cellulose nanofibers, making them more brittle and easier to divide. The cooling may occur before or during the dividing of the initial cellulose nanofiber material. Cooling may comprise cooling of the container in which the initial cellulose nanofiber material is held during the dividing step or cooling the initial cellulose nanofiber material by exposure to a coolant, such as liquid hydrogen, liquid helium and / or liquid nitrogen, or by keeping the initial cellulose nanofiber material in a fridge or freezer immediately before the dividing step.

[0275] Thus, an embodiment of the present invention relates to the method of preparing a nanofibrous cellulose scaffold as described herein, wherein the initial cellulose nanofiber material is cooled before or during the dividing step.

[0276] Cellulose sheets, if prepared by electrospinning, may be highly static and difficult to handle. Therefore, the initial cellulose nanofiber material may conveniently be provided as a liquid sample that is ready for processing, e.g. by dispersing.

[0277] Thus, an embodiment of the present invention relates to the method of preparing a nanofibrous cellulose scaffold as described herein, wherein the initial cellulose nanofiber material is provided as a liquid sample.

[0278] Another embodiment of the present invention relates to the method of preparing a nanofibrous cellulose scaffold as described herein, wherein the solvent of the liquid sample comprises water and / or ethanol. P97013PC

[0279] Yet another embodiment of the present invention relates to the method of preparing a nanofibrous cellulose scaffold as described herein, wherein the solvent of the liquid sample comprises ethanol.

[0280] A further embodiment of the present invention relates to the method of preparing a nanofibrous cellulose scaffold as described herein, 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%, with respect to the total weight of the liquid sample.

[0281] A common source of cellulose nanofibers is pulp, which may be mechanically defibrillated to generate cellulosic nanofibers. Mechanical treatment may include high-pressure homogenization, grinding and / or microfluidization. Mechanical defibrillation is commonly utilised in the pulp and paper industry, as well as the textile industry. Unfortunately, it can be difficult to precisely control cellulose nanofiber dimensions and properties when the starting material is raw pulp (that can vary in composition depending on source) which is subsequently exposed to the harsh mechanical treatment. Thus, it is preferred to avoid cellulose material derived directly from a raw plant source (e.g. without any treatment to extract and / or purify the cellulose nanofibers) as the source of the cellulose nanofibers. Raw plant sources for cellulose material may include wood, such as softwood (e.g. spruce, pine, fir, larch, or hemlock) or hardwood (e.g. birch, aspen, poplar, alder, eucalyptus or acacia), or non-wood sources such as agricultural residues, grasses or other plant substances (e.g. straw, leaves, bark, seeds, hulls, flowers, vegetables or fruits from cotton, corn, wheat, oat, rye, barley, rice, flax, hemp, manilla hemp, sisal hemp, jute, ramie, kenaf, bagasse, bamboo or reed).

[0282] Accordingly, it is advantageous to use initial cellulose nanofiber material derived from a sources, e.g. plant source, that has been treated to purify the cellulose nanofibers to ensure high quality and consistency of the cellulose nanofibers utilised for preparing the nanofibrous cellulose scaffold.

[0283] Therefore, an embodiment of the present invention relates to the method of preparing a nanofibrous cellulose scaffold as described herein, wherein the initial cellulose nanofiber material is not derived from a raw plant source.

[0284] Another embodiment of the present invention relates to the method of preparing a nanofibrous cellulose scaffold as described herein, wherein the initial cellulose nanofiber P97013PC material is not prepared by mechanical treatment, such as mechanical defibrillation, such as mechanical disintegration.

[0285] It is to be understood that a raw plant source is a source which has not been treated to extract or purify the cellulose nanofibers. Extraction may include chemical treatment of a cellulose source, such as a raw plant source. The chemical treatment can comprise treating the raw plant source with acetic acid and acetic anhydride in the presence of a catalyst, such as sulfuric acid. The chemical treatment can produce a cellulose acetate solution which can be further processed, e.g. by electrospinning, to yield cellulose nanofibers with highly controllable dimensions and properties.

[0286] Thus, the cellulose nanofibers may advantageously be obtained from electrospinning to allow precise control over nanofiber dimensions and quality. Electrospinning involves dissolving cellulose or its derivatives in a suitable solvent and then spinning it into nanofibers using an electric field. Electrospun nanofibers are spun as monofibers that can be aligned on a drum roll to yield a cellulose sheet that can subsequently be further processed.

[0287] Therefore, an embodiment of the resent invention relates to the method of preparing a nanofibrous cellulose scaffold as described herein, wherein the initial cellulose nanofiber material comprises monofibers.

[0288] Another embodiment of the resent invention relates to the method of preparing a nanofibrous cellulose scaffold as described herein, wherein said nanofibrous cellulose scaffold comprises monofibers.

[0289] The initial cellulose nanofiber material may be prepared from a cellulose acetate solution, e.g. by electrospinning of a cellulose acetate solution. However, the resulting cellulose acetate sheets are preferably regenerated to cellulose sheets in a sodium hydroxide bath before any further processing. This treatment opens up hydroxyl (-OH) groups that may subsequently be used for binding of functional moieties.

[0290] Therefore, an embodiment of the present invention relates to the method of preparing a nanofibrous cellulose scaffold as described herein, wherein the initial cellulose nanofiber material is prepared by electrospinning of a cellulose acetate solution to cellulose acetate sheets. P97013PC

[0291] Another embodiment of the present invention relates to the method of preparing a nanofibrous cellulose scaffold as described herein, wherein said initial cellulose nanofiber material is regenerated in a regeneration solution comprising sodium hydroxide.

[0292] Yet another embodiment of the present invention relates to the method of preparing a nanofibrous cellulose scaffold 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.

[0293] Regeneration of cellulose sheets is preferably performed in an ethanol solution comprising sodium hydroxide. It is possible to use varying amounts of ethanol, such as from 5% vol / vol to 99% vol / vol.

[0294] Accordingly, an embodiment of the present invention relates to the method of preparing a nanofibrous cellulose scaffold 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.

[0295] The content of ethanol in the regeneration solution may influence the mechanical properties of the resulting nanofibrous cellulose scaffold. Mechanical properties that can be impacted by the content of ethanol includes elasticity and brittleness. Without being bound by theory, it is contemplated that a more elastic and less stiff material is beneficial for interaction with the cells and to promote proliferation.

[0296] Thus, an embodiment of the present invention relates to the method of preparing a nanofibrous cellulose scaffold 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.

[0297] By regenerating the initial cellulose nanofiber material, e.g. by alkali treatment, the cellulose obtains a monoclinic crystal structure known as cellulose II. In contrast, native cellulose found in plants has the crystal structure cellulose I. Without being bound by theory, it is contemplated that the crystal structure cellulose II is advantageous for use as the initial cellulose nanofiber material, e.g. because it is more thermodynamically stable P97013PC than other polymorphs such as cellulose I. This may be due to the antiparallel polymer chain orientation and different hydrogen bonding of cellulose II.

[0298] Accordingly, an embodiment of the present invention relates to the method of preparing a nanofibrous cellulose scaffold as described herein, wherein said initial cellulose nanofiber material comprises regenerated cellulose.

[0299] Another embodiment of the present invention relates to the method of preparing a nanofibrous cellulose scaffold as described herein, wherein the crystal structure of said initial cellulose nanofiber material is cellulose II.

[0300] Subsequent to dividing of the initial cellulose nanofiber material, the resulting processed material is preferably handled to make it ready for functionalization. Part of the treatment can include filtration, washing and re-suspension of the processed cellulose nanofiber material. Filtration ensures that excess water is removed from the processed cellulose nanofiber material. Washing assists in removal of any acetate ions still present after the previous treatment. When re-suspending the processed cellulose nanofiber material, the concentration of cellulose nanofibers may be adjusted if desired.

[0301] Accordingly, an embodiment of the present invention relates to the method of preparing a nanofibrous cellulose scaffold as described herein, wherein the dividing step (ii) is followed by a step comprising filtration, washing and suspending the processed cellulose nanofiber material.

[0302] Prior to functionalization, the cellulose nanofibers are preferably mercerized to improve the substitution of functional moieties onto the cellulose nanofibers. Mercerization is a process in which the cellulose nanofibers are swollen by immersion in a concentrated aqueous NaOH solution. During the mercerization process, the crystal structure of the cellulose nanofiber is transformed from cellulose I to cellulose II. Under the action of concentrated alkaline solutions chemical, physicochemical and structural modifications of cellulose occur. Upon washing and neutralisation cellulose II is formed. As a result of the penetration of the base into the lattice, internal hydrogen bonds are broken and the number of available hydroxyl groups (-OH) in the cellulose nanofiber is increased. It is therefore contemplated that mercerization improves the degree of substitution (DS). The mercerization step may be performed before or after the dividing step. P97013PC

[0303] Therefore, an embodiment of the present invention relates to the method of preparing a nanofibrous cellulose scaffold as described herein further comprising a step of mercerization of said processed cellulose nanofiber material.

