EP2137298A1 - Improved three-dimensional biocompatible skeleton structure containing nanoparticles - Google Patents
Improved three-dimensional biocompatible skeleton structure containing nanoparticlesInfo
- Publication number
- EP2137298A1 EP2137298A1 EP08748985A EP08748985A EP2137298A1 EP 2137298 A1 EP2137298 A1 EP 2137298A1 EP 08748985 A EP08748985 A EP 08748985A EP 08748985 A EP08748985 A EP 08748985A EP 2137298 A1 EP2137298 A1 EP 2137298A1
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- EP
- European Patent Office
- Prior art keywords
- cell culture
- culture system
- nanoparticles
- cells
- cell
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/0068—General culture methods using substrates
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- A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
- A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
- A61F2/02—Prostheses implantable into the body
- A61F2/04—Hollow or tubular parts of organs, e.g. bladders, tracheae, bronchi or bile ducts
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- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
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- A61K47/6931—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer
- A61K47/6935—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer the polymer being obtained otherwise than by reactions involving carbon to carbon unsaturated bonds, e.g. polyesters, polyamides or polyglycerol
- A61K47/6937—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer the polymer being obtained otherwise than by reactions involving carbon to carbon unsaturated bonds, e.g. polyesters, polyamides or polyglycerol the polymer being PLGA, PLA or polyglycolic acid
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82B—NANOSTRUCTURES FORMED BY MANIPULATION OF INDIVIDUAL ATOMS, MOLECULES, OR LIMITED COLLECTIONS OF ATOMS OR MOLECULES AS DISCRETE UNITS; MANUFACTURE OR TREATMENT THEREOF
- B82B3/00—Manufacture or treatment of nanostructures by manipulation of individual atoms or molecules, or limited collections of atoms or molecules as discrete units
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
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- C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
- C12M25/16—Particles; Beads; Granular material; Encapsulation
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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- A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
- A61F2/02—Prostheses implantable into the body
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- C12N2533/90—Substrates of biological origin, e.g. extracellular matrix, decellularised tissue
Definitions
- the present invention relates to a cell culture system that includes a three-dimensional biocompatible scaffold structure and nanoparticles. Furthermore, the present invention also encompasses methods for producing such a cell culture system as well as methods for cultivating cells by means of this cell culture system.
- ECM extracellular matrix
- cytokines growth factors
- neighboring cells The extracellular matrix is present in all four basic tissue types, namely epithelial, muscle, nerve, connective and supporting tissues.
- ECM serves above all an interactive signal exchange through transmembrane receptors of the cells. This form of communication also affects the regulation of gene expression. This plays an important role in essential cell properties, such as adhesion, proliferation, differentiation, migration, apoptosis and remodeling processes.
- the ECM is not a static system, but ECM components are in a "steady state.” ECM components are secreted and synthesized by cells in the intercellular space (the space between cells), but can also be broken down by the cells at the same time
- the extracellular matrix consists of three major components: collagen fibers, anchor proteins, and bulky carbohydrates, which is a protein layer that can be considered a specialized extracellular matrix, providing a stabilizing layer and delimiting surface epithelia from the connective tissue. this prevents the cells of these layers from sliding apart.
- GAGs glycosaminoglycans
- ECM carbohydrate components include hyaluronic acid, heparan sulfate, dermatan sulfate, chondroitin sulfate and keratan sulfate.
- Adhesion proteins Another characteristic component of the extracellular matrix are adhesion proteins.
- Adhesion proteins, adapter proteins or other adhesive proteins can interact on the one hand with other constituents of the matrix, on the other hand, however, also with other cells by attaching to specific cell receptors.
- adhesion proteins are the protein family of laminins, vitronetin and fibronectin.
- the basement membrane as a specialized extracellular matrix contains several essential components.
- the first to be mentioned here are type I to IV collagens, with type IV being a special one Meaning.
- Laminins are another component of the basement membrane. They have a sword-like shape. Their ends are occupied by cell receptors that bind mainly to integrins.
- Laminin 1 is the most important adhesive component, furthermore laminin 5 as a crucial component of the basement membrane is of particular importance.
- entactin (nidogen) as a further component of the basement membrane, as it binds the collagen layers to the laminin layers.
- proteoglycans such as Perlecan link the individual components of the basement membrane, since Perlecan has in its structure binding sites for collagen, laminin, nidogen, and itself.
- Integrins are heterodimeric proteins that can be composed of different alpha and beta subunits. Depending on the composition of the alpha or beta subunits of the integrins, these can bind to the extracellular matrix, for example to laminin, vitronectin or fibronectin.
- nanoparticles and their use for identification and screening methods are known. According to these disclosures, the nanoparticles may also recognize or be inert to certain biologically active substances.
- emulsion polymerization is a special method of polymerization in which water-insoluble monomers are emulsified in water with the aid of emulsifiers and polymerized using water-soluble initiators, for example potassium persulfate.
- water-soluble initiators for example potassium persulfate.
