RU2785710C2 - Matrix structure for cell cultivation - Google Patents

Abstract

FIELD: cell biology; biotechnology.

SUBSTANCE: present invention relates to cell biology and biotechnology, in particular to matrix structures for cell cultivation, as well as bioreactors containing a chamber including such matrix structures. The specified matrix structures contain one or more layers for immobilization of cells having a surface for adhesion and cell growth and defining a cell immobilization section. Matrix structures also contain one or more separating layers containing a structure forming a winding path, which define a separating section adjacent to the specified cell immobilization section. The latter provides cell and medium passage along the surface of both the cell immobilization section and the separating section, but along the winding path. At the same time, the specified cells are effectively moved to layers for immobilization and stick in their depth. Layers for cell immobilization are made of woven or non-woven canvas. The ratio of a thickness of the immobilization section to the separating section is from 1:2 to 2:1. An outer layer of the structure is formed of one or more separating layers.

EFFECT: present invention allows for an increase in a surface for cell cultivation in a small volume, while providing circulation of a medium and cells, as well as improvement of reproduction and homogeneity of matrix structures.

28 cl, 12 dwg, 1 ex

Description

The technical field to which the invention belongs

The present invention relates to a matrix for culturing cells, in particular, to a structured matrix of high density for culturing cells. The present invention also relates to the use of a cell culture matrix and a bioreactor containing said matrix.

State of the art

Methods for culturing cells such as eukaryotic cells, animal cells, mammalian cells and/or tissues are difficult and complex because these cells are fragile and have nutrient and oxygen requirements during growth that are difficult and difficult to maintain. Given the growing need for cell culture in large quantities, bioreactors and culture devices have become an important tool in research and in the production of cells to obtain active proteins and/or antibodies and/or any cellular by-products.

Prior art bioreactors contain unstructured cell culture matrices and are provided with internal or external circulation mechanisms to circulate the culture medium. Cell culture matrices typically contain carriers, which may be in the form of regular or irregular beads, or may contain woven or non-woven microfibers of polymer or any other material compatible with cell culture. The carriers may have various shapes and sizes.

Some disadvantages may be inherent in known bioreactors and their unstructured cell culture matrices. The lack of reproducibility of uniformity of growth matrices in terms of volume during their loading, as well as their movement and redistribution in time under the influence of the flow of the nutrient medium, can lead to non-reproducible nutrient media and very different microenvironments in different parts of the bioreactor. Carriers can collect in this area inside the bioreactor, which significantly reduces the homogeneity in cell culture. Consequently, a portion of the cultured cells will have very limited or no access to nutrient medium and/or oxygen supply, resulting in different cell metabolism, differences in product quality, even cell death, and low cell production rates. In addition, inhomogeneous matrices are difficult to fabricate and load, given the variable sizes and shapes of supports that can stick to each other. Another disadvantage of prior art cell culture matrices is that their cell culture surface cannot be accurately determined. Finally, loading the carriers into the bioreactor is laborious and therefore costly.

US Pat. No. 3,948,732 describes a design that includes a spiral tube chamber assembly through which media and cells pass within the chamber and around the center of the helix and adhere to the inner wall of the chamber. The design includes a separating element provided with spaced protruding supporting elements, which alternates with the block of the tubular chamber. These protruding elements are designed to facilitate the flow of gas through the cross section of the helical structure in the longitudinal direction. Under no circumstances does the separating element come into contact with any cells or growth medium in the tube, nor does it in any way ensure the passage of the cells.

It is an object of the present invention to provide a cell culture matrix and a bioreactor containing said matrix that overcome at least some of the aforementioned disadvantages. Preferably, the cell culture matrix should provide a large surface area for cell culture in a small volume while still allowing medium and cells to circulate. Pressure drops in the system should be avoided, as this adversely affects the viability of some cells. Reproducibility and uniformity must be improved while maintaining manual control and minimum cost. It is necessary to provide a tortuous path for the movement of cells and growth medium in combination with a separating layer, which facilitates the passage along this path and parallel to the separating layer and layers for immobilizing cells.