[0304] Another embodiment of the present invention relates to the method of preparing a nanofibrous cellulose scaffold as described herein, wherein the mercerization step is immediately before or after the dividing step (ii).

[0305] A further embodiment of the present invention relates to the method of preparing a nanofibrous cellulose scaffold as described herein, wherein said mercerization step comprises addition of NaOH in a concentration of about 0.1 M to about 2 M, such as about 0.5 M to about 1.8 M, such as about 1 M to about 1.7 M, preferably about 1.4 M to about 1.6 M.

[0306] After functionalization of the processed cellulose nanofiber scaffold, the material is dried to yield the nanofibrous cellulose scaffold in its final form. Drying may be performed in two steps, such as freezing followed by lyophilization, or in a single step, such as by lyophilization. A lyophilizer performs a water removal process that can extend shelf life and / or make the material more convenient for transport. Lyophilizers work by freezing the material, then reducing the pressure and adding heat to allow the frozen water in the material to sublimate.

[0307] Accordingly, an embodiment of the present invention relates to the method of preparing a nanofibrous cellulose scaffold as described herein, wherein the drying step (iv) comprises freezing and / or lyophilization of the processed cellulose nanofiber material.

[0308] Another embodiment of the present invention relates to the method of preparing a nanofibrous cellulose scaffold as described herein, wherein the drying step (iv) comprises freezing of processed cellulose nanofiber material followed by lyophilization of the frozen processed cellulose nanofiber material.

[0309] The dried nanofibrous cellulose scaffold may be further processed to provide a dry product. This may be accomplished by grinding the dried product.

[0310] The method described herein provides a nanofibrous cellulose scaffold with large surface area that may advantageously be utilised as a support matrix for culturing of cells. The support matrix may be in the form of a dry product that is added to the cell culturing container, such as a bioreactor, to provide an optimal environment for the adherence and P97013PC proliferation of the cells to be cultured. The dry product may be reconstituted in phosphate-buffered saline (PBS), autoclaved, and transferred in culture medium before addition to the cell culturing container.

[0311] Thus, an aspect of the present invention relates to a nanofibrous cellulose scaffold obtainable from the method of preparing a nanofibrous cellulose scaffold as described herein.

[0312] The listing or discussion of an apparently prior published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.

[0313] Preferences, options and embodiments for a given aspect, feature or parameter of the invention should, unless the context indicates otherwise, be regarded as having been disclosed in combination with any and all preferences, options and embodiments for all other aspects, features and parameters of the invention. This is especially true for the description of the preparation of the nanofibrous cellulose scaffold and all its features, which may readily be part of the nanofibrous cellulose scaffold as such, or its utility in the method for passaging of cells. Embodiments and features of the present invention are also outlined in the following items.

[0314] Items

[0315] XI. A method of preparing a nanofibrous cellulose scaffold, said method comprising the steps of:

[0316] (i) providing an initial cellulose nanofiber material,

[0317] (ii) dividing said initial cellulose nanofiber material into a processed cellulose nanofiber material,

[0318] (iii) functionalizing the processed cellulose nanofiber material by addition of a reagent comprising a functional moiety, and

[0319] (iv) drying the processed cellulose nanofiber material, thereby providing the nanofibrous cellulose scaffold.

[0320] X2. The method according to item XI, wherein dividing said initial cellulose nanofiber material is achieved by dispersing.

[0321] X3. The method according to item X2, wherein dispersing is performed with a high-speed disperser. P97013PC

[0322] X4. The method according to any one of items X2 or X3, wherein dispersing is performed for at least 2 min, such as at least 5 min, such as at least 10 min, such as at least 15 min, such as at least 20 min, such as at least 30 min, such as at least 40 min, such as at least 50 min, such as at least 60 min, such as at least 90 min, such as at least 120 min.

[0323] X5. The method according to any one of items X2-X4, wherein dispersing is performed at a speed in the range of about 10000 rpm to about 30000 rpm, such as at about 12000 rpm to about 25000 rpm, such as about 15000 rpm to about 20000 rpm.

[0324] X6. The method according to any one of the preceding items, wherein the mean length of the cellulose nanofibers in said processed cellulose nanofiber material is shorter than the mean length of the cellulose nanofibers in said initial cellulose nanofiber material.

[0325] X7. The method according to any one of the preceding items, wherein the mean length of the cellulose nanofibers in said processed cellulose nanofiber material is less than 250 pm, such as less than about 200 pm, such as less than about 150 pm, such as less than about 120 pm, such as less than about 100 pm, such as less than about 80 pm.

[0326] X8. The method according to any one of the preceding items, wherein the mean length of the cellulose nanofibers in said processed cellulose nanofiber material is in the range of about 30 pm to about 250 pm, such as about 40 pm to about 200 pm, such as about 50 pm to about 175 pm, such as about 60 pm to about 150 pm, preferably about 70 pm to about 120 pm.

[0327] X9. The method according to any one of the preceding items, wherein the initial cellulose nanofiber material is provided as a liquid sample.

[0328] X10. The method according to item X9, wherein the solvent of the liquid sample comprises water and / or ethanol.

[0329] XI 1. The method according to any one of the preceding items, wherein the concentration of cellulose nanofibers in the processed cellulose nanofiber material is about 0.1 wt% to about 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%, with respect to the total weight of the liquid sample.

[0330] X12. The method according to any one of the preceding items, wherein the mean diameter of the cellulose nanofibers in said initial cellulose nanofiber material and / or processed P97013PC 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.

[0331] X13. The method according to any one of the preceding items, wherein the initial cellulose nanofiber material is prepared by a process selected from the group consisting of electrospinning, meltblowing, drawing, self-assembly, template synthesis, and thermal- induced phase separation.

[0332] X14. The method according to any one of the preceding items, wherein the initial cellulose nanofiber material is prepared by electrospinning of a cellulose acetate solution to cellulose acetate sheets.

[0333] X15. The method according to any one of the preceding items, wherein said initial cellulose nanofiber material is regenerated in a regeneration solution comprising sodium hydroxide.

[0334] X16. The method according to item X15, 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.

[0335] X17. The method according to any one of the preceding items further comprising a step of mercerization of said processed cellulose nanofiber material.

[0336] X18. The method according to item X17, wherein the mercerization step is immediately before or after the dividing step (ii).

[0337] X19. The method according to any one of the preceding items, wherein the functional moiety is selected from chemical moieties or biological molecules.

[0338] X20. The method according to item X19, wherein the chemical moieties are selected from the group consisting of negatively charged groups, positively charged groups, zwitterionic groups, hydrophobic groups, hydrophilic groups, amphiphilic groups, ligands, and combinations thereof.

[0339] X21. The method according to any one of items X19 or X20, wherein the chemical moieties are selected from the group consisting of quaternary ammonium (QA), carboxymethyl P97013PC

[0340] (CM), diethylaminoethyl (DEAE), 4-(l-bromoethyl)benzoic acid, 4- (dimethylamino)pyridine, epoxide, and combinations thereof.

[0341] X22. The method according to any one of items X19-X21, wherein the chemical moiety is quaternary ammonium (QA).

[0342] X23. The method according to any one of the preceding items, wherein the dividing step (ii) is followed by a step comprising filtration, washing and suspending the processed cellulose nanofiber material.

[0343] X24. The method according to any one of the preceding items, wherein the drying step (iv) comprises freezing and / or lyophilization of the processed cellulose nanofiber material.

[0344] X25. The method according to any one of the preceding items, wherein the drying step (iv) comprises freezing of processed cellulose nanofiber material followed by lyophilization of the frozen processed cellulose nanofiber material.

[0345] X26. The method according to any one of the preceding items, wherein the initial cellulose nanofiber material is not derived from a raw plant source.

[0346] X27. The method according to any one of the preceding items, wherein the initial cellulose nanofiber material is not prepared by mechanical treatment, such as mechanical defibrillation, such as mechanical disintegration.

[0347] Yl. A nanofibrous cellulose scaffold obtainable from a method according to any one of items X1-X27.

[0348] Zl. A nanofibrous cellulose scaffold comprising a cellulose nanofiber material with a mean length of the cellulose nanofibers of less than about 250 pm.

[0349] Z2. The nanofibrous cellulose scaffold according to item Zl, wherein the cellulose nanofibers have a mean length in the range of about 30 pm to about 250 pm, such as about 40 pm to about 200 pm, such as about 50 pm to about 175 pm, such as about 60 pm to about 150 pm, preferably about 70 pm to about 120 pm.

[0350] Z3. The nanofibrous cellulose scaffold according to item Zl, wherein the cellulose nanofibers have a mean length in the range of about 30 pm to about 250 pm, such as P97013PC about 40 pm to about 200 m, such as about 50 pm to about 150 pm, preferably about 60 pm to about 100 pm.