- the polymer dispersions obtained in the emulsion polymerization for example latex dispersions, can be used for a large number of applications.
- the US Pat. Nos. 4,521,317 and 4,021,364 show the possibilities and limits of emulsion polymerization.
- emulsion polymerization two types can basically be used: the oil-in-water emulsion and the water-in-oil emulsion, also called inverse emulsion.
- a monomer which is insoluble in the reaction medium is dispersed with stirring by means of emulsifier addition, and the reaction is started by addition of an initiator.
- the present invention is based on the technical problem of providing a cell culture system which, on the one hand, comes very close to the native extracellular matrix as a natural environment of the cells and, on the other hand, enables the controlled supply of growth factors and cytokines in order to specifically control cell growth and differentiation as well to customize tissue-specific. Furthermore, the present invention is based on the technical problem of providing cells, in particular also tissues and organs, which are suitable for transplantations. In addition, there is also a need for special tissues in the context of so-called tissue engineering as well as for research purposes.
- the present invention solves the underlying technical problem by providing a cell culture system comprising a three-dimensional biocompatible scaffold structure and biocompatible nanoparticles, as well as a method of culturing cells, providing cells or cell products and tissues, and cells or cell products and tissues in itself by means of such a cell culture system.
- the present invention accordingly provides a cell culture system which makes it possible to cultivate cells, in particular eukaryotic cells, in a particularly preferred embodiment, animal or human cells under conditions corresponding to the in vivo situation or a desired artificially oriented environment.
- the invention is based on the provision of a cell culture system which has biocompatible nanoparticles present together with a three-dimensional biocompatible scaffold structure.
- a cell culture system which has biocompatible nanoparticles present together with a three-dimensional biocompatible scaffold structure.
- Such a system makes it possible to expose the cells cultured in it to influences and conditions which are deliberately set by the presence of the nanoparticles and, depending on the purpose of the cultivation, the cells in their biological behavior, e.g. Influence growth or differentiation behavior.
- the nanoparticles can comprise active substances, for example by having them enclosed on the surface or in the nanoparticle itself.
- the active ingredients are present both on the surface and in the interior of the nanoparticles.
- the active substances associated in this way with the nanoparticles are controlled and / or regulated in the course of the cell culture system carried out cell culture system, in particular released with a time delay, and can be dosed and fed over a longer period of time controlled the cells. Coupling the nanoparticles with, for example, growth factors thus ensures targeted delivery of these growth factors over a defined period of time.
- nanoparticles for example of polymeric material, as carriers of, for example, growth factors have surprising advantages.
- nanoparticles in particular polymeric nanoparticles, control the controlled release of active substances, for example cytokines, as a carrier, ie with a certain, generally delayed release kinetics. This effect is also known as controlled release.
- active substances for example cytokines
- the release of the active ingredients is predeterminably regulated as a function of time, thus providing a desired and specific drug release kinetics.
- a desired active ingredient release kinetics is selected by selecting the nanoparticle material such that release of the active ingredient takes place cell-specifically.
- a constant release rate of the active ingredient based on the total release duration can take place.
- it can also be provided to provide, for example, in a first phase, an initially initially low drug release rate and an increased release rate starting after a certain time.
- the invention also provides, in an initial first phase, for a comparatively high release rate of the active ingredient, which is replaced after a certain time point by a lower release rate.
- the nanoparticles may have such an active ingredient loading that the active ingredient concentration is in a preferred range of 1 ng / ml to 10 ⁇ g / ml after release.
- the release of active ingredient can take place both by diffusion of the substance from or from the nanoparticles and by degradation of the nanoparticle itself, for example by hydrolysis of the polymer.
- cell culture system is to be understood as a system for cultivating cells, in particular in the form of the framework structure contained therein, adhesion sites for cells to be cultivated, preferably in a three-dimensionally structured structure, for example as a matrix or hydrogel, Typically, such a cell culture system, when in use, preferably in vitro use, is positioned in artificial containers capable of receiving, via the cell culture system itself, also the cells to be cultured and a culture medium.
- the cell culture system of the present invention is therefore particularly a system for culturing cells in vitro.
- the cell culture system according to the invention can be used for cultivating cells in conventional cell culture vessels such as petri dishes or cell culture bottles or in any other form.
- the cell culture system is used to culture primary cells.
- the cell culture system according to the invention is used in vivo, e.g. in animal experiments.
- framework structure means a structure for attaching cells
- a framework structure in the context of the present invention is in particular a preferred embodiment of a structure that not only allows a superficial adherence or adhesion of cells, but also in particular also ingrowth or integration of cells into the framework structure itself.
- a matrix preferably with pores or interstices.
- this matrix contains one or more components of the extracellular matrix, such as components of the basement membrane, adhesion proteins, fiber-forming proteins, carbohydrates, proteoglycans or cell receptors.