Disclosure of invention

In a first aspect, the present invention provides a cell culture matrix construct or a structured cell culture matrix according to claims. 1, 8, 14, 22, 29 and 30.

In a second aspect, the present invention provides the use of a cell culture matrix construct or matrix according to any embodiment of the invention for cell culture.

In a third aspect, the present invention provides a bioreactor comprising a cell culture matrix construct or matrix according to any embodiment of the invention.

The matrix construct or cell culture matrix of the present invention has several advantages over those known in the art. Instability in the cell culture method is avoided or minimized through the use of a structured design. The cell culture surface can be easily and accurately determined due to the known cell culture surface and the number of immobilization layers. The cell culture surface to be placed in the bioreactor can also be easily adapted without changing the matrix and/or model of the bioreactor.

The matrix provides an improved and organized flow of cells and growth media within the bioreactor.

In addition, the structured cell culture matrix provides efficient loading into the bioreactor, thereby optimizing the use of internal space and increasing the cell culture surface within said bioreactor. The matrix thus provides a culture surface with a high cell density. In addition, due to the structured design of the matrix, a consistent and reproducible production of bioreactors containing the specified matrix is ensured.

Brief description of the drawings

Figures 1A and 1B show cross sections of parts of a matrix structure in accordance with two embodiments of the present invention.

Figures 2A-D show non-limiting examples of a matrix design in accordance with embodiments of the present invention.

Figures 3A-B show a perspective and top view of a matrix structure, partially rolled up according to an embodiment of the invention; and an embodiment of the densely loaded configuration.

Figure 3C shows a view of a fully collapsed matrix in accordance with an embodiment of the invention.

Figures 4A-4D show examples of mesh separation layer structures according to an embodiment of the present invention.

Figure 5 shows a bioreactor chamber provided with a matrix structure according to an embodiment of the present invention in which fluid and cells flow longitudinally along surfaces of separating and immobilization layers between top and bottom or bottom and top.

Figure 6 shows a side view of a rotating bioreactor equipped with a matrix structure in accordance with an embodiment of the present invention, rotating around its axis during use.

Implementation of the invention

The present invention relates to a matrix construct for cell culture, the use of the latter for cell culture, and a bioreactor containing said matrix construct.

Unless otherwise indicated, all terms used to describe the invention, including technical and scientific terms, have the meaning generally accepted by a person skilled in the art to which this invention pertains. As follows, definitions of terms are included for a better understanding of the idea of the present invention.

The terms used in this document have the following meanings:

The indefinite and definite articles in this document refer to both the singular and the plural, unless the context clearly indicates otherwise. For example, "cell" refers to one or more cells.

The term "about" as used herein, which refers to a measurable value such as a parameter, magnitude, time range, and the like, is meant to cover deviations of ±20% or less, preferably ±10% or less, more preferably ± 5% or less, even more preferably ± 1% or less, and even more preferably ± 0.1% or less of the specified value, as long as such changes are suitable for implementation in the disclosed invention. However, it should be understood that the meaning to which the modifier "about" refers is itself also specifically disclosed.

The terms "comprise", "comprising" and "comprises" and "consisting of" as used herein are synonymous with the terms "comprise", "comprising", "includes" or "comprise", "consisting of ”, “consists” and are inclusive or non-limiting terms that define the presence of that element that follows, for example, a component, and do not exclude or eliminate the presence of additional, not listed components, features, elements, members, steps known in the art or disclosed in it.

A listing of numeric ranges by endpoints includes all numbers and fractions included in the range, as well as the listed endpoints.

The terms "construct", "matrix construct" and "matrix" are used interchangeably throughout the text.

In a first aspect, the present invention provides a cell culture matrix construct that comprises one or more cell immobilization layers having a surface that allows cells to adhere and grow on the surface and form a cell immobilization section. Adjacent to these cell immobilization layers are one or more separating layers, including a structure that forms a separating section allowing cells and media to pass through an open but tortuous path, provided that the structure or nature of the separating layers is chosen such that the separation layers formed a tortuous open path for the cells and growth medium to travel parallel to the surface of said separation and immobilization layers. The tortuous path or channel formed by the separating section creates turbulence which facilitates the penetration of the cell and cell medium into the layers for immobilization.