[0351] Z4. The nanofibrous cellulose scaffold according to any one of items Z1-Z3, wherein the mean length is the volume weighted mean value (D[4,3]) of the cellulose nanofibers.

[0352] Z5. The nanofibrous cellulose scaffold according to any one of items Z1-Z4, wherein the mean length is measured by light scattering.

[0353] Z6. The nanofibrous cellulose scaffold according to any one of items Z1-Z5, wherein the cellulose nanofibers have a mean diameter in the range of about 10 nm to about 2000 nm, such as about 50 nm to about 1500 nm, such as about 100 nm to about 1000 nm, preferably about 250 nm to about 750 nm.

[0354] Z7. The nanofibrous cellulose scaffold according to any one of items Z1-Z6, wherein the cellulose nanofiber material is functionalized with a functional moiety.

[0355] Z8. The nanofibrous cellulose scaffold according to item Z7, wherein the functional moiety is selected from chemical moieties or biological molecules.

[0356] Z9. The nanofibrous cellulose scaffold according to item Z8, wherein the chemical moieties are selected from the group consisting of negatively charged groups, positively charged groups, zwitterionic groups, hydrophobic groups, hydrophilic groups, amphiphilic groups, ligands, and combinations thereof.

[0357] Z10. The nanofibrous cellulose scaffold according to any one of items Z8 or Z9, wherein the chemical moieties are selected from the group consisting of quaternary ammonium (QA), carboxymethyl (CM), diethylaminoethyl (DEAE), 4-(l-bromoethyl)benzoic acid, 4- (dimethylamino)pyridine, epoxide, and combinations thereof.

[0358] Zll. The nanofibrous cellulose scaffold according to any one of items Z8-Z10, wherein the chemical moiety is quaternary ammonium (QA).

[0359] Z12. The nanofibrous cellulose scaffold according to any one of items Zl-Zll, wherein the nanofibrous cellulose scaffold is provided as a dry material.

[0360] Z13. The nanofibrous cellulose scaffold according to any one of items Z1-Z12, wherein the nanofibrous cellulose scaffold is provided as a lyophilized material. P97013PC

[0361] Z14. The nanofibrous cellulose scaffold according to any one of items Z1-Z13, wherein the nanofibrous cellulose scaffold has a BET surface area of at least about 40000 cm2 / g, such as at least about 50000 cm2 / g, such as at least about 55000 cm2 / g, such as at least about 60000 cm2 / g, such as at least about 70000 cm2 / g, such as at least about 80000 cm2 / g.

[0362] Z15. The nanofibrous cellulose scaffold according to any one of items Z1-Z14, wherein the initial cellulose nanofiber material is not plant-derived.

[0363] Z16. The nanofibrous cellulose scaffold according to any one of items Z1-Z15, wherein the initial cellulose nanofiber material is not prepared by mechanical treatment, such as mechanical defibrillation, such as mechanical disintegration.

[0364] Z17. The nanofibrous cellulose scaffold according to any one of items Z1-Z16, wherein the cellulose nanofibers are electrospun cellulose nanofibers.

[0365] Z18. The nanofibrous cellulose scaffold according to any one of items Z1-Z17, wherein the cellulose nanofibers comprise monofibers.

[0366] Z19. The nanofibrous cellulose scaffold according to any one of items Z1-Z18, wherein the cellulose nanofibers are derived from regenerated cellulose.

[0367] Z20. The nanofibrous cellulose scaffold according to any one of items Z1-Z19, wherein the cellulose nanofibers have the cellulose II crystal structure.

[0368] QI. A method for culturing of cells, said method comprising the steps of:

[0369] (i) providing a first container comprising a first cell culture and a support matrix;

[0370] (ii) passaging at least part of said first cell culture to a second container to provide a subculture;

[0371] (iii) adding an additional amount of support matrix to said second container; and

[0372] (iv) culturing said subculture; wherein said support matrix comprises cellulose nanofibers, and wherein said passaging does not comprise a step of detaching cells from the support matrix.

[0373] Q2. The method according to item QI, wherein the second container is a subculturing container. P97013PC

[0374] Q3. The method according to item Q2, wherein the first container and the subculturing container are culturing vessels.

[0375] Q4. The method according to any one of items Q2 or Q3, wherein the subculturing container comprises a larger volume than the first container.

[0376] Q5. The method according to any one of items Q1-Q4, wherein each passage of the cells is to a container comprising a larger volume than the previous container.

[0377] Q6. The method according to any one of items Q1-Q5, wherein the volume of the container(s) is in the range of about 15 mL to about 10000 L, such as about 50 mL to about 1000 L, such as about 100 mL to about 750 L, such as about 250 mL to about 500 L, such as about 500 mL to about 250 L, such as about 1 L to about 100 L, such as about 5 L to about 25 L.

[0378] Q7. The method according to any one of items Q2-Q6, wherein the volume of the subculturing container(s) is in the range of about 250 mL to about 10000 L, such as about 500 mL to about 1000 L, such as about 1 L to about 500 L, such as about 5 L to about 250 L, such as about 10 L to about 100 L.

[0379] Q8. The method according to any one of items Q1-Q7, wherein the volume of the second container(s) is at least 500 mL, such as at least 1 L, such as at least 2 L, such as at least 5 L, such as at least 10 L, such as at least 25 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, such as at least 10000 L.

[0380] Q9. The method according to any one of items Q1-Q8, wherein the container(s) are connected to or comprise an agitation system.

[0381] Q10. The method according to item Q9, wherein the agitation system is selected from the group consisting of impellers, baffles, blades, orbital shakers, bubble columns, magnetic stirrers, rocking board and perfusion systems.

[0382] Qll. The method according to any one of items Q1-Q10, wherein the container(s) comprise an agitation element.

[0383] Q12. The method according to item Qll, wherein the agitation element is selected from the group consisting of impellers, paddles, blades and magnetic stirring bars. P97013PC

[0384] Q13. The method according to any one of items Qll or Q12, wherein the rotational speed of the agitation system in the container(s) is in the range of about 100 rpm to about 400 rpm, such as about 110 rpm to about 300 rpm, such as about 120 rpm to about 200 rpm.

[0385] Q14. The method according to any one of items Qll or Q12, wherein the rotational speed of the agitation system in the container(s) is in the range of about 100 rpm to about 250 rpm, more preferably about 120 rpm to about 200 rpm.

[0386] Q15. The method according to any one of items Qll or Q12, wherein the rotational speed of the agitation system in the first container is in the range of about 25 rpm to about 400 rpm, such as about 50 rpm to about 300 rpm, such as 100 rpm to about 200 rpm.

[0387] Q16. The method according to any one of items Qll or Q12, wherein the rotational speed of the agitation system in the subculturing container(s) is in the range of about 50 rpm to about 500 rpm, such as about 75 rpm to about 400 rpm, such as 100 rpm to about 300 rpm, such as about 120 rpm to about 200 rpm.

[0388] Q17. The method according to any one of items Q1-Q16, wherein the container(s) comprise impellers.

[0389] Q18. The method according to any one of items Q12-Q17, wherein the rotational speed of the impeller in the container(s) is in the range of about 5 rpm to about 1000 rpm, such as about 10 rpm to about 750 rpm, such as about 20 rpm to about 500 rpm, such as about 25 rpm to about 250 rpm, such as about 50 rpm to about 200 rpm, such as 75 rpm to about 150 rpm.

[0390] Q19. The method according to any one of items Q12-Q18, wherein the rotational speed of the impeller in the first container is in the range of about 50 rpm to about 1000 rpm, such as about 100 rpm to about 750 rpm, such as 250 rpm to about 500 rpm.

[0391] Q20. The method according to any one of items Q12-Q19, wherein the rotational speed of the impeller in the subculturing container(s) is in the range of about 5 rpm to about 100 rpm, such as about 10 rpm to about 75 rpm, such as 20 rpm to about 50 rpm.

[0392] Q21. The method according to any one of items Q1-Q20, wherein no protectants against shear stress are added to the container(s) during cell culturing. P97013PC

[0393] Q22. The method according to item Q21, wherein said protectants against shear stress are selected from the group consisting of surfactants, protein-based protectants, antifoam agents, and encapsulation materials.

[0394] Q23. The method according to any one of items Q21 or Q22, wherein said protectants against shear stress are non-ionic surfactants.

[0395] Q24. The method according to any one of items Q1-Q23, wherein the method is for large scale industrial production.

[0396] Q25. The method according to any one of items Q1-Q24, wherein the first container and / or second container are bioreactors.

[0397] Q26. The method according to item Q25, wherein the bioreactors are suspension based bioreactors.