- a "primary cell” is a eukaryotic cell of human or animal origin derived from organs or from an embryo, and more preferably an embryonic stem cell is provided as the primary cell, preferably one derived from umbilical cord blood
- an embryonic stem cell is provided as the primary cell, preferably one derived from umbilical cord blood
- the stem cell employed in accordance with the present invention may be a totipotent, omnipotent or pluripotent cell
- the primary cell is an adult stem cell, eg an animal or human adult stem cell.
- primary cell thus means a eukaryotic cell of human or animal origin
- Primary cells can be obtained from organs such as skin, kidney or liver, or from whole embryos
- An example of a primary cell used according to the invention The primary cell is preferably omnipotent or pluripotent
- a primary cell can also be an adult stem cell
- the cells are treated by treatment with trypsin or by another protease and thereby isolated from the tissue and also
- Primary cell cultures of epithelial cells are one another example of primary cells.
- epithelia cover external and internal surfaces of tissues and organs.
- Epithelial cells have a very high degree of differentiation, which is expressed in the polarization of cells with an apical and a basolateral surface. The different surfaces perceive different functions.
- the apical surface of epithelial cells of the intestine serves to absorb nutrients, while the basolateral surface transfers these nutrients to the blood and forms connections to adjacent cells as well as to the basal membrane.
- Primary cells from the blood, bone marrow or spleen hardly adhere but can still be cultured in suspension.
- methods exist for further selection of a desired subtype from the isolated primary cells.
- a primary cell may also be a, preferably non-human, embryonic stem cell, preferably an embryonic stem cell, which in a particularly preferred embodiment has been obtained from umbilical cord blood.
- the embryonic stem cell is a cell isolated from an embryo that is in a stage of up to eight cells. This embryonic stem cell from the eight-cell stage is a totipotent cell and can be differentiated into all cell types of the main tissue types endodermal - like wall cells of the digestive tract - mesodermal - like muscles, bones, blood cells - and ectodermal - like skin cells and nerve tissue.
- the embryonic stem cell is a cell isolated from an embryo that is in the stage of Blastocyst is located.
- This embryonic stem cell from the blastocyst stage is a pluripotent cell and can be differentiated into all types of body cells of the main tissue types, ie endodermal, and laterally, with the exception of the formation of placental tissue, which can no longer be formed.
- a special feature of stem cells is that these cells are capable of self-replicating during cell division, thus preserving the pool of stem cells and simultaneously producing a differentiated cell, which then has a lower differentiation potential.
- Stem cells are distinguished by so-called markers of differentiated cells. Markers can be specific proteins that are amplified or expressed to a lesser extent by stem cells.
- the markers described are SSEA-1, stage-specific embryonic antigen-1, AP activity, alkaline phosphatase activity and the expression of the transcription factor Oct-3/4.
- the expression of the marker proteins depends on the origin of the stem cell.
- an adult stem cell is a cell that arises after the embryonic stage.
- Adult stem cells can be isolated from organs and tissues such as bone marrow, skin, adipose tissue, umbilical cord, umbilical cord blood, brain, liver or pancreas and are usually predeterminable, ie they have a lower potential for differentiation and are multi- or unipotent.
- Usually isolated primary cells from an embryo or an adult mammal have only a very limited ability to grow. In human fetal cells, after their isolation and cultivation, first a good cell growth takes place. Furthermore, primary cells can also be isolated from tumors and to be cultivated.
- tumor stem cells Genetic alterations, oncogenic transformations, override regulatory mechanisms such as apoptosis or potentiate positive growth signals such as overexpression of growth receptors. Therefore, many, especially primary tumor cells, often show unlimited growth. A special subpopulation of primary cells under tumor cells are so-called tumor stem cells. These could be detected as a very small cell fraction within certain tumors. Tumor stem cells have been isolated and cultured from breast cancer tumors. Characteristic marker proteins for these breast cancer stem cells are high CD44 expression, no or low CD24 expression and the absence of so-called line markers.
- an "adult stem cell” is to be understood as meaning, in particular, a cell which has arisen after the embryonic stage, has an undifferentiated differentiation potential compared to differentiated cells, is predeterminable and, in particular, epithelial cells, endothelial cells, dendritic cells, mesenchymal cells Cells, adipocytes or especially the corresponding progenitor cells from the heart, the musculature, adipose tissue, the skin and the brain are isolated.
- three-dimensional means a spatial extension into all three space coordinates.
- the extent can be substantially uniform in these three directions, so that, for example, there is a cylindrical shape, a column, cuboid or cube-shaped matrix structure. But it is also possible, for example, that the expansion is in two directions to a greater extent, in the third direction but only to a small extent, so that the three-dimensional structure acts flat, for example, represents a membrane or layer.
- biocompatible means that the framework structure and the nanoparticles, with regard to their material composition as well as their structure in the cells, tissues or in an organism, in particular an experimental animal, no toxic, apoptotic, unwanted immunological or other causes unwanted reaction and cellular or molecular processes, even after a possible intemalization of nanoparticles or degradation of the nanoparticles and / or scaffold structure does not or hardly disturbs.
- the invention preferably provides for the nanoparticles to be biodegradable or bioresorbable, that is to be degraded successively by biological influences, in particular the action of the cultured cells, and to thereby release the active substances preferably contained.