Said cell immobilization layers will define the cell immobilization section, while said separating layers define the separating section.

In a further or alternative embodiment, the present invention provides a structured cell culture matrix comprising one or more separation sections and one or more cell immobilization sections. Each separation section contains at least one separation layer, and each cell immobilization section contains at least one cell immobilization layer, wherein the ratio of the thickness of the immobilization section to the separation section is at least 0.1, and the thickness of the separation section is at least 0 .1 mm. In an embodiment, the separation section includes a structure that provides a tortuous path or channel for cells to move along the layers.

For purposes of the present invention, a tortuous path should be understood as a path with directional components that vary relative to and along the common path.

The matrix assembly is designed to allow fluid and cells to flow longitudinally along the surfaces of the separation layer and the cell immobilization layer in a stationary bioreactor or, in the case of a rotating bioreactor, tangentially along the surfaces of both layers. Thus, the structure of the separation layers should be chosen to create a tortuous but open path between the immobilization layers, thereby creating turbulence. This turbulence will place cells and media into layers for immobilization.

One skilled in the art will appreciate that such separation of the tortuous paths can be achieved in several ways that are well known in the art. In one embodiment, the separating layer consists of a carrier structure or spherical, nearly spherical or egg-shaped objects such as balls loaded in a three-dimensional structure on top of each other (see figure 1B). Under the carrier structure should be understood as a structure consisting of balls or spheres, optionally fixed on a surface support, which form a tortuous path through which the liquid can flow.

In another embodiment, the separating layer may be a mesh or contain a mesh structure. For the purposes of the present invention, a mesh structure or mesh is to be understood as a structure comprising a weave or mesh pattern of fibre, wire or thread, said weave defining pores, holes or channels formed by the three-dimensional weave. Examples of mesh structures are shown in figures 4A-4D. It should be understood that they should not be construed as limiting the present invention.

The matrix construct or cell culture matrix of the present invention provides a substrate that allows cells to grow at high density. Preferably, a high cell density bioreactor provides a maximum cell growth surface with a minimum volume. The model of the present cell culture matrix construct is optimized to meet these requirements. Through the separating section adjacent to the immobilization section, the flow of cells and media is provided. These separating sections promote turbulence and provide a substantially tangential flow between the cell adhesion sections, in addition to occasional perpendicular flow through the immobilization layers. This increases cell adhesion to the immobilization layers in the immobilization sections.

In addition, the cells are more evenly distributed within the bioreactor due to the separator sections, which provide space for the cells to move before they adhere and which prevent the immobilization sections from acting as filters. Separation further allows improved removal of toxic metabolites by providing space for said metabolites to move.

The separating section additionally provides a better distribution of the nutrient medium and thus nutrients within the bioreactor. All cells inside the bioreactor are equally provided with a nutrient medium. Given the structured matrix model, the preferred path of the growth medium within the bioreactor will not be provided. This is an improvement over prior art unstructured matrices which provide preferred growth media paths in bioreactor applications where the unstructured supports are driven by the flow of the growth medium. The result is a heterogeneous distribution of cells.

To ensure minimal pressure drop and volume loss, the separating section consists of a design incorporating a tortuous path for cell and fluid flow. In one embodiment, the structure is a mesh. Through the use of a mesh structure for the separating section, a minimum thickness of the physical barrier is maintained, which ensures a constant pressure and yet sufficient space between adjacent immobilization sections.

In an embodiment, the structure or cell size of the separating layer is between 0.05 mm and 5 mm. The choice of cell structure or size is important as it in turn affects the balance between providing a sufficient barrier between the immobilization sections, thereby allowing cell and media to pass through while maintaining adequate system pressure and achieving high cell density. The openings in the mesh or grid may be of any shape, such as round, elliptical, square or rectangular. In an embodiment, the hole size is at least 0.05 mm, at least 0.06 mm, at least 0.08 mm, at least 0.1 mm, at least 0.15 mm, at least 0 .2 mm or at least 0.25 mm. In another or additional embodiment, the hole size is 5 mm or less, 4.5 mm or less, 4 mm or less, 3.5 mm or less, 3 mm or less, or any value between the above values.