[0398] Q27. The method according to any one of items Q25 or Q26, wherein the bioreactors are selected from the group consisting of stirred tank bioreactors, fluidized bed bioreactors, wave and rocking bioreactors, and airlift bioreactors.

[0399] Q28. The method according to any one of items Q1-Q27, wherein the method is repeated for consecutive passages of the cells.

[0400] Q29. The method according to any one of items Q1-Q28, wherein the cells are passaged at least one time, such as at least two times, such as at least three times, such as at least four times, such as at least five times.

[0401] Q30. The method according to any one of items Q1-Q29, wherein steps (ii)-(iv) are repeated, such that the subculture is passaged to a further subculturing container and cultured to provide a further subculture.

[0402] Q31. The method according to any one of items Q1-Q30, wherein steps (ii)-(iv) are repeated at least one time, such as at least two times, such as at least three times, such as at least four times, such as at least five times.

[0403] Q32. The method according to any one of items Q1-Q31, wherein each passage of the cells involves transfer of the cell culture to a new container. P97013PC

[0404] Q33. The method according to any one of items Q1-Q32, wherein each passage comprises transfer of a number of cells in the range of about 5*104to about 3*106cells per mL of new culture, 7.5*104cells per mL of new culture to about l*106cells per mL of new culture, such as about l*105to about 5*105cells per mL of new culture, such as about 1.25*105to about 2.5*105cells per mL of new culture, preferably about 1.5*105cells per mL of new culture.

[0405] Q34. The method according to any one of items Q1-Q33, wherein cells are cultured under conditions suitable for cell growth.

[0406] Q35. The method according to any one of items Q1-Q34, wherein the container(s) comprises cell culture medium.

[0407] Q36. The method according to any one of items Q2-Q35, wherein the subculturing container(s) comprises fresh cell culture medium.

[0408] Q37. The method according to any one of items Q1-Q36, wherein the additional amount of support matrix comprises at least about 0.01 mg support matrix per mL new culture, such as at least 0.02 mg / mL, such as 0.05 mg / mL, such as 0.06 mg / mL, such as 0.075 mg / mL, such as 0.1 mg / mL, such as 0.2 mg / mL.

[0409] Q38. The method according to any one of items Q1-Q37, wherein the additional amount of support matrix comprises about 0.05 mg to about 2 mg support matrix per l*106passaged cells, such as 0.1 mg to about 1 mg support matrix per l*106passaged cells, 0.2 mg to about 0.5 mg support matrix per l*106passaged cells.

[0410] Q39. The method according to any one of items Q1-Q38, wherein the ratio between the number of passaged cells of step (ii) and the additional amount of support matrix of step (iii) is in the range of 0.5*106to 20*106cells / mg support matrix for each mL of new culture, 0.75*106to 10*106cells / mg support matrix for each mL of new culture, such as l*106to 3*106cells / mg support matrix for each mL of new culture.

[0411] Q40. The method according to any one of items Q1-Q39, wherein the at least part of said first cell culture is passaged to a second container together with the additional amount of support matrix.

[0412] Q41. The method according to any one of items Q1-Q40, wherein step (ii) is performed when the first cell culture has reached a confluency of at least about 50%, such as at least P97013PC about 60%, such as at least about 70%, such as at least about 80%, such as at least about 90%, such as at least about 95%, such as at least about 99%, such as about 100%.

[0413] Q42. The method according to any one of items Q1-Q41, wherein step (ii) is performed when the first cell culture has reached a confluency in the range of about 70%-90%.

[0414] Q43. The method according to any one of items Q1-Q42, wherein step (ii) is performed when the first cell culture has reached about l*105to about 10*106cells per mL culture, such as about 3*105to about 5*106cells per mL culture, about 5*105to about 2*106cells per mL culture.

[0415] Q44. The method according to any one of items Q1-Q43, wherein the cells grow as three- dimensional (3D) spheroids.

[0416] Q45. The method according to item Q44, wherein the diameter of the spheroids is in the range of about 25 pm to about 500 pm, such as about 30 pm to about 400 pm, such as about 40 pm to about 300 pm, such as about 50 pm to about 250 pm, such as about 75 pm to about 200 pm.

[0417] Q46. The method according to any one of items Q44 or Q45, wherein the diameter of the spheroids is in the range of about 20 pm to about 100 pm, such as about 30 pm to about 90 pm, such as about 40 pm to about 80 pm, at the time of the first passaging.

[0418] Q47. The method according to any one of items Q45 or Q46, wherein coefficient of variance (CV) of the diameter of the spheroids is less than 50%, preferably less than 40%.

[0419] Q48. The method according to any one of items Q1-Q47, wherein said step of detaching cells from the support matrix is selected from enzymatic treatment, chemical treatment and mechanical methods.

[0420] Q49. The method according to any one of items Q1-Q48, wherein said step of detaching cells is enzymatic treatment.

[0421] Q50. The method according to any one of items Q1-Q48, wherein said step of detaching cells is a mechanical method, such as pipetting or scraping.

[0422] Q51. The method according to any one of items Q1-Q50, wherein said first cell culture comprises adherent cells. P97013PC

[0423] Q52. The method according to any one of items Q1-Q51, wherein said first cell culture comprises mammalian cells.

[0424] Q53. The method according to item Q52, wherein the mammalian cells are selected from the group consisting of HEK293 cells, Vero cells, CHO cells, BHK cells, hybridoma cells, NSO cells, PER.C6 cells, MDCK cells, stem cells, smooth muscle cells, hepatocytes, endothelial cells, pancreatic cells, immune cells, epidermal cells, muscle cells, adipose cells, and neuron cells.

[0425] Q54. The method according to any one of items Q52 or Q53, wherein the mammalian cells are selected from the group consisting of HEK293 cells, CHO cells, and BHK cells.

[0426] Q55. The method according to any one of items Q52-Q54, wherein the mammalian cells are HEK293 cells.

[0427] Q56. The method according to any one of items Q1-Q55, wherein said step of providing a first container comprising a first cell culture and a support matrix comprises seeding an inoculum on a support matrix followed by culturing of said inoculum.

[0428] Q57. The method according to any one of items Q1-Q50, said method comprising the steps of:

[0429] (ia) seeding an inoculum on a support matrix;

[0430] (ib) culturing said inoculum to provide a first cell culture;

[0431] (ii) passaging at least part of said first cell culture to provide a subculture;

[0432] (iii) adding an additional amount of support matrix to the subculture;

[0433] (iv) culturing said subculture; wherein said support matrix comprises cellulose nanofibers, and wherein said passaging does not comprise a step of detaching cells from the support matrix.

[0434] Q58. The method according to item Q1-Q57, wherein said support matrix comprises a nanofibrous cellulose scaffold according to items Y1 or Z1-Z20.

[0435] The invention will now be described in further details in the following non-limiting examples.

[0436] Examples P97013PC

[0437] Example 1: Preparation of nanofibrous cellulose scaffold

[0438] In this example is given a non-limiting demonstration of how the nanofibrous cellulose scaffold can be prepared. The core properties of the material, such as surface area and degree of substitution, was characterized.

[0439] Method

[0440] Preparation of initial cellulose nanofiber material

[0441] A solution of 19% cellulose acetate was prepared by addition of cellulose acetate (6.38 g) to 12 mL of acetone in an Erlenmeyer flask, followed by addition of 12 mL DMF and 6 mL 96% ethanol. The solution was stirred using a magnetic stirrer overnight at room temperature.

[0442] The 19% cellulose acetate solution was electrospun to prepare cellulose acetate nanofiber sheets. The cellulose nanofibers were electrospun (Fluidnatek LE50) using a drum speed of 200 rpm and a flow of 10 mL / hour at 18 kV+ (emitter) and 10 kV- (collector). The cellulose acetate nanofibers were collected on an aluminium substrate, with temperature being 23°C and at a relative humidity of 63%. After the electrospinning process completed, the cellulose acetate sheet comprising cellulose nanofibers was removed from the drum.

[0443] The cellulose acetate sheets were regenerated to cellulose by submergence in a 0.5 M NaOH in 95% ethanol solution. The cellulose acetate sheets were left in the solution for 24 hours at room temperature followed by filtering through a Buchner filter and washing with copious volumes of distilled water. Filtering and washing were repeated until the conductivity of the final washing step was 0 pS / cm to make sure there were no residual NaOH, or acetate left. The washed sheets were put in the oven at 80°C for 12 hours to provide dry cellulose sheets. The cellulose sheets were weighed.

[0444] Processing of initial cellulose nanofiber material

[0445] The dry cellulose sheets were cut into rough pieces of approximately 2x2 cm squares using scissors. The size of the pieces does not have to be exact, but larger pieces should be avoided as they may hamper the dividing step. The rough pieces of cellulose were fully submerged in water and dispersed using a high-speed disperser (IKA T25 digital Ultra Turrax) at 18,000 rpm for 60 min (with intermittent stops to cool the disperser). The processed cellulose nanofiber material was transferred to a sieve, washed with water to remove any remaining acetate ions, and drained to remove excess water. Cellulose nanofiber material was transferred to a flask and fresh water was added to provide a cellulose concentration of 2 wt%. P97013PC

[0446] Functionalization of processed cellulose nanofiber material

[0447] The processed cellulose nanofiber material was functionalized with quaternary ammonium (QA).

