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    • 专利摘要:
    CELL SUPPLY OR CELL CULTURE COMPOSITION, CELL SUPPLY OR CELL CULTURE MATRIX, METHODS TO PRODUCE A COMPOSITION, TO REMOVE CELLULOSE NANOFIBERS AND NON-PHYSICAL USE, MICROOLOL USE, MICROOLOL USE, MICROOLOL USE refers to material that is useful in cell culture and transfer and also as a cell supplier. The material comprises plant-derived cellulose nanofibers or their derivatives, the cellulose nanofibers being in the form of a hydrogel or a membrane. The invention also provides methods for producing these materials and compositions and their uses.
    公开号:BR112013010377B1
    申请号:R112013010377-9
    申请日:2011-10-26
    公开日:2021-01-19
    发明作者:Marjo Yliperttula；Patrick Laurén；Madhushree Bhattacharya；Yanru Lou；Antti Laukkanen
    申请人:Upm-Kymmene Corporation；
    IPC主号:
    专利说明:

    FIELD OF THE INVENTION
    [001] The invention relates to plant-derived cell culture and cell supplier compositions comprising cellulose nanofibers (cellulose nanofibers, CNF) and / or their derivatives. BACKGROUND OF THE INVENTION
    [002] Health care remains at the most important frontiers for scientific research. The need to discover and develop cost-effective and safer medications is always growing. The ability to accurately model the organization of cells within an organ or tissue is of great importance. An exact copy of the system in vivo to in vitro would require cell growth in three dimensions (3D). The "cross-talk" achieved between cells in a 3D cell culture in vitro is an exact imitation of cell growth under physiological conditions. In fact, 3D cell culture has taken on significance in efforts aimed at regenerative medicine, a better understanding of chronic diseases and the provision of a superior in vitro model system for drug screening and toxicological testing. Its emergence is therefore being appropriately touted as the “new dimension of biology”.
    [003] Intense research efforts are appearing to identify and develop “factors and frameworks” that would favor the growth of cells in 3D in vitro. Cells under physiological conditions not only “cross-talk” with each other but also interact with the cellular microenvironment, the extra-cellular matrix (“ECM), with which they reside. ECM provides structural support to cells and also contributes to signaling and directing cell destiny. Predominantly, ECM is composed of glycosaminoglycans and fibrous proteins such as collagen, elastin, laminin and fibromectin self-assembled in a nanofibrillar network. An ideal framework for 3D cell growth should be able to imitate the structural component of native ECM, favor cell growth and cell maintenance, have the correctly sized network of interconnected pores for efficient cell migration and transfer of nutrients to cells. In essence, the mechanical and chemical properties of the frame must produce cellular function as in the native state.
    [004] Hydrogels, of both synthetic and natural origins, have emerged as frames suitable for 3D cell culture. The network of pores interconnected in hydrogels allows the retention of a large amount of biological fluid, facilitating the transport of oxygen, nutrients and waste. In addition, hydrogels can be formed mostly under mild cytocompatible conditions and biological properties can be modulated by surface chemistry. Engineered hydrogels with modified mechanical, chemical and physical properties have the potential to imitate ECM and therefore establish their usefulness in 3D cell culture. Commercial products for 3D cell culture are for example PuraMatrix ™ (3DM Inc.) and Matrigel (BD Biosciences). PuraMatrix ™ is a self-assembled peptide nanofiber hydrogel that looks like the natural fibrillar collagen structure in ECM with a fiber diameter of 5-10 nm. It also has a high water content, typically 99.5%. US 7,449,180 and WO 2004/007683 disclose peptide hydrogels. Matrigel is a mixture of gelatinous protein secreted by mouse tumor cells. The mixture looks like the complex extracellular environment found in many tissues and is used by cell biologists as a substrate for cell culture. MaxGel ™ ECM Matrix (Sigma-Aldrich), which includes a mixture of human ECM components, forms a gel at room temperature.
    [005] Bacterial cellulose ("bacterial cellulose", BC) has been used in wound healing membranes and as a framework in cell culture. The limitation in the use of bacterial cellulose is the inherent structure of the fermented material; under cultivation, BC is formed in very tight membranes at an air-water interface in the fermenter. The membranes formed are very watertight for many 3D cell culture tasks and several modifications are necessary to improve the porosity, which is necessary for the penetration of cells and the formation of cell clusters.
    [006] Hydrogel materials are also widely used in other types of culture tasks in which hydrophilic support material is required, for example hydrocolloids of the agar type are widely used in culturing plant cells, bacteria, and fungi for various microbiological purposes.
    [007] US 5,254,471 discloses a support for cell culture made of ultrafine fibers. WO 2009/126980 discloses cellulose-based hydrogel, which contains cellulose exhibiting an average degree of polymerization of 150-6,200.
    [008] It has been found that the solutions of the prior art are quite unsatisfactory in cell culture. All matrices and all cell and 2D cell culture methods require the use of animal-based chemical compounds or agents over biomaterials for the purpose of cells being maintained and multiplied. Maintenance of stem cells is especially demanding and there are no simple solutions for the matrix used with cell culture media that would keep stem cells alive. The presence of animal-based compounds in a cell culture environment poses a serious risk of immunoreactions, and different types of toxicity problems, which will irrevocably kill the cultured cells. Cell culture matrices containing animal-based additives are not suitable for use with stem cells, especially if the stem cells are to be used for tissue transplantation and tissue engineering (engineering). In addition, many of the polymers proposed for use in cell culture media do not tolerate a physiological temperature or are toxic to cells. BRIEF DESCRIPTION OF THE INVENTION
    [009] There is an evident need for improved cell culture material that is capable of providing two-dimensional or three-dimensional support for various types of cells. Those functional 3D cell models can be used as drug discovery tools replacing animal experiments and being more advanced than the 2D cell models used today. The transport of cultured cells is also highly desirable, for example when tissue transfers or cell therapy is the goal. Possibility to transfer cell clusters grown in a 3D matrix is desirable when different cell models in vitro are being developed. Biomaterials for existing 3D cell culture do not allow the transfer of the hydrogel matrix with a needle without seriously damaging the cultured cells.
    [0010] An objective of the present invention is therefore to provide a new approach to at least partially solve or alleviate the previously cited problems arising from the prior art. The objectives of the present invention are achieved by a cell supplier or cell culture composition comprising cellulose nanofibers or a derivative thereof which is characterized by what is stated in the independent claims. Preferred embodiments are disclosed in the independent claims.
    [0011] The present invention is based on the use of cellulose nanofibers and / or their derivatives in 2D and 3D cell culture matrix. The present invention provides the use of cellulose nanofibers and / or their derivatives in the cell culture matrix. The use of cellulose nanofibers and / or their derivatives as a cell culture matrix in 2D and 3D eliminates the need to use animal-based additives to multiply and proliferate cells on a matrix containing cellulose nanofibers and / or its derivatives.
    [0012] The present inventor has surprisingly discovered that plant-derived CNF hydrogel can be used without any modifications such as biomimetic human ECM for 3D cell culture. Cell proliferation and viability data suggest that the CNF hydrogel is an optimal biomaterial for 3D cell frames for advances in high-throughput screening functional tests based on drug development cells, drug toxicity testing and regenerative medicine and additionally for the provision of cells in vivo.
    [0013] The present inventors describe for the first time the physical and biocompatibility properties of plant-derived CNF hydrogel. Plant cellulose is used extensively in the textile and paper industries and is naturally abundant. The native cellulose nanofiber hydrogel is opaque. Chemical modification of the cellulose pulp before mechanical disintegration produces optically transparent hydrogels.