It should be understood that the thickness of the spacer section, which is comprised of one or more spacer layers, will affect the performance of the matrix structure. In an embodiment, the thickness of the separating section is at least 0.1 mm, more preferably 0.1 mm to 5 mm, more preferably 0.2 mm to 1 mm. In an embodiment, the thickness of the separating section is between 0.25 and 0.6 mm, such as 0.4 mm or 0.5 mm.

In an embodiment, the thickness of the separation section in millimeters is at least 0.1, at least 0.2, at least 0.3, at least 0.4, at least 0.5, at least 0.6 , at least 0.7, at least 0.8, at least 0.9, at least 1, at least 1.1, at least 1.2, at least 1.3, at least at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, at least 2, at least 2, 1, at least 2.2, at least 2.3, at least 2.4, at least 2.5, at least 2.6, at least 2.7, at least 2.8 , at least 2.9, at least 3, at least 3.1, at least 3.2, at least 3.4, at least 3.5, at least 3.6, at least at least 3.7, at least 3.8, at least 3.9, at least 4, at least 4.1, at least 4.2, at least 4.3, at least 4, 4, at least 4.5, by m less than 4.6, at least 4.7, at least 4.8, at least 4.9, at least 5, at least 5.5, at least 6, at least 6.5 , at least 7, at least 7.5, at least 8, at least 8.5, at least 9, at least 9.5, at least 10, or any value between the above values.

In a further or alternative embodiment, the thickness of the separation section in millimeters is 1000 or less, 950 or less, 900 or less, 850 or less, 800 or less, 750 or less, 700 or less, 650 or less, 600 or less, or less 550, max 500, max 450, max 400, max 350, max 300, max 250, max 200, max 190, max 180, max 170, max 160, max 150, no more than 140, no more than 130, no more than 120, no more than 110, no more than 100, no more than 95, no more than 90, no more than 85, no more than 80, no more than 75, no more than 70, no more than 65, no more 60, 55 max, 50 max, 45 max, 40 max, 35 max, 30 max 25 max, 20 max, 15 max, 12 max, or any value between the above values.

The thickness of the cell immobilization section is equally important to the functioning of the system. A cell immobilization section that is too thick will result in a poorly populated area, while a section that is too thin will adversely affect the available cell growth surface, also adversely affecting cell growth. Preferably, the thickness of the immobilization section is 0.1 mm to 15 mm, more preferably 0.1 mm to 10 mm, even more preferably 1 to 5 mm or 0.1 mm to 1 mm.

There is a need for a balance between sufficient flow of cells and media between cell growth surfaces and a large enough cell surface for cell growth. The present inventors have found that, ideally, the ratio between the thickness of the cell immobilization section and the separation section should be at least 0.1, and more preferably 0.1 to 5, even more preferably 0.1 to 2, such as, for example, 1:1. Thus, the needs of the system will be satisfied.

In an embodiment, the ratio of the thickness of the immobilization section to the separation section is at least 0.1, at least 0.2, at least 0.3, at least 0.4, at least 0.5, at least 0 .6, at least 0.7, at least 0.8, at least 0.9, at least 1, at least 1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, at least 2, at least 2.1, at least 2.2, at least 2.3, at least 2.4, at least 2.5, at least 2.6, at least 2.7, at least 2 .8, at least 2.9, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 or any value between the above values.

In a further or alternative embodiment, the ratio of the thickness of the immobilization section to the separation section is at most 50, at most 45, at most 40, at most 35, at most 30, at most 25, at most 20, at most 19, at most 18, 17 max, 16 max, 15 max, 14 max, 13 max, 12 max, 11 max, or any value between the above.

The separation layers and/or the cell immobilization layers which constitute respectively the separation section and the immobilization section in the matrix structure are preferably made of a biocompatible polymer selected from polyester, polyethylene, polypropylene, polyamide, plasma-treated polyethylene, plasma-treated polyester, plasma-treated polypropylene, or plasma-treated polyamide. Said layers may be hydrophilic or hydrophobic. The cell immobilization layers are preferably hydrophilic.