[0448] Briefly, 1 g of regenerated cellulose nanofiber material was resuspended in 50 mL of 1.5 M NaOH solution and mercerised for 2 hours under stirring at room temperature.

[0449] After the mercerization step, the temperature of the cellulose suspension was increased to 80°C and 2.5 mL of 3-chloro-2-hydroxypropyltrimethyl ammonium chloride (CHPTAC) was added dropwise. The reaction was continued at 80°C for 4 hours. After the reaction, the reaction mixture was cooling down to room temperature and filtered through a Buchner filter, immersed in 200 mL dHzO for 1 minute and filtered again. This process was repeated until the conductivity of the final washing step was 0 pS / cm to make sure there were no residual CHPTAC or NaOH left. Finally, the filtered processed and functionalized cellulose nanofiber material were resuspended in water to a 2% solution and freeze dried for further experimentation.

[0450] Drying

[0451] The functionalized cellulose nanofiber material was transferred to a -85°C freezer and left overnight. The frozen cellulose nanofiber material was then transferred to a lyophilizer and processed for 48 hours to yield a dry product. The dry product was grinded to provide a dry product of nanofibrous cellulose scaffold.

[0452] Microscopy

[0453] Samples of nanofibrous cellulose scaffold (diluted to 0.2 wt% cellulose) were added to a glass slide. Images of the network of cellulose nanofibers in the sample were captured on a light microscope (Leica) using 40x magnification.

[0454] Scanning electron microscopy (SEM)

[0455] Morphological studies of the nanofibrous cellulose scaffolds were carried out using scanning electron microscopy (SEM). The samples were dried and sputter coated with gold before carrying out the analysis. The micrographs were obtained in secondary electron (SE) imaging mode on a Hitachi SU3500 at an accelerating voltage of 5 kV and a working distance of 7 mm at varying magnifications.

[0456] Surface area measurements P97013PC

[0457] The Brunauer-Emmett-Teller (BET) model was employed to measure the specific surface areas using a molecule of nitrogen bearing molecular cross-sectional area of 0.162 nm2. Dried samples were degassed under vacuum for 6 hours before the BET surface area was measured. Nitrogen adsorption and desorption isotherms were acquired on an ASAP 2020 M analyzer (Micromeritics) at 77.3 K.

[0458] Elemental analysis and Degree of substitution

[0459] Elemental analysis of the nanofibrous cellulose scaffold was performed on an elemental analyzer FlashEA 1112 (Thermo Fischer Scientific) using 5 mg of sample. The samples were well dried before the analysis to remove any adsorbed moisture.

[0460] Degree of substitution (DS) of QA functionalized cellulose was calculated using the following formula:

[0461] DS = (162N / (1400-CAxN)) where 162 is the molecular weight of the anhydrous glucose unit (AGU); N is the percentage of nitrogen; CA is the molecular weight of the cationic reagent. For the present nanofibrous cellulose scaffold the cationic reagent is CHPTAC with a molecular weight of 188.1.

[0462] Results

[0463] Nanofibrous cellulose scaffolds with QA functional moieties were prepared and the dry product (Figure IB) was visualized by light microscopy (Figure 1A) and SEM (Figure 1C). The resulting nanofibrous cellulose material is highly homogenous in the sense that individual nanofibers are clearly visible without any large degree of entanglement or clusters. The homogenous distribution of nanofibers in the material ensures optimal exposure of the surface area for cell attachment and interaction.

[0464] Elemental composition and degree of substitution (DS) was assessed for the nanofibrous cellulose scaffold functionalized with QA. The content of carbon (C) was 37.71%, the content of hydrogen (H) was 6.59%, and the content of nitrogen (N) was 1.28%. Based on the content of N, the DS was calculated to 0.18. The amount of substituted QA may influence the cell growth as the positive charge facilitates electrostatic interactions with negatively charged cell membranes.

[0465] The surface area of the nanofibrous cellulose scaffold can be approximated in a theoretical calculation using the following equation:

[0466] Theoretical surface area per weight (cm2 / g) = 2 / (r x 6) P97013PC wherein m is the mass of nanofibrous cellulose material, r is the radius of the cellulose nanofibers, and 6 is the density of cellulose sheets.

[0467] The nanofibrous cellulose scaffold offers an increased surface area compared to the market standard microcarrier. The accuracy of the theoretical calculation was supported by a measurement of the BET surface area of a 0.5% cellulose sample, which gave a BET surface area of 58000 cm2 / g.

[0468] Conclusion

[0469] This example demonstrates that it is possible to produce a nanofibrous cellulose scaffold in a simple manner which is readily scalable for industrial usage. The nanofibrous cellulose scaffold has a large surface area which is available to the cells and does not comprise nanofibers collapsed in clusters or entangled in a fashion that exclude cells from gaining access to the surface.

[0470] Example 2: Passaging of cells cultured on support matrix

[0471] The purpose of this example was to evaluate if cells grown as 3D cultures using nanofibers could be passaged without enzymatic dissociation and with the addition of new support matrix. The criteria were that new spheroids formed had to contain nanofibers, and the cell growth and viability had to match that of spheroids before passage.

[0472] Method

[0473] Preparation of nanofiber microcarriers :

[0474] The support matrix comprising cellulose nanofibers (nanofibrous cellulose scaffold) was prepared according to Example 1 with the following adjustments:

[0475] - The cellulose acetate sheets were regenerated to cellulose in a 0.75 M NaOH in 95% ethanol solution for 3 hours.

[0476] - The cellulose nanofiber material was functionalized in a 0.75 M NaOH solution without the mercerization step for 1 hour at 45°C.

[0477] - The functionalized cellulose nanofiber material (100 mg) was freeze dried in autoclavable plastic vials.

[0478] Degree of functionalization

[0479] Ion exchange capacity

[0480] Samples were prepared by weighing up 0.05 g freeze dried cellulose samples in 50 ml tubes. For saturation of the exchange sites with chloride ions, the samples were incubated under agitation with 25 ml 0.1 M HCI for 15 minutes. After the incubation, the samples were filtered using a 0.2 pm Millipore filter unit and the supernatant discarded. To remove P97013PC unbound chloride ions, the support matrix was incubated with 25 ml dH2O 10 min. The support matrix was then filtered and the supernatant discarded. To displace the bound chloride ions with sulfate ions, the support matrix was incubated for 15 minutes with 40 ml of 1% (w / w) sodium sulfate solution. The support matrix was subsequently filtered using a 0.2pm Millipore filter unit and the filtrate was saved for silver nitrate titration.

[0481] Ion exchange capacity measurement

[0482] The ionic capacity for each sample was determined by AgNO3 titration using a Mettler Toledo T5 Titrator. 10 ml of each filtrate collected from the last sample preparation step above, was diluted in 30 mL distilled water. The samples were then titrated with 0.01 M AgNO3 to determine the amount of chloride ions that were bound to the functionalized cellulose fibers of the support matrix. From the equivalence point on each titration curve, the ion exchange capacity (mmol Cl- / g) was calculated using the LabX software.

[0483] Based on the ion exchange capacity measurement the produced support matrix comprising cellulose nanofibers had a charge between 0.2-0.4 mmol Cl / g.

[0484] Cell culturing

[0485] Overall, cells cultured on the support matrix comprising cellulose nanofibers were passaged (subcultured) without the requirement of a step for detachment of cells from the support matrix. A 1 mL sample was taken from the initial 3D culture and enzymatically dissociated for assessing cell count and viability. To establish the new culture in the subsequent culture vessel, for every mL of the new culture volume, 150.000 cells were transferred as spheroids from the previous culture to a subsequent culture vessel (e.g. flask or bioreactor) together with 0.075 mg of new support matrix.

[0486] First study - passaging from 3D culture to new 3D culture of similar volume

[0487] At day 0, cells from a 2D culture were inoculated in a 125 mL corning spinner flask. The inoculum was prepared from 22.5 mL culture media, 5.6 mg support matrix (nanofibrous cellulose scaffold) and 11.25 million HEK293T cells and incubated for 3 hours. After the 3 hours of incubation, additional culture medium was added to reach a final volume of 75 mL.

[0488] At day 4, cells from a 1 mL sample of the culture were counted and a volume of spheroid suspension corresponding to 11.25 million cells was transferred to a new 125 mL corning spinner flask and fresh culture medium was added to reach a working volume of 75 mL fresh culture medium comprising 5.6 mg of new support matrix (nanofibrous cellulose scaffold). This procedure corresponded to a single passage of cells (pl). P97013PC

[0489] The procedure was repeated for a total of four passages (pl-p4, 75 mL subcultures).