    [0014] The present invention is based on experimental studies on hydrogels composed of cellulose nanofibers (cellulose nanofibers, CNF), which are dispersed in an aqueous environment. Nanofibers are highly hydrophilic due to the hydroxyl functionalities of cellulose polymers and due to the fact that they are partially covered with hemicellulose polysaccharides.
    [0015] Consequently, the present invention provides as a first aspect a cell supplier or cell culture composition comprising cellulose nanofibers or a derivative thereof, the cellulose nanofiber being in a hydrogel or membrane form.
    [0016] A significant advantage of the present invention is that cells can be maintained (and proliferated) over biomaterials or within biomaterials without human or animal based chemical agents originating outside the cells. The cells are uniformly dispersed over or in the (o) matrix / medium containing the cellulose nanofibers or a derivative thereof. The cells divide on or in the media, start to proliferate and the clusters of cells start to grow spontaneously without the accumulation of cells on the bottom of the cell culture platform. The homogeneous division of cells in cellulose nanofibers or a derivative thereof is a prerequisite for the biomaterial to function as a cell culture medium in 3D.
    [0017] Other advantages of the present invention include: cellulose nanofibers and / or their derivatives are inert and do not give background fluorescence. Media comprising cellulose nanofibers or a derivative thereof can be injected. Injectability is explained by the rheological properties. The injection can be performed so that the cells remain stable within the matrix and are homogeneously dispersed in the matrix after the injection. BRIEF DESCRIPTION OF THE DRAWINGS
    [0018] Figure 1 depicts the cryo-TEM images of cellulose nanofiber hydrogels. Native CNF is on the left (A) and transparent CNF is on the right (B).
    [0019] Figure 2 depicts the viability of HepG2 cells in commercial cell culture materials [MaxGel ™ (Sigma-Aldrich), HydroMatrix ™ (Sigma-Aldrich) and PuraMatrix ™ (3DM Inc.)], in two nanofiber materials different cellulose (native CNF and transparent CNF) and CNF to which fibronectin (FN) was added. In an AB proliferation assay, cells were grown for 48 h and control cells were grown under equal conditions on a plastic surface.
    [0020] Figure 3 depicts the viability of ARPE-19 cells cultured in native CNF hydrogel after transferring the cells with a different sized syringe needle. Viability is shown as relative fluorescence intensity.
    [0021] Figure 4 depicts the diffusion of dextrans of different molecular weights (20 kDa, 70 kDa, and 250 kDa) through 1% native cellulose nanofiber hydrogel.
    [0022] Figure 5 depicts the light microscopy image of ARPE-19 cells on native CNF membrane. The CNF membrane supports the growth of cells on the top of the image, on the bottom of the image the cells grow on cell culture plastic. Magnification of 20x.
    [0023] Figure 6 depicts the images of the HepG2 cell confocal microscopy section on a cell culture plastic (A) and in the native cellulose nanofiber hydrogel (B).
    [0024] Figure 7 depicts the viscoelastic properties of 0.5% CNF hydrogel by dynamic oscillatory rheological measurements. The frequency dependencies of G '(the storage module) and G ”(the loss module) of a 0.5% native CNF hydrogel are shown.
    [0025] Figure 8 depicts the viscosity of 0.5% CNF hydrogels as a function of applied shear stress compared to 0.5% solution of water-soluble polyacrylamide polymers (5,000 kDa) and CMC (250 kDa) .
    [0026] Figure 9 depicts the viscosity of 0.5% CNF hydrogels as a function of the shear rate measured compared to 0.5% polyacrylamide and 0.5% CMC. Shear regions typical of different physical processes are marked in the Figure with arrows.
    [0027] Figure 10 depicts the schematic representation of a CNF hydrogel containing cells flowing inside a needle. High shear rate region (low viscosity) is located at the gel-needle interface and low shear region (very high viscosity) is located in the center of the needle.
    [0028] Figure 11 depicts the evolution of the shear rate and viscosity when 0.7% native CNF hydrogel was sheared in a concentric cylindrical geometry rheometer at a constant tension of 40 Pa.
    [0029] Figure 12 depicts the structure recovery of a 0.7% native CNF hydrogel dispersion after high shear rate shear compared to mild mixing with a glass rod.
    [0030] Figure 13 depicts the stability of two gravel suspensions in 0.5% native CNF hydrogel (upper row) and in transparent 0.5% CNF hydrogel (lower row) over a period of 17 days. The gravel was CEN Standard sand (EN 196-1) with an average particle size of 1-2 mm and 2-3 mm. The samples were stored at room temperature.
    [0031] Figure 14 depicts the influence of enzymatic hydrolysis on the suspension capacity of cellulose nanofiber gels. The gravel was CEN Standard sand (EN 196-1) with an average particle size of 1-2 mm.
    [0032] Figure 15 depicts the image by confocal microscopy of liver progenitor cells derived from human ES cells, which are immersed in native CNF hydrogel. Scale bar: 70 μm. DETAILED DESCRIPTION OF THE INVENTION
    [0033] The present invention relates to a cell supplier or cell culture composition comprising cellulose nanofibers and / or their derivatives, in which the cellulose nanofibres or their derivatives are in the form of a hydrogel or a membrane. Cellulose nanofibers or their derivatives can be obtained from non-animal based material such as raw material comprising plant material.
    [0034] Unless otherwise stated, the terms, which are used in the specification and in the claims, have the meanings commonly used in cell culture. Specifically, the following terms have the meanings indicated below.
    [0035] The term "cell supplier or cell culture composition" refers to a material comprising cellulose nanofibers or cellulose nanofiber derivatives and the material of which is used as a cell culture medium or for supplying cells. Said composition can also be used for the transfer of cells or groupings of cells. Cellulose nanofibers can be in the form of a hydrogel or a membrane. Said composition may additionally contain various additives such as special extracellular matrix components, serum, growth factors, and proteins.
    [0036] The term "cellulose raw material" refers to any source of cellulose raw material that can be used in the production of cellulose pulp, refined pulp, or cellulose nanofibers. The raw material can be based on any plant material that contains cellulose. Plant material can be wood. Wood can be from softwood tree such as spruce, pine, pine, larch, douglas or Canadian pine, or from hardwood tree such as birch, aspen, aspen, poplar, eucalyptus or acacia, or from a mixture of soft woods and hard woods. Non-wood material can be from agricultural residues, grasses or other plant substances such as straw, leaves, cork, seeds, husks, flowers, vegetables or cotton fruits, corn, wheat, oats, rye, barley, rice, flax , hemp, manila hemp, sisal fiber, jute, ramie, kenaf, bagasse, bamboo or reed.
    [0037] The term “cellulose pulp” refers to cellulose fibers, which are isolated from any cellulose raw material using chemical, mechanical, thermo-mechanical, or chemo-thermo-mechanical pulping processes. Typically the diameter of the fibers varies between 15 and 25 μm and the length exceeds 500 μm, but the present invention is not intended to be limited to these parameters.
    [0038] Cellulose in the present invention is structurally type I cellulose.
    [0039] The term "cellulose nanofiber" refers to a collection of isolated cellulose nanofibers (cellulose nanofibers, CNF) or bundle of nanofibers derived from cellulose raw material or cellulose pulp. Nanofibers have a typically high aspect ratio: the length can exceed one micrometer while the average numerical diameter is typically less than 200 nm. The diameter of the nanofiber bundles may be larger but is generally less than 1 μm. The smaller nanofibers are similar to the so-called elementary fibrils, which are typically 2-12 nm. The dimensions of the fibrils or bundles of fibrils are dependent on the raw material and the disintegration method. Cellulose nanofibers can also contain some hemicelluloses; the amount is dependent on the plant source.