The thickness of both layers is preferably 0.05 to 3 mm, more preferably 0.1 to 2 mm or 0.1 to 1 mm.

A suitable material for the cell immobilization layer may be a woven or non-woven material. Preferably, a non-woven material is used. Nonwoven fabric, unlike woven fabric, is a fabric that is not created by weaving or knitting and does not require the conversion of fibers into yarn. Nonwovens are broadly defined as sheet or roll structures bonded to each other by interlacing fibers or filaments (and by perforating films) mechanically, thermally or chemically. The nature of the nonwoven material used in this application can be any, including natural or synthetic fibers. Preferably, the nonwoven material is made from a polymer such as polyester or polypropylene. The cell immobilization layers used in the present invention may be selected from non-woven polyethylene terephthalate. The nonwoven fabric can be plasma treated to improve cell adhesion and flow.

The separating layers may be composed of a (biocompatible) cell sized polymer as described above. In one embodiment, the release layer is a synthetic woven fabric or structure. In another embodiment, the separating layer is a carrier structure. Such a structure can be obtained from a biopolymer (eg alginate). Another suitable material for this task is silicon dioxide, polystyrene, agarose, styrene-divinylbenzene, polyacrylonitrile or latex.

The separating layer can be treated with gamma radiation to reduce the bioburden.

The matrix design model can take various forms depending on the application and type of bioreactor.

In an embodiment of the present invention, the immobilization section and the separating section are arranged alternately. The sequencing means that each separation section is followed by a cell immobilization section, which is immediately followed by a separation section. Sections arranged alternately can alternate in a vertical position, as shown in the figures (see below), or in a horizontal position in accordance with the use of the matrix and/or the bioreactor into which the matrix will be introduced.

In this embodiment, one or more cell immobilization layers are superimposed on one or more separating layers (or vice versa). This configuration may be repeated several times if considered necessary to create a block of multiple immobilization sections and separating sections. Ideally, the final configuration may contain from 1 to 500 interlaces of the above layers. The stacked layers may be arranged in a frame or cassette, or sealed/bonded around their circumference. In another embodiment, the resulting block may be rolled around an axis or core to form a helical configuration.

The number of layers used in both the immobilization section and the separating section can be selected based on the application, the characteristics of the layers (dimensions, size, etc.) and the desired result. Therefore, the number of layers in any immobilization section or separating section can range from 1 to 20, more preferably 1 to 10, even more preferably 1 to 5.

As already mentioned, the presence of separating sections creates a space within the matrix through which the nutrient medium flows. This provides improved circulation of the growth medium through the matrix, thereby reaching all cultured cells. This effect is even stronger in the embodiment in which the separating section contains one separating layer and the immobilization section contains two immobilization layers. The nutrient medium flowing inside the matrix through the separating sections is oriented tangentially relative to the cell immobilization sections.

The spacer sections improve the rigidity of the matrix due to the rigidity of the spacer layers. The matrix in accordance with any embodiment of the invention can be compressed by any method known to the person skilled in the art. The size of the compressed matrix is reduced by a maximum of 20%, preferably by a maximum of 15%, more preferably by a maximum of 10% compared to the size of the uncompressed matrix.

In an embodiment, one surface of at least one separating layer or section is at least partially not covered by any cell immobilization layers or another layer or section. The bare bed section or bed is designed to be placed in contact with the inner wall of any bioreactor. This design prevents the piston effect inside the bioreactor.

In an alternative embodiment of the present invention, the matrix structure consists of a configuration of one or more cell immobilization layers, as described above, forming an immobilization section, and one or more separation layers, as described above, forming a separation section, located adjacent to (for example, above and /or under) the specified layers for immobilization of cells. Optionally, the layering can be repeated to form an alternating block-shaped configuration of one or more cell immobilization layers and one or more spacer layers. Said resulting configuration is subsequently helically or concentrically wound along the axis or core. The thickness of the separation section may be from 0.1 mm to 5 mm, more preferably from 0.2 mm to 1 mm, while the thickness of the immobilization section may be from 0.1 to 5 mm. The ratio of the thickness of the immobilization section to the thickness of the separation section is preferably 1:2 to 2:1, most preferably 1:1. Although the number of layers to achieve this can be varied depending on the characteristics of the layers used and thus chosen at will as described above, it has been found that a good result was achieved using two immobilization layers and one release layer. The thickness of the separating and immobilization layers is 0.05 to 3 mm, more preferably 0.1 to 2 mm or 0.1 to 1 mm. Although the material of the immobilization layer and the separation layer should not be considered limiting, the immobilization layer is preferably made from a woven or non-woven fabric, or material as described above for other embodiments. Said separating layer may also be formed from a woven or non-woven material and may comprise a mesh structure.