- nanoparticles are understood as meaning particles having a diameter of from 1 to 1000 nm.
- Such nanoparticles can be composed of various materials, for example inorganic or organic substances.
- their surfaces may comprise chemically reactive functional groups which fertilize affine bonds, ie covalent and / or non-covalent binders, with complementary functional groups of active substances to be bound, and in this way can stably fix the active substances to their surfaces.
- the present invention provides that the nanoparticles can also form bonds with the framework structure. Such bonds are preferably electrostatic interactions.
- the present invention provides a cell culture system in which the nanoparticles have a diameter of from 50 to 1000 nm, preferably from 50 nm to 900 nm, preferably from 60 to 600 nm. In a further preferred embodiment, the present invention provides a cell culture system in which the nanoparticles have a diameter of 80 to 150 nm, preferably 50 to 150 nm.
- the cell culture system according to the invention contains nanoparticles composed of inorganic substances, for example gold or other noble metals or metals or metal oxides, calcium phosphate and calcium hydrogen phosphate or other mixed phosphates, silicon-based oxidic materials, such as silicates, silicon oxides , such as silicon dioxide, are built up.
- the nanoparticles can also be DynaBeads.
- the cell culture system according to the invention contains nanoparticles which are composed of organic materials, in particular organic polymers.
- the nanoparticles can preferably be prepared by way of emulsion polymerization.
- Nanoparticles are preferably used in the cell culture system according to the invention, which are constructed from biodegradable polymers. Also preferred is a use of nanoparticles which have a diameter of 50 nm and a biodegradable matrix.
- the cell culture system according to the invention contains nanoparticles composed of polylactides (PLA), poly (lactide-co-glycolides) (PLGA), poly (lactic acid) PCL / PGA-di-block systems, polyorthoesters (POE), polyanhydrides, polyhydroxyalkanoates (PHA), polypyrroles (PPy), polypropylenecarbonate, Polyethylene carbonate, polyalkylcyanonitrile or polyethylene glycol are constructed.
- the nanoparticles have polymers of different molecular weight and variable polarity.
- the selection of the material to be used for the construction of the nanoparticle can be carried out according to which form and kinetics the active substance is to be released.
- the invention provides a preferred embodiment, according to which a nanoparticle of different materials, in particular different polymers, constructed in order to achieve a particularly high variability in the control of the release profile of the drug to be released.
- the cell culture system according to the invention comprises nanoparticles of different materials which, in a further preferred embodiment, in turn have different active substances. In this way too, a temporally and / or spatially specifically defined release of active ingredients can take place.
- the nanoparticles are carriers of at least one active substance.
- active ingredients are understood to mean those substances which exert an effect on the cells to be cultivated.
- Preferred such active substances are substances which can be cultivated in the regulation of growth and differentiation processes.
- Fourth cells are involved, in particular control, regulate, determine, initiate or finalize these agents growth and differentiation processes.
- agents may also be involved in migration, invasion, redifferentiation or division processes.
- the active compounds are those which are to be supplied to the cells to be cultivated in a desired and particular particular application kinetics.
- the cell culture system contains nanoparticles which have active substances enclosed in the framework structure and / or applied to their surface.
- the inclusion of active substances for example growth factors, the water-in-oil-in-water technique as the preferred method for the preparation of active substance-loaded nanoparticles, in particular PLA and PLGA Particles, is used.
- active substances for example growth factors
- the water-in-oil-in-water technique as the preferred method for the preparation of active substance-loaded nanoparticles, in particular PLA and PLGA Particles, is used.
- the present invention furthermore preferably provides a cell culture system in which the active substances enclosed in the nanoparticles are growth factors, cytokines, cell adhesion proteins such as integrins, dyes such as fluoresceneamines, chemokines, vitamins, minerals, fats, proteins, nutrients, fiber-forming proteins, carbohydrates, Adhesion proteins, cell receptors, pharmaceuticals, DNA, RNA, aptamers, angiogenic factors, lectins, antibodies, antibody fragments or inhibitors.
- growth factors cytokines
- cell adhesion proteins such as integrins
- dyes such as fluoresceneamines
- chemokines chemokines
- Adhesion proteins cell receptors
- pharmaceuticals DNA, RNA, aptamers, angiogenic factors, lectins, antibodies, antibody fragments or inhibitors.
- the cell culture system contains nanoparticles which Have stabilizers.
- the stabilizers preferably represent carbohydrates, for example trehalose, proteins, polyethylene glycols or detergents.
- the cell culture system contains nanoparticles which have been functionalized by coupling with functional groups.
- the nanoparticles themselves have functional groups on their surface.
- the present invention provides, in particular, for a first functional group 1A to be applied to the surface of the nanoparticles, which can form an affine, preferably covalent bond with a complementary group 2A of an active substance to be immobilized.
- the first functional group 1A is selected from the group consisting of amino group, carboxy group, epoxy group, maleimido group, alkyl ketone group, aldehyde group, hydrazine group, hydrazide group, thiol group and thioester group.