    [0040] Mechanical disintegration of cellulose nanofibers from the raw material cellulose, cellulose pulp, or refined pulp is carried out with suitable equipment such as a refiner, a grinder, a homogenizer, a colloidal material coagulator, friction grinder, ultrasound sonifier, fluidizer such as microfluidizer, macrofluidizer or fluidizer-type homogenizer. Preferably "cellulose nanofibers" are mechanically disintegrated material.
    [0041] "Cellulose nanofibers" or "cellulose nanofibers and / or their derivatives" can also be any chemically or physically modified derivative of cellulose nanofibers or bundles of nanofibers. The chemical modification can be based on, for example, a carbomethylation reaction, oxidation, such as TEMPO oxidation, esterification, or etherification of cellulose molecules. Modification can also be carried out by physical adsorption of anionic, cationic, or non-ionic substances or any combination of these on the cellulose surface. The described modification can be carried out before, after, or during the production of cellulose nanofibers. Certain modifications can produce CNF materials that are degradable within the human body.
    [0042] Suitably the cellulose raw material such as cellulose pulp is pre-treated with acid or base before mechanical disintegration. Pre-treatment is carried out by subjecting the cellulose pulp to acid treatment, preferably with hydrochloric acid to remove any positively charged ions having a charge greater than +1, followed by treatment with an inorganic base containing positively charged ions having a charge + 1, preferably NaOH, where Na + ions replace the initial ions. This pre-treatment provides “cellulose nanofibers” with excellent gelling and transparency properties. This pretreated product is called “cellulose nanofibers” pretreated with acid-base or ion-exchanged.
    [0043] Microbial purity of "cellulose nanofibers" is essential for the performance of cell culture. Therefore, "cellulose nanofibers" are sterilized before cell culture experiments in the form of a hydrogel or a membrane. In addition, it is important to minimize the microbial contamination of the product before and during fibrillation. Before fibrillation, it is advantageous to aseptically collect the cellulose pulp from the pulp mill immediately after the bleaching stage when the pulp is still sterile.
    [0044] There are several synonyms widely used for cellulose nanofibers. For example: nanocellulose, nanofibrillated cellose (CNF), nanofibrillary cellulose, cellulose nanofiber, nanoscale fibrillated cellulose, microfibrillary cellulose, microfibrillated cellulose (CNF), or cellulose microfibrils. Cellulose nanofibers produced by certain microbes have several synonyms, such as bacterial cellulose, microbial cellulose (microbial cellulose, MC), biocellulose, coconut cream (NDC), or coconut cream.
    [0045] Cellulose nanofibers described in this invention are not the same material called cellulose whiskers, which are also known as: cellulose nanowhiskers, cellulose nanocrystals, cellulose nanocarriers, stick-like cellulose microcrystals, or cellulose nanowires. In some cases, similar terminology is used for both materials, for example by Kuthcarlapati et al. (Metals Materials and Processes 20 (3): 307-314, 2008) where the material studied was called “cellulose nanofiber” although they referred to cellulose nanowhiskers. Typically, these materials do not have amorphous segments along the fibrillar structure like cellulose nanofibers, which leads to a more rigid structure. Cellulose whiskers are also shorter than cellulose nanofibers; typically the length is less than a micrometer.
    [0046] The dimensions of individual cellulose nanofibers are quite close to the previously mentioned dimensions of collagen fibers in ECM, i.e. 4-10 nm. Therefore, CNF-based hydrogels can be used as a cell culture matrix in 3D.
    [0047] In the cell culture experiments of the present invention, two types of cellulose nanofibers were used: opaque native CNF and optically transparent CNF, which was TEMPO oxidized cellulose. Detailed description of the materials is presented in the Examples, Materials and Methods section.
    [0048] The term "cellulose nanofiber hydrogel" refers to the aqueous dispersion of cellulose nanofibers.
    [0049] The term "cellulose nanofiber membrane" refers to a formation of cellulose fibers similar to a dry or wet sheet. The membranes are typically produced by filtering the dispersion of diluted cellulose nanofibers with a vacuum filtration apparatus with an appropriate filter. The solvent leak can also be used to obtain the aforementioned membrane structures. The membrane obtained can be used as such in a wet state or be dried before use.
    [0050] Cellulose nanofibers or a derivative of the present invention may comprise chemically or physically modified derivatives of cellulose nanofibers or bundles of nanofibers.
    The drug [sic] or cell culture delivery composition of the present invention may further comprise suitable additives selected from the group consisting of special extracellular matrix components, serum, growth factors, and proteins.
    [0052] The present invention also relates to a cell supply or cell culture matrix, in which the matrix comprises living cells and the cell supply or cell culture composition forms a hydrogel and in which the cells are present in the matrix in a two-dimensional or three-dimensional arrangement.
    [0053] The cells can be any cells. Any eukaryotic cell, such as animal cells, plant cells and fungal cells are within the scope of the present invention and also prokaryotic cells such as bacterial cells.
    [0054] Depending on the cell line, the experiments are carried out in 2D or 3D, i.e. the cells are grown on the CNF membranes or gels or the cells are homogeneously dispersed in the CNF hydrogels or in the CNF membranes. Specific examples of the present invention disclose that spontaneously apparent retinal pigment epithelial cells (“spontaneously arising retina pigment epithelium”, ARPE-19) form monolayer, whereas human hepatocellular carcinoma (HepG2) cells produce both monolayer and cell colonies .
    [0055] The cells can be detected using any known detection medium or dye known in the art.
    [0056] The present invention also relates to a method for producing a composition according to any one of the preceding claims, comprising the steps of obtaining cellulose nanofibers and / or their derivatives; optionally mixing together said cellulose nanofibers and / or their derivatives with water; and transferring or positioning the cellulose nanofibers and / or their derivatives or the mixture obtained to the (in) environment suitable for supplying or culturing cells.
    [0057] Cellulose nanofiber membranes or hydrogels or their derivatives or the composition of the present invention can be used as a cell supply material.
    [0058] Cellulose nanofiber membranes or hydrogels or their derivatives or the cell or cell culture supply composition may be used to supply material for clinical use.
    [0059] The present invention relates to the microbiological use of cellulose nanofibers or a derivative thereof or the composition according to the present invention for laboratory and / or industrial purposes as a means or a compound of a means for keeping cells inactive. vitro.
    [0060] The composition comprising cellulose nanofibers or their derivatives can be used to immobilize cells or enzymes.
    [0061] The present invention also relates to a method of culturing cells, in which the method comprises the steps of obtaining cells; contacting the cells with a cell culture composition comprising cellulose nanofibers or a derivative thereof to form a matrix; and cultivating the cells within said matrix in a two-dimensional or three-dimensional arrangement.
    [0062] The present invention additionally relates to a composition, method, or use, in which the cells are eukaryotic cells.
    [0063] The present invention additionally relates to a composition, a method or a use, in which the cells are prokaryotic cells. Prokaryotic cells comprise microorganisms such as aerobic or anaerobic bacteria, viruses, or fungi such as yeasts and molds.
    [0064] The present invention additionally provides a composition, method or use, in which the cells are stem cells.
    [0065] The removal of cellulose nanofibers can be carried out, for example, with enzymes using enzymatic degradation of cellulose molecules. Suitable enzymes are, for example, commercially available cellulases. Cultured cell lines can also be genetically engineered to produce the necessary enzyme protein into the culture system.
    [0066] The present invention also relates to a method for removing cellulose nanofibers or a derivative thereof from cell culture or cell growth material, the method comprising the steps of obtaining material comprising cell growth medium and cells and optionally a drug; diluting said material with aqueous or non-aqueous liquid; and remove the cellulose nanofibers by decantation. Moderate centrifugation can be used to pellet cells and cell aggregates before decanting.