Preferably, the outer layer of the structure, which is the side facing the wall of the bioreactor when placed in the bioreactor, is a separating layer. This prevents cells from growing against the wall of the reactor. Such bioreactors can be heated and cell growth in this area can affect cell yield and/or quality. In a further embodiment, several spacer layers are provided at the outer boundary of the structure, again to provide good insulation. In another or additional embodiment, an insulating layer is provided which is made of a different material than the separating layer.

The matrix design is simple and reproducible compared to prior art counterparts. The present invention further provides scalable matrices, allowing for easy production of matrices having specific cell dimensions and/or growth surfaces. The scalability of the design/matrix of the invention does not affect the uniformity and/or quality of the nutrient flow provided.

In another aspect, the present invention provides the use of a construct/matrix according to any embodiment described above for cell culture.

In another aspect, the present invention provides a bioreactor comprising a construct or matrix according to any of the embodiments described above. The bioreactor may contain more than one construct or matrix. In an additional embodiment, the design/matrix and/or bioreactor containing said matrix are disposable. The structure/matrix can be positioned vertically or horizontally in the bioreactor.

Preferably, the structure/matrix makes up at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% of the interior of the bioreactor. The specified matrix is not more than 100%, not more than 90%, not more than 80%, not more than 70% or not more than 60% of the internal space of the bioreactor.

The bioreactor can be any type of bioreactor known to the person skilled in the art, such as perfusion bioreactor, wave bioreactor, cylindrical bioreactor, rotary bioreactor, bagged bioreactor, moving bed bioreactor, packed bed bioreactor, fibrous bed bioreactor, membrane bioreactor, batch bioreactor or continuous bioreactor. The bioreactor may be of any shape and may be made of any material such as stainless steel, glass or plastic.

It is assumed that the present invention is not limited to any form of implementation described previously, and that some modifications can be added to the presented example without re-evaluating the appended claims.

Blueprints

Figures 1A and 1B show cross sections of parts of a matrix structure in accordance with two embodiments of the present invention. The provision of one or more separation layers 3 or 3' between one or more cell immobilization layers 2 (forming the immobilization section 10) promotes turbulence (shown by black arrows) and random perpendicular flow (horizontal open arrows). The structure provides a fluid passageway that provides sufficient flow distribution for fixed bed applications. Thanks to this model, a uniform flow of fluid is ensured, and, as a result, the cells in the structure are distributed equally uniformly. By providing a separation that creates a tortuous open path, the flow of cells and media is able to pass over the surface of both layers. The turbulence will push the cells and media deeper into the layers to immobilize the cells. Overall, the model provides reproducibility and uniformity when used to obtain cells.

Figure 1A shows an embodiment in which the separating section 11 consists of one or more separating layers 3 which are made of a mesh web. Examples of possible mesh webs that can be used for this purpose are shown in Figures 4A-4D showing various hole shapes.

Figure 1B shows an alternative implementation in which the separating section is made of spherical or nearly spherical objects such as balls.

Both the mesh structure and the beads are examples of structures or layers that provide a tortuous open path as described above.

Figures 2A-2D show possible arrangements of a number of layers for immobilization relative to the separating layers, which respectively constitute the cell immobilization section 10 and the separating section 11.

The thickness of each section corresponds to the sum of the thicknesses of the layers contained in it. In one embodiment, the separation section 11 contains one separation layer, and the cell immobilization section 10 contains two cell immobilization layers, as shown in figures 2A and 2B. In this configuration, at least one surface of each immobilization layer is in contact with one surface of the release layer. Figures 2C and 2D show alternative examples in which the separating section 11 contains one separating layer and the immobilization section contains three immobilization layers.