[0490] Cell culturing on bead-based microcarriers

[0491] 8 million HEK293T cells were inoculated with 120 mg Cytodex 1 in 20 mL culture media for 3 hours with intermittent stirring (20 rpm for 2 minutes and static conditions for 20 minutes) in a 125 mL Corning spinner flask. After 3 hours, the culture was topped up to reach 40 mL of final working volume corresponding to an initial cell density of 2xl05cells / mL and the stirring was set to 50 rpm. The cells culture was counted daily by sampling 1 mL of the culture. After 72 hours, the culture was passaged by transferring 8 mL of the culture, corresponding to 8 million cells, into a new 125 mL Corning spinner flask containing 32 mL of fresh culture media and 120 mg Cytodex 1 (3mg / mL) keeping the rotational speed at 50 rpm. The passaged culture was counted every 24 hours.

[0492] Second study - passaging of a 3D culture with upscaling to large volume

[0493] An inoculum was prepared in a 125 mL corning spinner flask as described for the first study.

[0494] At day 3, the spheroid suspension was passaged into either a new 125 mL corning spinner flask (pl, 75 mL subculture) as described for the first study or upscaled into a new 1 L corning spinner flask (pl, 600 mL subculture) by transferring 90 million cells and 45 mg new support matrix into 600 mL of cell culture medium.

[0495] In one experiment, the pl 75 mL subculture was further passaged at day 7 to a new 125 mL corning spinner flask (p2, 75 mL subculture).

[0496] Third study - passaging of a 3D culture from lab scale to industrial scale volume

[0497] In a scalability study, 3D passaging of spheroids from spinner flasks to 2.4 L and 10 L stirred-tank bioreactor systems was conducted.

[0498] At day 0, cells from a 2D culture were inoculated in a 1000 mL Corning spinner flask. The inoculum for the culture to passage into the 2.4 L bioreactor was prepared from 167 mL culture media, 37.5 mg support matrix (nanofibrous cellulose scaffold) and 75 million HEK293T cells and incubated for 3 hours. After the 3 hours of incubation, additional culture medium was added to reach a final volume of 500 mL.

[0499] The inoculum for the cell culture to passage into the 10 L bioreactor was prepared from 300 mL culture media, 75 mg support matrix (nanofibrous cellulose scaffold) and 150 P97013PC million HEK293T cells and incubated for 3 hours. After the 3 hours of incubation, additional culture medium was added to reach a final volume of 1000 mL.

[0500] At day 3, cells from a 1 mL sample of the 500 mL culture were counted and a volume of spheroid suspension corresponding to 360 million cells was transferred to a 2.4 L bioreactor comprising 2050 mL fresh culture medium alongside 180 mg new support matrix (nanofibrous cellulose scaffold) reaching a 2.4 L working volume.

[0501] Cells from a 1 mL sample of the 1000 mL culture were counted and a volume of spheroid suspension corresponding to 1500 million cells was transferred to a 10 L bioreactor comprising 9 L of fresh culture medium alongside 750 mg of new support matrix (nanofibrous cellulose scaffold) reaching 10L working volume.

[0502] Imaging of spheroids

[0503] Spheroid images were acquired using an inverted microscope at 24 hours intervals, with the same magnification for each image allowing for direct comparison of spheroid size and morphology in the different culture formats.

[0504] Results

[0505] 3D cultures were passaged multiple times to subsequent culture vessels of the same culture volume without enzymatic dissociation from the nanofibers and the subcultures proliferated at a rate equal to the former culture (Figure 2A). Passaging of cells to culture vessels of larger size was also achieved without the need to detach cells from the support matrix (Figure 2B-C).

[0506] In contrast, the growth of cells passaged together with bead-based microcarriers started to slow down after 24 hours compared with the initial culture (Figure 2D). The cell growth reached a peak at 48 hours after passage with 570000 cells / mL and after 72 hours the cell culture had 400000 cells / mL.

[0507] Images were taken every 24 hours after the passage (Figure 2E). The images verified that the cells continued to grow on already populated beads instead of colonizing / populating the new added beads. The beads with cells aggregated and formed clusters. The results concluded that the passaging protocol used with Cellevate nanofiber microcarriers did not work using bead-based microcarriers.

[0508] The real challenge of up-scaling in particular comes with larger volumes. Surprisingly, cells were efficiently transferred to bioreactors of industrial relevance without detachment of P97013PC cells from the support matrix (Figure 3A-B), and with subsequent proliferation of the cells in the bioreactors being on par with lab-scale shake flasks and spinner flasks.

[0509] Upon passage of a 2D culture (non-spheroids) to a 3D culture (spheroids), the size of the spheroids is retained over a culture period of 144 hours (Figure 4A). In contrast, upon passaging from 3D culture to a new 3D culture, the spheroids may grow in size (Figure 4B-C) indicating that after passage the cells keep growing on the preexisting spheroids in addition to the formation of new spheroids.

[0510] In this regard, the culture volume and the rotational speed also influence the size of the spheroids, with large culture volume and fast agitation promoting the formation of smaller spheroids (Figure 5).

[0511] Conclusion

[0512] This example demonstrates that cells (e.g. spheroids) cultured on a support matrix comprising cellulose nanofibers can be passaged without enzymatic dissociation. In particular, after 3D passage the cells keep growing on the preexisting spheroids in addition to the formation of new spheroids. The spheroid size after passing can be controlled by adjusting rotational speed in correlation culture volume. The advantage of passaging cells without detachment is of great importance due the simple workflow and practical aspects when up-scaling.

[0513] Example 3: Processing of the initial cellulose nanofiber material

[0514] In this example various methods of dividing the initial cellulose nanofiber material were tested and their influence on the nanofibrous cellulose scaffold was assessed.

[0515] Method

[0516] Preparation of the initial cellulose nanofiber material was performed as described in example 1, with the exception that different means of dividing the nanofibers were tested.

[0517] Processinq / dividinq protocols

[0518] Different methods of dividing the initial cellulose nanofiber material were evaluated. Thus, samples were prepared according to the following:

[0519] Mechanical cutting (by scissor):

[0520] 2.5 g Cellulose sheets were cut up in 10x10 mm pieces with a pair of scissors and assayed directly as fragmented cellulose sheets. P97013PC

[0521] Laser cutting:

[0522] Cellulose sheets were cut with a laser cutter (Epilog laser, Zing 24) in three different sizes (0.75x0.75mm, 1.5x1.5mm and 3x3mm). The cut sheets were assayed as is.

[0523] Blending:

[0524] 2.5 g cellulose sheets were cut up in 20x20 mm pieces with a pair of scissors and added together with 250 mL water to a lab blender (LB20, Waring Laboratory). The cellulose nanofiber material was processed at 7000 rpm for 1 (Bl), 5 (B5), 15 (B15) or 60 (B60) minutes.

[0525] Dispersing:

[0526] 2.5 g cellulose sheets were cut up in 20x20 mm pieces with a pair of scissors and added together with 250 mL water to an Erlenmeyer flask. The sample was processed using a disperser (IKA T25 digital Ultra-Turrax with S25 NB - 25 G disperser tool) at 18000 rpm for 1 (DI), 5 (D5), 15 (D15) or 60 (D60) minutes.

[0527] Fiber length measurements

[0528] Fiber length of the cellulose nanofibers were determined either by SEM (Hitachi SU3500) or by light scattering (Malvern Mastersizer S).

[0529] Samples of processed cellulose nanofiber material were diluted 1000-10000X in water and a droplet of sample is applied to a SEM fixture. The samples were dried and sputter coated with gold before capturing images on a Hitachi SU3500. Images were captured at different magnifications.

[0530] SEM images were evaluated manually by visual inspection to ensure that all measured nanofibers had both endings visible. The nanofiber lengths were determined by use of Image! software with several SEM images being analysed to get a larger dataset.

[0531] Fiber lengths were also determined using light scattering. Briefly, 1 mL of sample was added in water to the Malvern Mastersizer S and measurements were performed with the settings described under the definition of "mean length". Sample was added to the sample container until the obscuration value was between 15-20%. From each sample a fiber length histogram displaying the fiber length distribution was generated. The statistics of the distribution are calculated from the results using the derived diameters D[m,n] - an internationally agreed method of defining the mean and other moments of particle size. D(v, 0.5), D(v, 0.1) and D(v, 0.9) are standard "percentile" readings from the analysis. D(v, 0.5) is the fiber length at which 50% of the sample is smaller and 50% is larger than P97013PC this length. This value is also known as the Mass median diameter (MMD) when used for particles. D(v, 0.1) is the fiber length for which 10% of the sample is below this length. D(v, 0.9) gives a fiber length for which 90% of the sample is below this length. The volume weighted mean fiber length D[4,3] was also determined.