- the functional group 2A of the active substance is selected from the group consisting of amino group, carboxy group, epoxy group, maleimido group, alkyl ketone group, aldehyde group, hydrazine group, hydrazide group, thiol group and thioester group.
- a nanoparticle according to the invention thus has on its surface a first functional group 1A which is covalently linked to a functional group 2A of the active substance to be immobilized, the functional surface group 1A being a group other than the functional protein group 1A. group 2A.
- the two bonding moieties 1A and 2A must be complementary to each other, that is, able to form a covalent bond with each other.
- the surface of the nanoparticle according to the invention functional groups 1B and an active substance to be immoblisierende the functional groups 1 B binding complementary groups 2B, wherein the functional groups 1 B and 2B according to the invention in particular a non- Covalent bond can enter.
- the second functional group 1B of the surface of the nanoparticle is selected from the group consisting of oligohistidine group, Strep-Tag I, Strep-Tag II, Desthiobiotin, Biotin, Chitin, chitin derivatives, Chitinbinddomäne, metal ion chelate complex, streptavidin, streptactin, avidin and Neutravidin.
- the functional group 2B of an active substance to be immobilized is selected from the group consisting of oligohistidine group, Strep-Tag I, Strep-Tag II, Desthiobiotin, biotin, chitin, chitin derivatives, Chitinbinddomäne, metal ion chelate complex, streptavidin, streptactin, avidin and Neutravidin.
- a nanoparticle according to the invention has in its surface a functional group 1B which is non-covalently linked to a functional group 2B of a molecule, the functional surface group 1B of the nanoparticle being a group other than the functional molecular group 2B.
- the two non-covalent bond-forming groups must be complementary to each other, that is, able to form a non-covalent bond with each other.
- the surface of the nanoparticle according to the invention and the active substances to be immobilized optionally have both functional groups 1A and 1B as well as 2A and 2B.
- the three-dimensional framework structure contains one or more of the following components, namely fiber-forming proteins such as collagens, elastic fiber-forming fibrillins and / or elastins, carbohydrates such as glucosaminoglycans, long-chain polysaccharides, in particular hyaluronic acid, heparan sulfate, dermatan sulfate, chondroitin sulfate and keratan sulfate, adhesion proteins, for example adapter proteins or other adhesive proteins, for example laminins, vitronectin and fibronectin, components of the basal membrane such as laminins, entactin and proteoglycan and cell receptors for ECM components such as cell membrane proteins.
- fiber-forming proteins such as collagens, elastic fiber-forming fibrillins and / or elastins
- carbohydrates such as glucosaminoglycans, long-chain polysaccharides, in particular hyaluronic acid, heparan
- the three-dimensional framework structure contains or consists of components of the extracellular matrix selected from the group consisting of laminins, glycosaminoglycans (GAG), proteoglycans, elastin, type I collagen, II, III and IV, entactin (nidogen), vitronectin, hyaluronic acid, heparan sulfate, dermatan sulfate, chondroitin sulfate, keratan sulfate, perlecan, adhesion proteins and fibronectin.
- laminins glycosaminoglycans (GAG), proteoglycans, elastin, type I collagen, II, III and IV, entactin (nidogen), vitronectin, hyaluronic acid, heparan sulfate, dermatan sulfate, chondroitin sulfate, keratan sulfate, perlecan, adhesion proteins and fibro
- the framework structure can be constructed from a framework structure, for example collagen, in particular collagen fibers, this framework structure being advantageously and optionally designed with further components from the aforementioned group of components forming the framework structure.
- the Framework basic structure in particular the collagen scaffold structure, for example, be additionally equipped with components that promote cell properties such as adhesion and proliferation, for example anchor proteins and / or carbohydrates.
- the framework structure in particular of collagen, is in fiber form and / or in net shape.
- the framework structure in particular of collagen, is present in a three-dimensional network-like branched form.
- the framework structure may be in a preferred embodiment in hydrogelartiger or sponge-like form.
- the three-dimensional biocompatible scaffold structure in the cell culture system according to the invention represents an extracellular matrix.
- the concentration of active ingredients in the nanoparticle is in a range of 1 ng / ml to 10 ug / ml.
- the cell culture system contains nanoparticles which are distributed in the cell culture system integrally in the framework structure.
- the nanoparticles can be distributed homogeneously in the framework structure. In a preferred embodiment, however, there may also be a heterogeneous, non-uniform distribution of the nanoparticles.
- the nanoparticles are present in the form of at least one layer above and / or below the framework structure. In a In a preferred embodiment, the nanoparticles are present in the form of at least one layer above the framework structure.
- the nanoparticles in the cell culture system according to the invention are arranged in several layers below the framework structure.
- the nanoparticles in the cell culture system according to the invention are preferably arranged in several layers above the framework structure.