    [0067] The present inventors surprisingly found that the plant-derived CNF hydrogel can be used even without any modification as a biomimetic human ECM for 3D cell culture. Cell viability and proliferation data suggest that the CNF hydrogel is an optimal biomaterial for 3D cell frames for high throughput screening functional assays based on advanced cells in drug development, drug toxicity testing and in regenerative medicine and additionally in cell supply in vivo.
    [0068] The present invention discloses for the first time the physical and biocompatibility properties of plant-derived CNF hydrogel. Plant cellulose is used extensively in the textile and paper industries and is naturally abundant. The hydrogel of native cellulose nanofibers is opaque. Chemical modification of cellulose pulp before mechanical disintegration produces optically transparent hydrogels.
    [0069] Cellulose nanofibers of the present invention can be used in the form of hydrogel or wet or dry membrane. The gel resistance of the CNF hydrogel can be easily changed by dilution. Cellulose nanofibers or a derivative thereof having similar properties are non-toxic to cells.
    [0070] If cellulose nanofiber hydrogels are compared with UV crosslinkable cell culture hydrogels, such as PEG or hyaluronic acid hydrogels, CNF materials are considered much less toxic. In UV-crosslinkable gels, harmful photoinitiators are required to initiate gelation while CNF hydrogels are formed spontaneously. The non-covalent nature of CNF hydrogels also allows for the adjustment of porosity by dilution.
    [0071] The cells are evenly spread across cellulose nanofiber hydrogels and can automatically initiate duplication and growth in 3D cell clusters without sedimentation to the bottom of the cell culture platform. All commercial 3D cell culture media currently used require the addition of adhesion peptide causing the cells to form a 3D structure on the cell culture platform.
    [0072] Cellulose nanofibers according to the present invention or a derivative thereof can be used without adhesion peptide. The cells attach themselves to the platform and spontaneously distribute homogeneously into the cellulose nanofiber hydrogel. The cells are suspended homogeneously in the continuous phase due to the mechanical support provided by the cellulose nanofiber fibers. The extraordinarily high flow limit stabilizes cells and cell clusters grown against sedimentation.
    [0073] Cellulose nanofiber hydrogels originating from plants work without adhesion peptide and / or adjusted porosity, whereas bacterial cellulose nanofibers require adhesion peptide. Bacterial cellulose has been used directly after fermentation, in which case the resulting membrane structure is considerably firmer than that of the hydrogel of the present invention i.e. a cellulose nanofiber hydrogel. Therefore, prior art methods have required additional processes to make the hydrogel matrix more porous.
    [0074] The firmness of cell culture media containing cellulose nanofibers in gel form can be adjusted without influencing the properties of cell culture. Cellulose nanofibers from bacteria are also thicker than cellulose nanofibers from other sources and are therefore not freely modifiable for cell culture.
    [0075] The cells grow inside the matrix in 3D or on the matrix. Said material can be injectable or sheet-like membrane with appropriate surface topology.
    [0076] The properties of CNF are close to ideal for cell culture in 3D: transparent, non-toxic, highly viscous, high suspending power, high water retention, good mechanical adhesion, non-animal based, it seems if with the dimensions of ECM, insensitive to salts, temperature or pH, non-degradable, without auto-fluorescence. CNF has negligible background fluorescence due to the chemical structure of the material. In addition, the CNF gel is non-toxic to cells.
    [0077] The cells can be grown or grown on CNF gels for a long time, for example 2 to 7 days or even longer. The cells can also be cultured or just suspended in the hydrogel for a short time, for example minutes to several hours. The cells use the cellulose nanofiber matrix as a support / growth frame used as a platform. The cells form clusters thus indicating the usefulness of cellulose nanofibers as a framework for culturing cells in 3D. The cells grow as cell layers or aggregates on or within the CNF gel, depending on the method of deposition and the type of cell.
    [0078] The non-toxic CNF hydrogel is likewise good ECM for the cells studied as the MaxGel ™ based on human ECM. The viability of the cells is even higher than the viability in PuraMatrix ™ or HydroMatrix ™. Human and animal based ECM components are not added to the CNF hydrogels. However, the addition of fibronectin or collagen IV can be beneficial in some cases. Based on the diffusion studies, the CNF hydrogel is highly permeable and freely facilitates the exchange of oxygen, nutrients and water-soluble metabolites of the cells.
    [0079] Transmission electron microscopy shows that the CNF hydrogel is composed of a mixture of individual cellulose nanofibrils and fiber bundles. The dimensions of CNF are similar to those of native human collagen, which is a natural component of ECM and commonly used as a cell support. The CNF hydrogel resistance (elasticity) remains almost constant as a function of the frequency used from 0.01 to 1 Hz. Rheology data reveal shear viscosity of about several hundred kilogramsPascals at rest (low shear stress) to lower to a few Pascals within a Pascal's shear stress. This behavior is somewhat unusual for biomaterial hydrogels. It allows extremely good support and suspension capacity of cells and the shear thinning behavior allows for the easy dispensing and injection of cells in CNF hydrogel regardless of the size of the needles used, whose behaviors are not obtained before for other biomaterial hydrogels for cell culture. The mechanical properties of elasticity and firmness are optimal for CNF hydrogels for growth in 3D cell culture and cell injection.
    [0080] The advantage of the present invention is that the dimensions of the fibrillar network of cellulose nanofibers or a derivative thereof are very close to the dimensions of the natural ECM network of collagen nanofibers. In addition, cellulose nanofibers or a derivative thereof are of non-animal based material, i.e. there is no risk of disease transfer. Currently, commercial products are mostly isolated from animals. In addition, the invention provides possibilities to adjust the physical form because CNF materials from hydrogels to membranes can be used.
    [0081] Injectable hydrogel forms a support matrix around the cells due to the very high shear stress. CNF membranes are transparent and highly porous. Mass production is easy compared to the alternatives.
    [0082] Native cellulose nanofibers are non-toxic to cells. The proliferation of cells is almost double in the case of cellulose nanofibers or a derivative thereof compared to the control (cells only). Cells can be controlled on CNF hydrogels for a long time (for example for 2-7 days). The cells use the cellulose nanofiber matrix as a growth platform. The cells form clusters, which indicates the usefulness of cellulose nanofibers or a derivative thereof as a framework for culturing cells in 3D. The cells grow as layers within the CNF gel. Cellulose nanofibers or a derivative thereof have negligible background fluorescence. The cellulose nanofiber hydrogel has excellent elasticity, firmness, shear stress, mechanical adhesion and porosity to be used as a cell culture matrix in 2D and 3D.
    [0083] In an aqueous environment, cellulose nanofibers form a continuous hydrogel network of dispersed nanofibers or bundles of dispersed nanofibers. The gel is formed by highly hydrated fibrils that are entangled in each other, even at very low concentrations. Fibrils can also interact via hydrogen bonds. The macroscopic structure is easily destroyed with mechanical agitation, i.e. the gel begins to flow at high shear stress. Cellulose nanofiber hydrogels and / or their derivatives used as material for cell culture have not been previously described.
    [0084] Applications of the present invention include obtaining material for cell culture for research in biotechnology. Cell culture or cell growth media containing CNF can be used for the maintenance and growth of cells and also for cell transfer. The present invention obtains cell culture media which can be used for example in tissue engineering or wound healing. Other applications include, for example, drug dosing applications, biotechnological or biological drugs and their dosage and also functional drug cell testing assays in 3D. The unique rheological properties of the CNF hydrogel allow for various applications that are based on the injectability of the hydrogel, such as injection of cells or drugs in CNF hydrogel in intraocular, intramuscular, or subcutaneous treatments.