[0532] Pipettinq / floatinq test

[0533] Samples of processed cellulose nanofiber material were tested for their ability to be pipetted. 1 mL of sample was pipetted out of the sample container and subsequently expelled from the pipette tip into a tube with water. The ease of pipetting was evaluated, including the propensity for processed cellulose nanofiber material to clog the pipette.

[0534] Processed cellulose nanofiber material transferred to a tube containing water was then assessed for its propensity to float. Samples were vigorously shaken and it was observed by visual inspection whether the material floated immediately after and 24 hours after shaking.

[0535] Results

[0536] The pipetting test of scissor cut and laser cut cellulose sheets showed that these cut pieces are too large to pipette as they will clog the pipette tip. Also, cellulose nanofiber material processed in this manner generates pieces of dimensions that trap air bubbles and float in solution (Figure 6A). Furthermore, cutting with a laser makes the cellulose nanofibers melt, burn and stick together and is therefore not a suitable operation for dividing the cellulose nanofibers (Figure 6B-C).

[0537] None of the cellulose nanofiber materials processed by dispersing trapped air bubbles or had any tendency to float in solution. Pipetting of these samples was least challenging for samples that had been dispersed for longer durations of time. As such it was preferred to disperse for at least 10 min to improve flow through the pipette.

[0538] SEM measurements were used to determine cellulose nanofiber diameter (Figure 7A-B) and length (Figure 8A-H + Figure 9).

[0539] More than 2000 individual cellulose nanofibers were measured using Image! software and gave a mean cellulose nanofiber diameter of 500 nm (Figure 7C).

[0540] It is clear from the SEM images (Figure 8A-H) that the disperser quickly provides a homogenous nanofiber population with relatively few long nanofibers and without any significant entanglement. While large chunks of uncut nanofibers are present in the P97013PC samples after only 1 min of dividing (Figure 8A-B), the disperser presents a finer population of nanofibers with only few smaller chunks of entangled nanofibers already after 5 min of dispersing (Figure 8D). After 15 and 60 min of dispersing the presence of entangled nanofibers is almost completely eliminated (Figure 8F and 8H). In contrast, the blended samples comprise large chunks of entangled fibers even after 60 min of blending (Figure 8G).

[0541] The data are summarised for each of the samples in Figure 9A-B. It is clear that the frequency of long nanofibers is higher in the blended samples compared to the dispersed samples. Furthermore, there is a tendency towards longer processing times producing less long nanofibers.

[0542] This relative trend is supported by measurements of the nanofibers using light diffraction. These data are summarised in Table 1. Shorter fiber lengths were obtained for longer dispersing times.

[0543] Table 1. Fiber length measurements of cellulose nanofibers prepared by blending (Bl, B5, B15, B60) and dispersing (DI, D5, D15, D60). Measurements were performed on a Malvern Mastersizer S.

[0544] Overall, it is desired that the mean length of the cellulose nanofibers is reduced. It is also preferred that the population of nanofibers does not contain many long nanofibers as these may act as nucleus for larger clusters of nanofibers and entanglements. These clusters or entanglements of nanofibers are undesirable in the nanofibrous scaffold as large fractions of the surface area remains inaccessible to the cells seeded thereupon and increase the risk of clogging process equipment. Moreover, an inhomogeneous population of nanofibers comprising clusters and entanglements are undesirable because the variability between P97013PC batches of the nanofibrous scaffold becomes inconsistent and therefore unreliable for cell growth.

[0545] Conclusion

[0546] This example demonstrates that not every method for dividing the initial cellulose nanofiber material is equally effective and suitable for preparation of the nanofibrous cellulose scaffold. In particular, dispersing is advantageous because it rapidly removes longer fibers and yields a homogeneous population of cellulose nanofiber material without generation of entanglements or clusters.

[0547] Example 4: Processing nanofibers of material different from cellulose

[0548] In this example the processing of a variety of nanofibers different from cellulose were assessed with the aim of identifying other nanofibers suitable for preparation of nanofibrous scaffolds for growing cell cultures on. The electrospun nanofibers were characterized by scanning electron microscopy (SEM) and by visual inspection (following dividing of the nanofibers).

[0549] Method

[0550] Electrospinning

[0551] Nanofibers of polycaprolactone (PCL), poly-L-lactic acid (PLA), a mixture of PCL and PLA (PLA / PCL) and cellulose were electrospun. Electrospinning was performed on a Fluidnatek LE50 apparatus as described in Example 1.

[0552] PCL fibers were obtained by dissolving 8% Polycaprolactone pellets (Sigma Aldrich, MW 80000), in ChloroforrmMethanol, 1: 1 solution. Needle to collector distance was set to 20 cm, flow rate to 3mL / h, and voltage to 18kV.

[0553] PLA fibers were obtained by dissolving 8% PLLA pellets (Goodfellow, MFR: 24), in ChloroforrmMethanol, 3:2. Needle to collector distance was set to 24cm, flow rate to 4.5 mL / h and voltage to 35kV.

[0554] PLA / PCL fibers were obtained by dissolving PLA pellets (Goodfellow, MFR: 65) and PCL pellets (Sigma Aldrich, MW 80000) 1 :2 to an 8% polymer solution in Chloroform: Methanol, 3:2. Needle to collector distance was set to 20cm, flow rate to 3 mL / h and voltage to 18kV

[0555] 1 mL of polymer solution was spun for each sheet of fibers. P97013PC

[0556] Scanning electron microscopy

[0557] The electrospun material of PCL, PLA, mixed PCL / PLA and cellulose were imaged on a Hitachi SU3500 as described in Example 1. Images of the nanofiber materials were obtained for both undivided and divided nanofibers.

[0558] Processing of PCL, PLA and PCL / PLA nanofiber material

[0559] Electrospun sheets of PCL, PLA and mixed PLA / PCL were cut into smaller lOxlOmm pieces and mixed with two different mixing tools for 5 minutes. The cut sheets were mixed either with a blender at 7000 rpm (LB20E Laboratory blender, Waring) or with a disperser at 18000 rpm (IKA T25 digital Ultra Turrax).

[0560] Results

[0561] Nanofibers could be electrospun from all materials (PCL, PLA, PLA / PCL, and cellulose) and formed nanofibrous sheets (see Figure 10A-D).

[0562] The sheets made from nanofibers different from cellulose were subjected to two individual modes of dividing the nanofibers, namely blending (Figure 11A-F) and dispersing (Figure 12A-F). The tests showed that it was not possible to uniformLy blend or disperse either PCL, PLA, or PLA / PCL nanofibers into a homogenous mixture of short strand nanofibers.

[0563] Both the PCL nanofibers (Figure 11A-B and Figure 12A-B) and the PLA / PCL nanofibers (Figure 11E-F and Figure 12E-F) melted or deformed during the process. In particular, the nanofibers melted together to form either large pieces of solid polymer or large entangled clusters of nanofibers with semi-melted nanofibers.

[0564] The PLA nanofibers (Figure 11C-D and Figure 12C-D) did not melt in the same way as the PCL fibers, but all materials did easily get stuck in the mixing tool or got entangled on the blades of the blender and halted the processing.

[0565] Conclusion

[0566] This example demonstrates that it is not possible to uniformly blend or disperse nanofibers of all materials. Accordingly, not all nanofiber materials can be easily transformed into shorter strands followed by formation of a nanofibrous scaffold as described herein. Thus, it is preferred to use cellulose nanofibers for the preparation of the nanofibrous scaffolds.

[0567] Example 5: Evaluating cell growth under elevated shear stress P97013PC

[0568] This example evaluates the persistence of cells to shear stress when cultured on cellulose nanofibers. In particular, the rotational speed in the culture container was investigated and cell growth, cell viability and spheroid size was recorded.

[0569] Method

[0570] Preparation of nanofiber microcarriers

[0571] The support matrix comprising cellulose nanofibers (nanofibrous cellulose scaffold) was prepared according to Example 2.

[0572] Cell cultivation on support matrix at different rotational speeds in IL spinner flasks

[0573] 100 mg of the support matrix comprising cellulose nanofibers was hydrated in Ca2+and Mg2+free PBS, washed once and then resuspended in 20 mL of PBS to make a stock solution of 5 mg / mL. The stock solution was autoclaved at 121°C for 15 minutes (liquid program).

[0574] For the 50 rpm sample, 11.25 million HEK293T cells (150000 cells / mL) were inoculated with 5.625 mg support matrix (0.075 mg / mL) in 75 mL DMEM culture media in 125 mL spinner flasks.

[0575] For high rotational speed samples (120-200 rpm), 150 million HEK293T cells (150000 cells / mL) were inoculated with 75 mg support matrix (0.075 mg / mL) in IL DMEM culture media in IL spinner flasks.