- the average diameter of the nanoparticles is always less than or equal to the average thickness of the framework structure. In a further preferred embodiment, the average diameter of all nanoparticles is always smaller than the average thickness of the framework structure. In a preferred embodiment, all nanoparticles, preferably predominantly or completely, are preferably embedded in the framework structure.
- the ratio of the dimensions of nanoparticles to the thickness of the framework structure is preferably 1: 1 or less, preferably 1:10 or less, more preferably 1: 100 or less, more preferably 1: 1000 or less.
- the nanoparticles in the cell culture system according to the invention are in the form of a gradient within the framework structure.
- the preferably provided active substance is present in the cell culture system according to the invention in the form of a gradient within the framework structure.
- the term "gradient" can thus mean the formation of various concentrations of nanoparticles and / or active substance within the cell culture system according to the invention, in particular the framework structure, in particular a spatially graduated, increasing or decreasing concentration of nanoparticles and / or agents.
- an active ingredient gradient is formed by the use of nanoparticles which have different concentrations of an active substance.
- the active substance can be enclosed or applied in different concentrations in the nanoparticles.
- the active compound can also be applied to the nanoparticles in different concentrations by binding via functional groups. Depending on the order of these nanoparticles with different concentrations within the framework structure, for example a homogeneous distribution, an increasing concentration or a concentration gradient of the active substance in the cell culture system is thereby achieved.
- a drug gradient is formed by having nanoparticles homogeneously distributed within the scaffold structure, wherein the nanoparticles have different drug concentrations and are arranged such that a drug gradient is formed. It is also provided in a preferred embodiment to distribute nanoparticles with different concentrations of active substance in a spatially uneven form, ie heterogeneously, in the framework structure, in particular to introduce such nanoparticles in the form of a nanoparticle gradient into the framework structure.
- the drug gradient is formed in the cell culture system by forming a nanoparticle gradient, namely by nanoparticles in which a certain and in the nanoparticles used the same concentration of an active ingredient is incorporated or applied to the surface thereof , spatially heterogeneous, that is differently distributed in the framework structure, are present.
- a lower number of these nanoparticles in, for example, an upper region of the framework structure followed by a higher number of these nanoparticles in an underlying region of the framework structure leads to an increase in concentration within the framework structure towards the bottom.
- a higher number of these nanoparticles in an upper region of the framework structure and a smaller number of these nanoparticles in an underlying region of the framework structure lead to a decrease in concentration within the framework structure downwards.
- a controlled and delayed release of active substance takes place in the cell culture system, in particular through the use of biodegradable nanoparticles.
- the controlled release of active substances which is preferred according to the invention can additionally or alternatively also be ensured by diffusion from the nanoparticles into the surrounding cell culture system, in particular into the fibrous or network-like framework structure.
- the nanoparticles are connected to the framework structure.
- the binding is carried out so that a culture medium change does not lead to detachment of the nanoparticles from the framework structure.
- the bond is stable to washing, ie the nanoparticles are not detached from the framework structure even when changing the culture medium and optionally washing steps with conventional washing media, such as buffers.
- the nanoparticles are connected via electrostatic interactions with the framework structure, in particular by ionic bonding.
- the nanoparticles are connected to the framework structure by UV crosslinking.
- the cell culture system is used for carrying out migration experiments, in particular migration experiments in vivo.
- the invention also relates to a method for producing a cell culture system comprising a three-dimensional biocompatible scaffold structure and biocompatible nanoparticles, wherein the scaffold structure with the nanoparticles, e.g. in accordance with one of the following methods.
- the cell culture system is produced by a "contactless printing process".
- the term "contactless printing process” means a process in which nanoparticles are transferred to the substrate without contact with the surface, and there are various possibilities for achieving this - called drop on demand provided by ink jet method.
- a method by means of printhead or individual needles is provided.
- one or more drops of suspension are transferred to the desired location.
- commercially available machines from Dimatix - FujiFilm- are used for inkjet processes.
- the delivery of the nanoparticle suspension takes place via a pneumatic method, a vacuum method or a piezoelectric method
- the penetration depth of the nanoparticles into the substrate, in particular into the framework structure of the cell culture system is controlled by the drop volume or the dropping speed in the respective embodiments of the method according to the invention
- the contactless printing method preferably leads to a layer-like structure wrestle the nanoparticles within the framework structure.
- a cell culture system which is produced by impregnation, in particular by using a framework structure with a nanoparticle-containing suspension is penetrated, for example by impregnation with a nanoparticle-containing suspension.
- a cell culture system which is produced by a laser induced forward transfer - LIFT method.
- material to be transported in particular one or more components of the extracellular matrix, is cut out by laser energy and applied to a target material.
- a further preferred embodiment of the present invention provides that the cell culture system is produced by electrospinning (spincoating).
- electrospinning preferably polymeric structures having preferably a fiber diameter of preferably 2 to 20 ⁇ m, which are very similar to the natural environment of the cells, namely the extracellular matrix, can be produced.
- the invention thus relates to methods for producing a cell culture system, wherein it is provided in a particularly preferred embodiment to produce the biocompatible nanoparticles, for example, by means of solvent evaporation of the Spontanoues Emulsifaction Solvent Diffusion (SESD) method, salting out, spray drying or phase separation.