The separation sections and the cell immobilization section may be of any shape and may be of similar dimensions. The matrix can be formed by alternating different sections, thereby obtaining a three-dimensional (3D) matrix. Said matrix may have any geometric shape, such as cylindrical, triangular, rectangular, or any irregular three-dimensional shape. The matrix may also have other shapes obtained by further forming the 3D matrix, such as rolling the 3D matrix to form a helix.

An example of a folded matrix is shown in Figures 3A-3B showing a perspective view and a plan view of the folded matrix, respectively. These figures show a matrix or design that is "loosely" coiled to illustrate the helical structure. It should be understood that the degree of matrix folding is variable depending on the application of the design and/or the desire of the user. The structure or matrix is tightly rolled into a structure such as the helical structure shown in Figure 3C.

Figure 3C shows an embodiment of the present invention, according to which one or more cell immobilization layers 2 are adjacent to one or more separating layers 3 made of a mesh structure, and, as a result, if necessary, the layering can be repeated several times to achieve a configuration in block form. The embodiment shown in Figure 3C is shown as being subsequently wound helically or concentrically along an axis or core, provided that all layers are tightly wound. Examples of mesh structures are shown in Figures 4A-4D (enlarged view). Preferably, the network structure included in the separating layers of the present invention forms a tortuous path for cells and fluid to flow when they are layered between two immobilization layers. Alternatively, other spacer structures can be used which form such winding paths. The core diameter, length and/or number of layers will ultimately determine the size of the structure or matrix. Preferably, the thickness of the layers is between 0.1 and 5 mm. In the embodiment shown in figure 3D, the thickness of the layers is from 0.25 to 0.7 mm, with two layers of cell immobilization alternating with one separating layer. Preferably, the outer layer of the resulting helical configuration will be one or more spacer layers. Alternatively, an insulating layer can be provided as the outer layer, which is different from both the separating layer and the cell immobilization layer. The separating layers are preferably a woven mesh fabric with a mesh size of 0.05 to 5 mm. The cell immobilization layers are preferably a non-woven material.

Figures 5 and 6 show, respectively, a stationary bioreactor and a rotating bioreactor equipped with a matrix structure in accordance with embodiments of the present invention.

In figures 5 and 6, the matrix shown in figure 3C is included in the chambers of the bioreactor. In both embodiments, the flow of cells and media will follow the path created by the separation layers.

In the embodiment shown in Figure 5, media and cells flow through the matrix structure in a longitudinal direction over the surface of the separating and immobilization layers (top down or bottom up).

The figure 6 shows a rotating bioreactor, in which the matrix structure is partially immersed in the environment, and the bioreactor and the matrix rotate along their axis. In this embodiment, the flow of media and cells will be in a tangential direction along the surfaces of the separating layer and layers for immobilization (in a spiral path).

Claims (28)