    [0085] The following examples are provided to further illustrate the invention and are not intended to limit its scope. Based on the description, a person skilled in the art will be able to modify the invention in many ways. EXAMPLES Materials and methods Preparation of CNF hydrogels
    [0086] The opaque native CNF hydrogel (1.7% by weight) was obtained by high pressure homogenization of wet cellulose pulp fibers. Thus, the direct product of the process is a diluted cellulose nanofiber hydrogel. The transparent CNF hydrogel (0.9% by weight) was obtained by a similar homogenization process of a chemically treated cellulose pulp (TEMPO oxidized cellulose pulp). The samples were sterilized in an autoclave. For cell studies, the CNF hydrogel was diluted to the appropriate concentration and homogenized with sonification or mechanical mixing. Cryo-TEM images of native CNF and transparent CNF are shown in Figure 1. Hydrogel of native cellulose nanofibers is composed of a mixture of individual cellulose nanofibrils and bundles of fibers (Figure 1A). The diameter of smaller fibers is approximately 7 nm, however most of the cellulose material forms beam structures of 50-100 nm. The exact length scale cannot be estimated from the images due to the tangled and bundled nature of the material, but it seems evident that the individual nanofibers are several micrometers in length. The cryo-TEM image of the optically transparent CNF hydrogel shows a homogeneously distributed network of individual cellulose nanofibers. The diameter of these nanofibers is approximately 7 nm and the length exceeds one micrometer. Nanofibers have segments of length 100-200 nm followed by sharp folds along the fiber axis. The straight segments are composed of highly crystalline cellulose domains - the fold sites are formed by the amorphous parts. Preparation of CNF membranes
    [0087] CNF membranes were prepared by vacuum filtration of a native 0.2% by weight aqueous dispersion of CFN. After filtration, the wet membranes were dried under weight in an oven at 55 ° C for 48 h. The dry films were smooth and opaque with a weight of 70-80 g / m2. Enzymatic hydrolysis
    [0088] Enzymatic degradation of CNF hydrogels has been demonstrated by hydrolysis of 0.5% hydrogels containing gravel with Celluclast 1.5 LFG, CCN0367 (Novozymes, optimum pH of 5), Prot. 90 mg / ml. Degradation of native CNF was conducted at pH 5 at 50 ° C for 4 days and the degradation of clear CNF at pH 7 at 21 ° C for one hour. The enzyme dosage was 5 mg of enzyme per gram of CNF. HepG2 cells Origin of HepG2 cells
    [0089] Human hepatocellular carcinoma (HepG2) cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). HepG2 cell maintenance culture
    [0090] HepG2 cells were maintained in Dulbecco's modified Eagle's medium (Dulbecco's modified Eagle's medium, DMEM, Gibco) supplemented with 10% fetal bovine serum, penicillin / streptomycin (Gibco), 2mM L-glutamine (Gibco), Pyruvate of 100 mM sodium (Gibco). The cells were kept in 75 cm2 culture flasks at 37 ° C in an incubator with 95% relative humidity at room temperature (RT) in an atmosphere containing 5% CO2. The cells were raised to 1:10 concentration by trypsinization twice a week with a 1: 4 division rate. The medium was changed every 48 h and the cells were subcultured to the confluence of 90%. 3D culture of HepG2 cells over CNF hydrogel
    [0091] Cellulose nanofiber hydrogel was positioned at the bottom of a 96 well tissue culture plate and the HepG2 cell suspension in growth medium containing 25,000-50,000 cells per well either was seeded over the top of the CNF hydrogel or was mixed with him. The hydrogel concentration of CNF ranges from 0.01% to 1%. ARPE-19 cells Origin of ARPE-19 cells
    [0092] Spontaneously apparent retinal pigmented epithelial cells (ARPE-19) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). Maintenance culture of ARPE-19 cells
    [0093] ARPE-19 cells were grown in Dulbecco's modified Eagle's medium (DMEM): F12 Nutrient Mix, 1: 1 mix supplemented with 10% fetal bovine serum (fetal bovine serum, FBS), 2mM L-glutamine, penicillin 100 U / mL and streptomycin 100 U / mL. The cells were grown at 37 ° C in an atmosphere containing 7% CO2. The growth medium was changed every 2-3 days and the cultures were used in 24-30 passages. Culture of ARPE-19 cells on CNF membrane
    [0094] Cellulose nanofiber hydrogel was positioned at the bottom of a 96 well tissue culture plate and the suspension of ARPE-19 cells in growth medium containing 25,000-50,000 cells per well either was seeded over the top of the hydrogel or was mixed with him. The hydrogel concentration varies from 0.01%, 1%. Liver progenitor cells derived from human ES cells Maintenance culture of human embryonic stem cells
    [0095] The H9 human embryonic stem cell line (HES) (Wisconsin International Stem Cell Bank, the “WISC Bank” with the WiCell research Institute, Madison, WI, USA) was used for the attendees studies. H9 cells were routinely cultured on tissue culture plates coated with Matrigel mTeSR1 medium and passed through the use of Dispase 1 mg / mL (StemCell Technologies). In this condition, stem cells formed two-dimensional (2D) monolayer colonies. 3D culture of hES cells in CNF
    [0096] H9 cell colonies were mixed with either 0.3% native CNF or 0.3% clear CNF and cultured in mTeSR1 medium. CNES hES cells form 3D cell clusters. In some experiments, CNF 0.3% was mixed with 58 μg / mL of human fibronectin (Sigma-Aldrich). 3D culture of liver progenitor cells derived from H9 cells in CNF
    [0097] H9 cells were differentiated into liver progenitor cells for 11 days following the published protocol [Hay DC, Zhao D, Fletcher J, Hewitt ZA, McLean D, Urruticoechea-Uriguen A, Black JR, Elcombe C, Ross JA, Wolf R , Cui W. “Efficient differentiation of hepatocytes from human embryonic stem cells exhibiting markers recapitulating liver development in vivo.” Stem Cells. 2008 Apr; 26 (4): 894-902)]. The derived liver progenitor cells were cultured in a 3D environment using either native CNF 0.3% or transparent CNF 0.3% for two. In some experiments, CNF 0.3% was mixed with 13 μg / mL of mouse type IV collagen (Sigma-Aldrich). Staining of living cells / dead cells (‘Live / Dead’)
    [0098] Clusters of H9 cells and liver progenitor cells in CNF were co-stained with CellTracker Blue CMAC (20 μM) for live cells and with propidium iodide (25 μg / mL) for dead cells. Cell images were acquired by confocal laser scanning microscopy (Leica TCS SP5 MP SMD FLIM) at an excitation wavelength of 405 nm for CellTracker Blue CMAC and 514 nm for propidium iodide. AlamarBlue assay for cell proliferation / viability
    [0099] Cell viability has been quantified by the AlamarBlue ™ Cell Viability Assay Kit (Biotium Inc., Hayward, CA, USA). Cellulose nanofiber hydrogel was positioned on the bottom of a 96 well tissue culture plate and HepG2 / ARPE-19 cell suspension in growth medium containing 25,000-50,000 cells per well whether it was sown on top of the hydrogel or mixed with him. The hydrogel concentration ranges from 1 to 0.01%. Cell viability and proliferation were measured as a function of days after cell culture on cellulose nanofiber hydrogel in an incubator at 37 ° C in 5% CO2 and 95% relative humidity.
    [00100] After 48 hours, AlamarBlue was added directly to the culture medium in 96-well plates at a final concentration of 10%. The plates were incubated for 5 h and were exposed to an excitation wavelength of 530 nm, and an emission wavelength of 590 nm to measure fluorescence. The percentage viability was expressed as fluorescence counts in the presence of CNF hydrogel as a percentage of that in the absence of cellulose nanofiber hydrogel (cells growing on a plastic surface).