- SESD Emulsifaction Solvent Diffusion
- this method it is particularly preferably possible according to the invention to incorporate the most diverse hydrophilic active ingredients into the nanoparticles.
- the active substance in the water The substance is emulsified in a polymer-containing oil phase. This mixture is emulsified in a further water phase and the organic solvent removed, for example by reducing the pressure.
- the oil phase acts equally as a solvent for the polymer as well as the active ingredient.
- the novel nanoparticles in particular by the solvent evaporation based on Spontanoues Emulification Sovent Diffusion SESD method.
- it is provided to dissolve both the polymer and the active ingredient in an organic mixture, preferably in dichloromethane and acetone, and to transfer this solution into an aqueous phase with stabilizer and to emulsify it by stirring.
- the hydrophilic solvent, preferably acetone diffused into the surrounding water phase and the nanoparticles according to the invention are formed by stirring under reduced pressure.
- the particle size is reduced by an increase in the proportion of water-miscible solvent.
- the nanoparticles according to the invention in particular by salting out.
- the polymer preferably PVA
- the polymer is added to a saturated solution, in particular of magnesium chloride or magnesium acetate, so as to obtain a viscous gel as an aqueous phase.
- a biodegradable polymer with an active ingredient preferably in acetone as the organic phase.
- the organic phase is emulsified in the resulting two-phase system by stirring in the gel.
- the nanoparticles according to the invention precipitate into the aqueous phase by adding sufficient water and after diffusion of preferably acetone.
- nanoparticles according to the invention in particular by spray drying. It is preferably provided that nanoparticles according to the invention are produced by sputtering or atomizing solutions and emulsions in which biodegradable polymers and active substances are dissolved, in particular with the aid of a two-substance nozzle in a hot air stream.
- the nanoparticles according to the invention are produced in particular by phase separation - coacervation.
- phase separation - coacervation it is provided in particular to dissolve biodegradable polymers in preferably dichloromethane and to disperse or emulsify an active substance therein.
- silicone oil which does not mix with the organic polymer phase, is added and carries out a phase separation. This mixture is stirred further with the addition of preferably heptane, whereby nanoparticles according to the invention can be obtained.
- the method for producing a cell culture system according to the present invention relates to a cell culture system manufactured by contactless printing method.
- the present invention relates to a process for the preparation of a cell culture system prepared by impregnation.
- the present invention relates to a method for producing a cell culture system produced by an LIFT method.
- the present invention relates to a method for producing a cell culture system in which the cell culture system is produced by electrospinning (spin coating).
- the present invention also relates to a method for cultivating cells by introducing the cells into a cell culture vessel containing a cell culture system of the present invention and a suitable nutrient medium and cultured there under suitable conditions allowing cultivation.
- the cultured cells are preferably eukaryotic cells, in particular of human or animal origin. More preferably, the cultured cells are primary cells.
- the cell to be cultivated is a tumor cell, in particular a tumor cell line such as MCF-7, HeLa, HepG2, PC3, CT-26, A125, A549, MRC-5, CHO, MelJuso28, Nalm ⁇ or Jurkat.
- a tumor cell line such as MCF-7, HeLa, HepG2, PC3, CT-26, A125, A549, MRC-5, CHO, MelJuso28, Nalm ⁇ or Jurkat.
- the cell to be cultivated is an adherent cell, in particular an established cell line such as HUVEC or HaCaT.
- this relates to a method for cultivating cells by means of the cell culture system according to the invention, wherein the cell to be cultivated is a differentiated cell.
- the present invention relates to a method, in particular a non-therapeutic method, for producing differentiated cells or cell aggregates from undifferentiated cells, wherein the undifferentiated cells are cultivated in the cell culture method according to the invention and thus differentiated cells or cell aggregates are obtained.
- the present invention relates to a method, in particular a non-therapeutic method, for maintaining the differentiation stage of predifferentiated or differentiated cells, wherein the predifferentiated or differentiated cells are cultured in the cell culture method according to the invention.
- the present invention relates to an aforementioned, preferably non-therapeutic method, wherein undifferentiated cells are cultured in the cell culture method according to the invention and kept in this undifferentiated state.
- the cell aggregates represent transplants or test systems.
- the cell aggregates can also be organs, organ parts, in particular a trachea or parts thereof.
- the present invention relates to differentiated cells and cell aggregates, which are obtained according to the invention cultivation of differentiated or undifferentiated cells or cell aggregates, for example, grafts, test systems, organs, organ parts, in particular a trachea, or parts thereof.
- the present invention in particular the present cell culture method, allows the long-term cultivation of stem and progenitor cells, which can be used for a variety of applications, for example for transplants and test systems, but also in basic research.
- the present invention particularly the present cell culture method, enables the proliferation of functional cells, especially primary cells.
- the present invention in particular the present cell culture method, allows the directed differentiation to functional cells and tissues, for example in the transplant production or for test systems in drug development.