1. A cell culture matrix construct comprising one or more cell immobilization layers having a surface for cell adhesion and growth, said one or more cell immobilization layers defining a cell immobilization section, and one or more separating layers comprising a tortuous path-forming structure , said one or more separating layers define a separating section adjacent to said cell immobilization section, which allows cells and media to pass over the surface of both the cell immobilizing section and the separating section, but in a tortuous path, wherein said cells effectively move into the layers for immobilization and adhere in their depth, wherein one or more layers for immobilization of cells is made of woven or non-woven fabric, and the ratio of the thickness of the immobilization section to the separating section is from 1:2 to 2:1, while the outer layer of the structure is formed from one or more dividing lines oev. 2. A cell culture matrix construct according to claim 1, said matrix construct comprising one or more separation sections and one or more cell immobilization sections. 3. A cell culture matrix construct according to claim 1 or 2, wherein said separating layer comprises a mesh structure. 4. Matrix construction for cell cultivation according to claim 3, characterized in that the cell size of the separating layer is from 0.05 mm to 5 mm. 5. A cell culture array structure according to any one of the preceding claims, characterized in that the thickness of the separation section is at least 0.1 mm, preferably between 0.1 and 5 mm. 6. Matrix structure for cell culture, containing a configuration of one or more layers for immobilizing cells, made of woven or non-woven fabric, and one or more separating layers, made of a mesh structure with a mesh size of 0.05 mm to 5 mm, adjacent to said layers for immobilization of cells, wherein said configuration has a spiral or concentric winding along the axis, characterized in that the thickness of the separation layers and layers for immobilization is from 0.05 mm to 3 mm, and the ratio of the thickness of the immobilization section to the separation section is from 1:2 to 2:1, while the outer layer of the structure is formed from one or more separating layers. 7. The use of a matrix design according to any one of paragraphs. 1-6 for cell culture. 8. Bioreactor for cell culture, containing a chamber, including a matrix design, as described in any of paragraphs. 1-6. 9. The bioreactor according to claim 8, having an internal space, and in which the matrix structure is at least 10% and not more than 100% of the specified internal space. 10. A bioreactor according to any one of the preceding claims, wherein the outer layer of the cell culture matrix structure, facing the wall of the bioreactor, consists of one or more separating layers. 11. A bioreactor according to any one of the preceding claims, wherein the bioreactor and/or matrix construct are disposable. 12. Matrix structure for cell culture, having a spiral configuration, containing one or more layers for immobilizing cells, having a surface that provides adhesion and cell growth, and one or more separating layers containing a structure forming a tortuous path, adjacent to said layers for immobilization cells, and forming a channel that ensures the passage of cells and the environment over the surface of one or more layers for immobilization of cells, and the structure of each separating layer forming a tortuous path is adapted to move cells into one or more layers for immobilization and sticking in their depth, and one or more layers for cell immobilization is made of woven or non-woven fabric; moreover, the ratio of the thickness of at least one of the one or more immobilization layers to at least one of the one or more separating layers is from 1:2 to 2:1. 13. The cell culture matrix construct of claim 12, wherein the ratio of the thickness of at least one of the one or more immobilization layers to at least one of the one or more separation layers is 2:1. 14. The cell culture matrix construct of claim 12, wherein the thickness of each of the one or more spacer layers is between 0.05 mm and 3 mm. 15. The cell culture matrix construct of claim 12, wherein the thickness of each of the one or more immobilization layers is between 0.05 mm and 3 mm. 16. The cell culture matrix construct of claim 12, wherein each spacer layer comprises a mesh structure. 17. A cell culture matrix construct according to claim 16, wherein the cell size of each separation layer is between 0.05 mm and 5 mm. 18. The cell culture matrix construct of claim 12, wherein the one or more immobilization layers are made of a biocompatible polymer selected from polyester, polyethylene, polypropylene, polyamide, plasma-treated polyethylene, plasma-treated polyester, plasma-treated polypropylene, or plasma-treated polyamide . 19. The cell culture matrix construct of claim 12, wherein the one or more spacer layers are made of a biocompatible polymer selected from polyester, polyethylene, polypropylene, polyamide, plasma-treated polyethylene, plasma-treated polyester, plasma-treated polypropylene, or plasma-treated polyamide. 20. Matrix construction according to claim 12, wherein one or more immobilization layers are hydrophilic. 21. Matrix construction according to claim 12, in which one or more separating layers are hydrophilic. 22. The matrix structure of claim 12, wherein at least one surface of each immobilization layer is in contact with one surface of at least one separating layer. 23. Matrix construction according to claim 12, in which each separating layer forms a channel that allows cells and media to pass over the surface of the adjacent layer to immobilize the cells. 24. Bioreactor for cell culture, containing a chamber, including a matrix design, as described in any of paragraphs. 12-23. 25. The bioreactor according to claim 24, having an internal space, and in which the matrix structure is at least 10% and not more than 90% of the specified internal space. 26. The bioreactor of claim 24, wherein the bioreactor and/or matrix construct are disposable. 27. Bioreactor according to claim 24, wherein the bioreactor is adapted to provide flow through the matrix structure in a longitudinal direction from bottom to top. 28. Bioreactor according to claim 24, wherein the bioreactor is adapted to provide flow through the matrix structure in a longitudinal direction from top to bottom.

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