    [00101] Background fluorescence measurements (negative control) were determined from wells containing hydrogel and dye reagent in culture medium but without cells. The mean and standard deviation for all fluorescence measurements were calculated and subsequently corrected for background fluorescence and expressed as relative fluorescence. Confocal laser microscopy
    [00102] The viability of HepG2 cells cultured over hydrogel and the formation of HepG2 spheroids in 3D were evaluated with the Live / Dead® Viability / Cytotoxicity Assay Kit (Invitrogen) consisting of AM calcein and ethidium homodimer.
    [00103] Briefly, HepG2 cells were suspended in 1% transparent and native CNF hydrogel with or without fibronecin. The cell suspension in hydrogel was transferred to each cell well. The cell culture medium was added to each well. The hydrogel-encapsulated HepG2 cells were cultured for 5 days and the medium was renewed every 48 h. After 5 days, the medium was removed from the wells and the encapsulated cells were washed with PBS and incubated in 'Live / Dead' solution containing 0.2 μM AM calcein and 1.0 μM ethidium homodimer for about 45 min at room temperature . Living cells had their image obtained using a confocal laser scanning microscope (CLSM, Leica SP2 inverted microscope, Zurich, Switzerland) equipped with an argon laser (488 nm / 35mW), HC PL APO 10x / 0.4 CS and HC PL APO 20x / 0.7 CS (air), incubator box with an air heater system (Life Imaging / Services, Switzerland), and CO2 mixer (Okolab). The images were acquired from two detectors (one for Calcein and the other for Ethidium Homodimer). The images were created and edited with Imaris 7.0 (Bitplane). No deconvolution was carried out. Example 1 Comparison of cell viability of HepG2 cells in different cell culture materials
    [00104] Cellulose nanofiber hydrogels were positioned at the bottom of a 96 well tissue culture plate and HepG2 cell suspension in maintenance growth medium containing 25,00050,000 cells per well was either seeded on top of the hydrogel or mixed with him. The hydrogel concentration ranges from 1-0.01%. The fluorescence intensity that indicates cell viability and proliferation was measured as a function of days after culturing the cells on the cellulose nanofiber hydrogel in an incubator at 37 ° C in 5% CO2 and 95% relative humidity.
    [00105] Three commercially available cell culture materials were used as reference 3D culture materials: MaxGel ™ (Sigma-Aldrich), HydroMatrix ™ (Sigma-AIdrich) and PuraMatrix ™ (3DM Inc.). The experimental configuration was identical in all materials studied.
    [00106] The viability of HepG2 cells was quantified by the AlamarBlue ™ Cell Viability Assay Kit (Biotium Inc., Hayward, CA, USA) as presented above in Materials and methods, Alamar Blue assay for cell viability / proliferation.
    [00107] The percentage viability of HepG2 cells for the studied materials is shown in Figure 2.
    [00108] Both types of cellulose nanofiber hydrogels, i.e. native and transparent CNF show higher viability values than those of commercial reference materials Hydromatrix ™ or PuraMatrix ™. If fibronectin is added to CNF hydrogels, viability is close to viability in commercial MaxGel ™. In addition, cell proliferation and viability increase linearly as a function of cell concentration in both hydrogels. This observation confirms the hypothesis that the hydrogel mimics the human ECM components. It has all the main composition of ECM. Example 2 Transfer of ARPE-19 cells with a syringe needle
    [00109] ARPE-19 cells (25,000 cells per well) were seeded and cultured in a CNF matrix on the 96 well plate bottom. The viability of ARPE-19 cells after transferring the cells with a syringe needle of different sizes is shown in Figure 3. The same phenomenon can also be obtained with other cell types such as HepG2 and ES cells.
    [00110] More detailed explanation of the transfer of ARPE-19 cells with a syringe needle is given below: In Transfer 1 in Figure 3 the cells were incubated for 48 hours with 1.66% CNF, and after that the cells were transferred with a syringe (20G-27G) about 100 μL into a 96-well plate. After transferring with the syringe, the cells were incubated for 24 hours, and the viability of the cells in CNF was measured.
    [00111] In Transfer 2, the cells incubated for 24 hours in 1.66% CNF were transferred with a 27G syringe (about 2 mL) into a new medium. The cells were incubated for 24 hours after the transfer, after which the cells were transferred again with a syringe (20G-27G) about 100 μL into the 96-well plate, incubated again for 24 hours and the viability of cells doubly transferred in CNF 1.66%.
    [00112] These experiments prove that it is possible to transfer the cells in CNF hydrogel, the transfer process was successful and the cells were alive and remained alive during the transfer with a syringe. This phenomenon was obtained even with the smallest needle size of 27G, and there was no interruption regarding the size of the needle used in the transfer process. Samples that were transferred twice (Transfer 2) showed lower proliferation rates most likely due to the shorter 24-hour incubation time at the beginning of the experiment. The transfer of cells in the CNF hydrogel proves that the cells were in fact inside the hydrogel and remained there because the cells that were attached to the plate were not transferred (without trypsinization). These experiments showed that the cells remained viable during the transfer. Example 3 Stem Cells Live / dead staining of hepatic progenitor cells derived from hES cells
    [00113] Liver progenitor cells derived from human ES cells were immersed in CNF hydrogel (Figure 15) and cultured for 7 days with and without IV collagen. Background fluorescence was not detected. Liver progenitor cells derived from ES cells were immersed in a transparent CNF hydrogel and cultured for 7 days with and without IV collagen. No background fluorescence was detected, which makes this material extremely easy to use in this context. Usually other materials used e.g. MatriGel and MaxGel have significant background fluorescence, and therefore are difficult to work with those matrices. It is possible to maintain ES cells in CNF hydrogel, they survive and so this material is able to keep them alive. In addition, ES cells also form a 3D structure, which has not been seen before with any other material. Example 4 Diffusion of dextrans through CNF hydrogels
    [00114] Detailed knowledge about the diffusion properties of a cell culture material is important. The cell culture material must be sufficiently porous to allow diffusion of nutrients and oxygen to cultured cells and also to allow efficient diffusion of cell metabolites. The diffusion properties of CNF hydrogel were demonstrated with dextrans of different molecular weights in the following manner: 400 μL of transparent or opaque CNF (1%) was deposited by filter on the apical compartment in Transwell ™ filter cavity plates (size of 0.4 µm filter pore). 1 mL of PBS was added on the basolateral side and 100 μL (25 μg) of fluorescently labeled dextrans were added on top of the hydrogels (20k, 70k and 250k PM). Plate was firmly fixed and left undisturbed on a well plate shaker. Samples of 100 μL were taken from the basolateral side and an equal amount of PBS was replaced. First samples were taken at 15 minute intervals, other samples were taken at different time points ranging from 30 minutes to 2 hours and the final samples at 24 hours. A total of 168 samples were taken. Target plate (OptiPlate ™ -96 F) was measured at excitation and emission wavelengths of 490 nm and 520 nm respectively.
    [00115] Diffusion of dextrans of different molecular weights through 1% native cellulose nanofiber gel is shown in Figure 4. The diffusion of the model compounds occurs at constant speed and is highly dependent on the molecular weight (size) of the compound. It is evident that in CNF hydrogels the molecules are able to diffuse efficiently although the gel structure is sufficiently firm to stabilize the cell suspension.
    [00116] The observed diffusion profile can also be used in various applications and drug release formulations. The diffusion of drugs can be controlled as a function of the size of the drug or protein molecule (used as a drug) or as a concentration of the CNF hydrogel. The evident extended release profile is especially beneficial for certain treatments where longer release is preferred. Example 5 Proliferation of ARPE19 cells over CNF membrane
    [00117] Native CNF membrane was positioned on the bottom of a 96 well cell culture plate and the cell suspension in the maintenance growth medium containing 25,000-50,000 cells per well was seeded over the top of the membrane. The concentration of the membrane varies from 1.6 to 0.01%. Cell viability and proliferation were measured as a function of days after culturing cells on the native CNF membrane in an incubator at 37 ° C in 5% CO2 and 95% relative humidity.