- the present invention particularly the present cell culture method, enables the production of grafts for healing chronic / diabetic wounds, for example by cell migration induced by nanoparticles.
- the present invention also relates to a method for the production of expression products of a cell, wherein the cell are cultured according to the cell culture method according to the invention and the expression product is obtained from this cell culture.
- the present invention also relates to expression products of a cell which is cultured by the method according to the invention and whose expression products are obtained.
- FIG. 1 shows an SEM micrograph of biodegradable nanoparticles
- FIG. 2 shows the release result of the 8% BSA-loaded PLGA particles from Example 1.
- Figure 3 shows cells on a PAH + carboxy nanoparticle coated slide on day 1; A) gives experiment 1, B) gives experiment 2 again.
- Figure 4 shows cells cultured for control in a cell culture flask from Experiment 2; A) on day 1 and B) on day 3.
- FIG. 5 shows cells which have been cultivated on surfaces with nanoparticles with amino functionalization and subsequent coating with carbo xy nanoparticles with a planar and globular surface;
- FIG. 6 shows cells which have been cultivated on surfaces with nanoparticles with amino functionalization and subsequent coating with carbo-xy nanoparticles with a planar and globular surface;
- A) PAH, Day 1, Approach 3, B) PAA + Amino Nanoparticles, Day 1, Approach 3, C) PAH, Day 3, Approach 2, D) PAA + Amino Nanoparticles, Day 3, Approach 2 Abbreviations:
- PCL polycaprolactone
- PGA polyglycolide
- PAA polyacrylic acid
- PAH polyallylamine hydrochloride
- EGF epidermal growth factor
- BMP bone morphogenic protein
- SESD morpholinoethanesulfonic acid
- the prepared W / O phase is dispersed in a further water phase (100 mL water + 500 mg polyvinyl alcohol) by means of ultrasound, so that a W / O / W emulsion is formed.
- the organic solvent of the polymer-containing O-phase is removed by evaporation, thereby precipitating the polymer as a nanoparticle.
- the size of the particles is about 150 to 350 nm.
- the inclusion of active ingredient is 5-8 wt .-%. By use a larger amount of active ingredient can also be achieved a higher load.
- This W1 / O phase is dispersed in a second aqueous phase (10 mL of a 0.5% PVA solution) by means of ultrasound.
- the removal of the dichloromethane / acetone mixture of the W1 / 0 / W2 emulsion is carried out by evaporation under reduced pressure. In this case, nanoparticles with a size of 100-250 nm precipitate.
- the drug loading is about 10-15 wt .-% (Fig. 1).
- a 1% by weight aqueous suspension of the silica particles is mixed with 10% by volume of 25% ammonia. Then 20% by weight of aminopropyltriethoxysilane, based on the particles, are added and the mixture is stirred for 1 h at room temperature. The particles are purified by repeated centrifugation and carry functional amino groups on their surface (zeta potential in 0.1 M acetate buffer: + 35 mV).
- the modified surfaces include, in addition to the nanoparticles, a fibrous framework structure.
- the inoculated nanoparticles are at a distance of 10 to 1000 nm.
- Ojekt breed (OT) is first cleaned in a water bath at 40 0 C with a 2% (v / v) Hellmanex Il solution. Subsequent hydroxylation of the surfaces occurs at 70 ° C. in a 3: 1 (v / v) NH 3 (30%) and H 2 O 2 (25%) solution to produce a negative charge. Before or after the hydroxylation is carried out a coating with, for example, a collagen solution, for example, collagen type I 1 in a concentration of 0.1 to 6 mg / ml, in a conventional manner.
- a collagen solution for example, collagen type I 1 in a concentration of 0.1 to 6 mg / ml
- the polyelectrolyte coating of the surfaces is produced by the so-called layer-by-layer technique.
- the freshly purified and negatively charged OT are first added to a cationic poly (allylamine hydrochloride) solution (PAH solution) (0.01 M, based on the monomer) or poly (diallyldimethylammonium chloride).
- PAH solution cationic poly (allylamine hydrochloride) solution
- PDADMAC solution 0.02 M, based on the monomer
- Table 1 shows observations on the morphology of human keratinocytes on the carbonyl and amino nanoparticles.
- modified surfaces with triplicates with n 3 samples each were carried out. Both the colony density and thus the adherent cell number and proliferation as well as the morphology and thus the differentiation status of the primary cells were evaluated.
- Fig. 3 On the modified surfaces with a fibrous framework structure, there is a much faster adhesion and spreading (Fig. 3) of the keratinocytes in contrast to the culture flask (Fig. 4).
- the surfaces modified with nanoparticles induce rapid spreading, which is visible within a few hours.
- the morphology of the primary epidermal cells was comparable to the cell culture flask on these modified substrates. Furthermore, a faster differentiation of the primary cells on the amino-functionalized surfaces (Fig. 6) was seen, whereas the differentiation of the keratinocytes on the carboxy surface corresponded to that of the controls (Fig. 5). Table 1
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