[0576] The flasks were put on magnetic stirrers at different rotational speeds; 50, rpm, 120 rpm, 150 rpm, 180 rpm and 200 rpm. The viable cell density (VCD) was counted, and the diameter (pm) of approximately one hundred spheroids were measured to calculate the median spheroid diameter, at different time points up to 72h of culturing.

[0577] Cell cultivation with standard bead-based microcarriers at higher rotational speeds

[0578] 1000 mg of standard bead-based microcarriers (Cytodex 1) were hydrated in 50 mL Ca2+and Mg2+free PBS for 2h. The microcarriers were then washed with PBS, the supernatant was discarded and replaced with fresh PBS to make a stock solution of 20 mg / mL. The stock solution was autoclaved at 121°C for 15 minutes (liquid program).

[0579] 11.25 million HEK293T cells (150000 cells / mL) were inoculated with 225 mg standard bead-based microcarriers (3 g / L) in 75 mL DMEM culture media in 125 mL spinner flasks. To enable cell attachment to the bead-based microcarriers, the flasks were placed on a magnetic stirrer with intermittent agitation, consisting of continuous stirring at 50 rpm for P97013PC

[0580] 5 min followed by no agitation for 15 min, for a total duration of 2h. Thereafter, one spinner flask was placed on a magnetic stirrer and agitated at 120 rpm, while the second spinner flask was agitated at 180 rpm, both at 37°C with 5% CO2. The viable cell density (VCD) (cells / mL) was counted after 72h of culturing.

[0581] Passaging in IL spinner flask

[0582] For investigation of passaging of cells at elevated rotational speed, a sample cultured at 120 rpm for 96h, while recording VCD, cell viability, and spheroid diameter as described above.

[0583] After 96h of culturing, 150 million cells (150 000 cells / mL) in the form of spheroids from the previous 3D culture, were passaged into a new IL spinner flask containing fresh medium and 75 mg support matrix comprising cellulose nanofibers (0.075 mg / mL). The VCD was counted, and the spheroid diameter was measured at the time points 72h and 96h post passaging.

[0584] Results

[0585] The cell growth curves and viability (%) were similar between all four tested high rotational speeds (120 rpm, 150 rpm, 180 rpm, 200 rpm). The VCD (cells / mL) was measured at all time points up to 72h. At 72h, 1.4 x 106cells / mL was reached for 150 rpm, 180 rpm and 200 rpm, whereas 120 rpm had a slightly higher VCD of 1.58 xlO6cells / mL at the same time point (Figure 13A).

[0586] The median spheroid size was approximately 50 pm in diameter, with a coefficient of variation (CV) value between 24 to 26%, after 72h of culturing at the rotational speeds of 150 rpm, 180 rpm and 200 rpm. At 120 rpm, the spheroid size was slightly larger, 66 pm with a CV value of 28%, at the same time point compared to the higher speeds (Table 2).

[0587] Table 2. Cell culturing at varying rotational speeds. P97013PC

[0588] Cells cultured on cellulose nanofibers at a rotational speed of 50 rpm yielded a spheroid diameter of 242 pm and a CV value of 21% after 72h of culturing. Accordingly, these data confirm that the slower rotational speed results in larger spheroid size. The corresponding VCD was 1,3 x 106, i.e. close to the numbers achieved at higher rotational speeds.

[0589] Cells cultured on standard bead-based microcarriers at a rotational speed of 120 rpm or 180 rpm yielded VCD of 0.26 x 106and 0.18 x 106, respectively, after 72h of culturing. Both numbers were close to the seeding density (0.15 x 106), demonstrating that standard bead-based microcarriers are not suitable for culturing cells at high rotational speed.

[0590] Passaging (Pl) of spheroids at a rotational speed of 120 rpm was successful and resulted in corresponding VCDs, 2.2 x 106cells / mL, before and post passaging at the 96h time point (Figure 13B). The study also demonstrated that the cell viability was not affected by a higher rotational speed or volume (Figure 13B). Interestingly, the spheroids kept growing after passaging while retaining viability (Table 3), suggesting that cells remain attached to the support matrix upon passaging and continue growth in the new container.

[0591] Table 3. Cell growth at a rotational speed of 120 rpm, including after passaging (Pl).

[0592] Conclusion

[0593] This example demonstrates that cells can grow at high rotational speed when cultured utilizing a support matrix comprising cellulose nanofibers. The cells grow controllable as spheroids with consistent coefficient of variation (CV) and can be passaged effectively without need for detachment from the support matrix.

[0594] At increased rotational speed, the spheroids are kept smaller which ensures sufficient nutrient supply during industrial scale production. P97013PC

[0595] In contrast, it was not efficient to culture cells on standard bead-based microcarriers at high rotational speed.

[0596] References

[0597] - WO 2024 / 133700 A2

Claims

P97013PCClaims1. A method for culturing of cells, said method comprising the steps of:(i) providing a first container comprising a first cell culture and a support matrix;(ii) passaging at least part of said first cell culture to a second container to provide a subculture;(iii) adding an additional amount of support matrix to said second container; and(iv) culturing said subculture; wherein said support matrix comprises cellulose nanofibers, and wherein said passaging does not comprise a step of detaching cells from the support matrix.

2. The method according to claim 1, wherein said step of detaching cells from the support matrix is selected from enzymatic treatment, chemical treatment and mechanical methods.

3. The method according to any one of claims 1 or 2, wherein the second container is a subculturing container.

4. The method according to any one of the preceding claims, wherein the method is repeated for consecutive passages of the cells.

5. The method according to any one of the preceding claims, wherein the method is for large scale industrial production.

6. The method according to any one of the preceding claims, wherein the first container and / or second container are bioreactors.

7. The method according to any one of the preceding claims, wherein the volume of the second container(s) is at least 500 mL, such as at least 1 L, such as at least 2 L, such as at least 5 L, such as at least 10 L, such as at least 25 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, such as at least 10000 L.

8. The method according to any one of the preceding claims, wherein the additional amount of support matrix comprises about 0.05 mg to about 2 mg support matrix per l*106passaged cells, such as 0.1 mg to about 1 mg support matrix per l*106passaged cells, 0.2 mg to about 0.5 mg support matrix per l*106passaged cells.P97013PC9. The method according to any one of the preceding claims, wherein the cells grow as three-dimensional (3D) spheroids.

10. The method according to claim 9, wherein the diameter of the spheroids is in the range of about 25 pm to about 500 pm, such as about 30 pm to about 400 pm, such as about 40 pm to about 300 pm, such as about 50 pm to about 250 pm, such as about 75 pm to about 200 pm.

11. The method according to any one of claims 9 or 10, wherein the diameter of the spheroids is in the range of about 20 pm to about 100 pm, such as about 30 pm to about 90 pm, such as about 40 pm to about 80 pm, at the time of the first passaging.

12. The method according to any one of the preceding claims, wherein the container(s) are connected to or comprise an agitation system.

13. The method according to claim 12, wherein the rotational speed of the agitation system in the container(s) is in the range of about 100 rpm to about 400 rpm, such as about 110 rpm to about 300 rpm, such as about 120 rpm to about 200 rpm.

14. The method according to any one of the preceding claims, wherein the container(s) comprise an agitation element.

15. The method according to any one of the preceding claims, wherein said first cell culture comprises adherent cells.

16. The method according to any one of the preceding claims, wherein said first cell culture comprises mammalian cells.

17. The method according to claim 16, wherein the mammalian cells are selected from the group consisting of HEK293 cells, Vero cells, CHO cells, BHK cells, hybridoma cells, NS0 cells, PER.C6 cells, MDCK cells, stem cells, fibroblasts, smooth muscle cells, hepatocytes, endothelial cells, pancreatic cells, immune cells, epidermal cells, muscle cells, adipose cells, and neuron cells.

18. The method according to any one of claims 16 or 17, wherein the mammalian cells are selected from the group consisting of HEK293 cells, CHO cells, and BHK cells.

19. The method according to any one of the preceding claims, wherein the cellulose nanofibers have a mean length of less than about 250 pm.67P97013PC20. The method according to any one of the preceding claims, wherein the cellulose nanofibers have a mean length in the range of about 30 pm to about 250 pm, such as about 40 pm to about 200 pm, such as about 50 pm to about 175 pm, such as about 60 pm to about 150 pm, preferably about 70 pm to about 120 pm.

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

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

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

24. The method according to any one of the preceding claims, wherein the cellulose nanofiber material is functionalized with a functional moiety.

25. The method according to claim 24, wherein the functional moiety is selected from the group consisting of quaternary ammonium (QA), carboxymethyl (CM), diethylaminoethyl (DEAE), 4-(l-bromoethyl)benzoic acid, 4-(dimethylamino)pyridine, epoxide, and combinations thereof, preferably quaternary ammonium (QA).68

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