    [00118] ARPE-19 cells on native CNF membrane had their image acquired by light microscopy. The CNF membrane supports cell growth as shown at the top of the image (Figure 5) showing that ARPE-19 cells can be grown in 2D on the CNF membrane and that the CNF membrane is useful as a growth matrix for 2D cells.
    [00119] ARPE-19 cells proliferated well in hydrogels regardless of the concentration of cells used. There is no significant difference between hydrogels. Cell proliferation increased ~ 2-fold when grown on hydrogel compared to cells grown in the absence of hydrogel. Example 6 Morphology of clusters of HepG2 cells cultured in 3D Confocal laser microscopy
    [00120] Confocal laser microscopy was used to acquire the image of living cells. The spheroidal shape of the HepG2 cells encapsulated in the CNF hydrogel clearly suggests that the cells are trapped within the hydrogel and are growing in three dimensions (Figure 6). Images obtained from the cells after 5 days of culture are shown in Figure 6 showing that the cells are viable within 3D spheroids in both hydrogels. The viability of the cells was independent of the concentration of the cells in hydrogels and the size of the spheroids increased as a function of time in all cultures (Figure 6).
    [00121] The medium was renewed after every 48 h and the spheroid size increases as a function of time in the culture. When fibronectin was added to the CNF hydrogel, the viability of the cells within the 3D spheroid was increased. Live / dead staining confocal microscopy images revealed that the cells remained viable during the 5-day culture period. These findings are related to the results of the Alamar Blue cell proliferation assay (Figure 2). This observation confirms our hypothesis that the CNF hydrogel mimics the components of the human ECM. It has all the main ECM compositions except fibronectin. Therefore, the addition of fibronectin improves the viability of cells in 3D clusters. Fibronectin facilitated the fixation and viability of HepG2 cells. It has previously been shown that fibronectin increases hepatocyte survival and decreases apoptosis via β1 integrin binding.
    [00122] By this means it is possible to show the 3D structure of the HepG2 cells obtained without any support material or other ECM components than just the CNF hydrogel. This proves the usefulness and ease of use of the CNF hydrogel as a 3D cell culture matrix. Example 7 Resistance of the gel
    [00123] An important function of a cell culture medium in 3D is to keep cells homogeneously suspended in the matrix and prevent sedimentation. CNF meets this requirement because of its ability to form a gel network in very low concentration (0.5%). The CNF gel-like structure was shown by determining its viscoelastic properties by dynamic oscillatory rheological measurements. The results of the frequency scans show typical gel-like behavior. The storage module (G ') is several orders of magnitude higher than the loss module (G') and almost independent of frequency, which means that the elastic properties (similar to that of solid) are more pronounced than those viscous characteristics (similar to those of liquid) (Figure 7). Typical for gels is also that both G ’and G’ are relatively frequency independent. The viscoelastic properties of CNF gels were determined with an oscillatory frequency sweep measurement on a rheometer (AR-G2, TA Instruments) at a 0.1% strain. Example 8 Flow properties of the CNF hydrogel
    [00124] The rheological flow properties of CNF hydrogels show several characteristics that are beneficial in use in cell culture. Hydrogels have a high viscosity at low shear (or resting) for optimal cell suspension capacity but also show shear thinning behavior at higher shear rates to allow for dispensing and injection. The ability of CNF to provide these types of rheological properties was demonstrated in a series of tests in which the viscosity of CNF dispersions was measured over a wide range of shear stress (shear rate) on a rotary rheometer (AR-G2, TA Instruments, UK).
    [00125] CNF dispersions show very high zero-shear viscosities (the region of constant viscosity at small shear stresses) different from water-soluble polymers, as shown in Figure 8. The zero-shear viscosity of CNF is greatly increased by the diameter of minor nanofibril induced by the preceding chemical pretreatment of the starting material. The stress at which the shear thinning behavior starts ("yield strength") is also considerably high for CNF dispersions. The suspension capacity of a material is better the higher the yield limit. The cells are effectively stabilized against sedimentation by the combined effects of high viscosity at zero shear and high yield limit and high storage modulus. The gravitational force applied by the cells is much weaker than the yield limit. Thus, the suspended cells are "frozen" within the gel matrix if mixed with CNF or "frozen" on the gel if deposited on top of the gel.
    [00126] In Figure 9 the viscosity is presented as a function of the measured shear rate. From this Figure 9 it is obvious that the viscosity of the CNF dispersions falls at relatively small shear rates and reaches a level similar to that measured for the reference materials at shear rates of about 200 s-1.
    [00127] The CNF network structure breaks under shear (Figure 7). Under the application of a certain tension, the viscosity of the system drops dramatically and a transition from solid-like to liquid-like behavior occurs. This type of behavior is beneficial because it allows the cells to mix homogeneously within the CNF suspension by moderate mechanical shear. When two-phase liquids, such as flocculated CNF dispersions, are sheared (eg, in a rheometer or in a tube), the dispersed phase tends to move away from the solid boundaries, which leads to the creation of a viscosity layer lower level of liquid on the walls of the container (Figure 10). This phenomenon means that resistance to flow, i.e. viscosity is lower at the solid limits than in the dispersion region away from the solid limits (Barnes, 1995). Respectively, the injection of the CNF hydrogel with a syringe and needle or with a pipette is easy even at high concentrations (1-4%). The phenomenon also allows for the easy dispensing of cell suspensions with minimal disturbance of the cells, i.e. most of the cells are located in the center of the needle and are practically at rest (Figure 10).
    [00128] Easy injectability of CNF hydrogels is an important feature when injectable formulations are considered. As described in Example 6, CNF hydrogels have release profiles that could be used in controlled and prolonged drug delivery applications. These two discoveries for CNF hydrogels allow for several potential drug treatment applications, such as intraocular, intramuscular, subcutaneous treatments or for example viscoelastic ophthalmic formulations. Example 9 Structure recovery after shear has ceased
    [00129] An additional important rheological property of CNF hydrogels is that the high viscosity level is maintained after shearing (e.g. injection or mixing) has ceased. The structure recovery of a CNF dispersion was demonstrated by a series of tests in which the material was first sheared in a rheometer (StressTech, Reologica Instruments Ab) at a high shear rate and after the shear interruption the recovery of the gel strength (G ') was monitored with an oscillatory time scan measurement. The shear cycle was carried out in a concentric cylindrical geometry at a constant stress of 40 Pa for 61 s. The evolution of the shear rate and viscosity during this test is shown in Figure 10. The material was sheared at a relatively high shear rate (1000 s-1) for a period of time of at least 40 s, during which the viscosity of the material has dropped below 40 mPa s.
    [00130] After the end of the shear, the evolution of G '(a measurement of the resistance of the gel) was allowed by an oscillatory measurement at constant frequency (1 Hz) and small voltage (0.5 Pa). The measurement was started exactly 10 s after the end of the shear. From Figure 11 it is obvious that a gel network is formed very quickly when the CNF dispersion is allowed to rest after it has been sheared at high shear rates. Substantial structure recovery is observed already 10 s after the shear cessation (equal to the zero time in Figure 11). A constant storage module level (G ') is achieved after keeping the CNF dispersion at rest for less than 10 min. The level of G 'that the extensively sheared CNF dispersion developed was comparable to that of a CNF dispersion that was only gently mixed with a glass rod prior to the structure recovery test.
    [00131] Evolution of the shear rate and viscosity when a dispersion of native CNF 0.7% was sheared in a concentric cylindrical geometry rheometer at a constant tension of 40 Pa is shown in Figure 11.
    [00132] The structure recovery of a 0.7% native CNF dispersion after shear at high shear stress compared to after mild mixing with a glass rod is shown in Figure 12.
    [00133] Rapid structure recovery is important for hydrogel-type cell culture materials for two reasons. First, it allows cells to be homogeneously distributed in the CNF hydrogels after mixing them with the hydrogel. Second, if CNF hydrogels are used to transport cultured cells, rapid recovery of the gel structure effectively traps the cells in the desired location and migration is minimal, for example when cell transplantation is considered. Rapid recovery is also essential in injectable drug release formulations. Example 10 Stability
    [00134] As shown in Example 1, even very diluted dispersions of CNF have a very high viscosity at low shear rates. The structure of the hydrogel is also recovered when shearing, such as injection, ceases. Under static conditions, CNF forms a hydrogel network with a high elastic modulus and exceptionally high flow limit. Due to these properties, CNF has a very high suspending power of solid particles even at very low concentration.
    [00135] The ability to suspend under static conditions is demonstrated with gravel suspensions. Dispersions 0.5% of native CNF and transparent CNF are able to stabilize even 2-3 mm gravel particles for very long periods of time, see Figure 13. It should be noted that the transparent CNF is able to stabilize the suspensions of particles in lower concentration than native CNF. Example 11 Enzymatic hydrolysis
    [00136] It is commonly known that certain enzymes, cellulases, are capable of hydrolyzing β- (1 ^ 4) bonds in cellulose. For example endo-1,4-p-glycanases (EGs) that select cellulose chains at random locations away from the chain ends; exoglycanases or exocelobiohydrolases (CBHs) that degrade cellulose by breaking molecules from both ends of the chain producing cellobiose dimers; and β-glycosidases (BGLs) that hydrolyze the cellobiose units (produced using attack by EG and CBH) to glucose. Respectively, cellulose nanofibers can be enzymatically hydrolyzed to glucose with the aid of cellulases (Ahola, S., Turon, X., Osterberg, M., Laine, J., Rojas, OJ, Langmuir, 2008, 24, 11592-11599 ).
    [00137] Enzymatic cellulose hydrolysis can be used in cell culture systems containing cellulose nanofibers due to several reasons. Under hydrolysis of the CNF hydrogel, the viscosity of the medium is drastically lowered and the cultured cell structures are easily accessible e.g. for staining. Also, after hydrolysis, cell structures can be transferred or transplanted without cellulose-containing material. The degradation product, glucose, is generally non-toxic to cells and can be used as a nutrient in cell culture.
    [00138] The enzymatic hydrolysis of cellulose nanofibers can be carried out with the aid of different cellulases in a different environment. In Figure 14, the effect of commercial Celluclast enzymes on the suspending power of gels is demonstrated. Both hydrogels of native CNF and clear CNF lose their suspending power due to enzymatic degradation of the gel structure. Cultured cell lines can also be genetically engineered to produce the necessary enzyme protein into the culture system.
    权利要求:
    Claims (19)
    [0001]
    1. Cell supplier or cell culture composition, characterized by the fact that the composition comprises plant-derived, cellulose nanofibers mechanically disintegrated and / or their derivatives, in the form of a hydrogel or a membrane in a wet state.
    [0002]
    2. Composition according to claim 1, characterized by the fact that the diameter of the cellulose nanofibers or the bundles of nanofibers in the cellulose nanofibers and / or their derivatives is less than 1 μm, preferably less than 200 nm, more preferably less than 100 nm.
    [0003]
    Composition according to either of Claims 1 or 2, characterized in that the cellulose nanofibers and / or their derivatives comprise chemically or physically modified derivatives of a cellulose nanofiber or bundles of nanofibers.
    [0004]
    Composition according to any one of claims 1 to 3, characterized in that the mechanically disintegrated cellulose nanofibers are pretreated with acid-base.
    [0005]
    Composition according to any one of claims 1 to 4, characterized in that the composition additionally comprises additives selected from the group consisting of components of extracellular matrix, serum, growth factors and proteins.
    [0006]
    6. Composition according to any one of claims 1 to 5, characterized by the fact that the cells are at least one among eukaryotic cells, prokaryotic cells and stem cells.
    [0007]
    7. Cell supply or cell culture matrix, characterized by the fact that the matrix comprises living cells and the composition as defined in any one of claims 1 to 6, forming a hydrogel and the cells are present in the matrix in a two-dimensional arrangement or three-dimensional.
    [0008]
    8. Matrix according to claim 7, characterized by the fact that the cells are eukaryotic cells or prokaryotic cells or stem cells.
    [0009]
    9. Method for producing a composition as defined in any one of claims 1 to 6, characterized by the fact that it comprises the steps of: - obtaining plant-derived cellulose nanofibers, mechanically disintegrated and / or their derivatives; and, - mix the cellulose nanofibers and / or their derivatives together with water.
    [0010]
    Method according to claim 9, characterized in that it further comprises combining the mixture with a suitable medication.
    [0011]
    Method according to either of claims 9 or 10, characterized in that the mechanically disintegrated cellulose nanofibers are pretreated with acid-base.
    [0012]
    12. Method for removing plant-based cellulose nanofibers and / or their derivatives from a cell culture material, characterized by the fact that it comprises the steps of: - obtaining a material comprising cell and cell culture medium; - dilute the material with aqueous or non-aqueous liquid; - optionally centrifuge the material to sediment the cells and cell aggregates; and, - remove the cellulose nanofibers by decantation.
    [0013]
    13. Method for removing cellulose nanofibers based on plants and / or their derivatives from a cell culture material, characterized by the fact that it comprises the steps of: - obtaining a material comprising cell and cell culture medium; - contacting the cell culture material with a degrading enzyme; - optionally centrifuge the material to sediment the cells and cell aggregates; and, - remove the cellulose nanofibers by decantation.
    [0014]
    14. Method for culturing cells, characterized by the fact that it comprises the steps of: - obtaining cells; - contacting the cells with the composition as defined in any one of claims 1 to 5 to form a matrix; and, - cultivating the cells within the matrix in a two-dimensional or three-dimensional arrangement.
    [0015]
    Method according to claim 14, characterized in that before the culture the matrix is transferred to or positioned in an environment for the culture of cells or for the supply of cells.
    [0016]
    16. Method according to any one of claims 9 to 15, characterized by the fact that the cells are at least one among eukaryotic cells, prokaryotic cells and stem cells.
    [0017]
    17. Microbiological use of plant-derived, mechanically disintegrated cellulose nanofibers and / or their derivatives or composition as defined in any of claims 1 to 6, characterized by the fact that it is for laboratory or industrial purposes as a medium or a compound of a means to maintain IN VITRO cells.
    [0018]
    18. Use of the composition as defined in any one of claims 1 to 6, characterized in that it is for immobilizing cells or enzymes.
    [0019]
    19. Use according to either of claims 17 or 18, characterized by the fact that the cells are at least one among eukaryotic cells, prokaryotic cells and stem cells.
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    法律状态:
    2018-04-03| B06F| Objections, documents and/or translations needed after an examination request according art. 34 industrial property law|
    2019-06-04| B06T| Formal requirements before examination|
    2020-12-08| B09A| Decision: intention to grant|
    2021-01-19| B16A| Patent or certificate of addition of invention granted|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 26/10/2011, OBSERVADAS AS CONDICOES LEGAIS. |
    优先权:
    申请号 | 申请日 | 专利标题
    FI20106121A|FI123988B|2010-10-27|2010-10-27|Cell Culture Materials|
    FI20106121|2010-10-27|
    PCT/FI2011/050939|WO2012056109A2|2010-10-27|2011-10-26|Plant derived cell culture material|
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