WO2013007224A1

WO2013007224A1 - Cell culture substrate and a method for producing thereof

Info

Publication number: WO2013007224A1
Application number: PCT/CZ2012/000066
Authority: WO
Inventors: Marcela MUNZAROVA; Martina JUKLICKOVA; Tomas FLORIS; Barbara KUBESOVA; Lucie STRAJTOVA; Eva MATEJKOVA; Lukas SCHUREK; Jakub SCHUREK
Original assignee: Primecell A.S.; Elmarco S.R.O.; Centrum Bunecne Terapie A Diagnostiky A.S.
Priority date: 2011-07-14
Filing date: 2012-07-13
Publication date: 2013-01-17
Also published as: CZ303453B6; CZ2011424A3

Abstract

The invention concerns the cell culture substrate containing a nanofiber layer from a biologically compatible polymer deposited on a bearing stratum wherein the bearing stratum is formed by a reticule from a biologically compatible material and the nanofiber layer fills up the space of its meshes. The invention also concerns the method for producing said substrate wherein the nanofiber layer from a biologically compatible polymer is deposited on a bearing stratum formed by a reticule from a biologically compatible material by means of an electrostatic spinning of the polymer system in an electric field created between a collecting electrode and an oblong spinning electrode arranged against said electrode, whereas the nanofiber layer being deposited covers also the reticule meshes. ˙

Description

Cell culture substrate and a method for producing thereof

Field of the invention The invention concerns a cell culture substrate layer containing a nanofiber layer from a biologically compatible polymer deposited on a bearing stratum.

The invention also concerns a method for producing said substrate.

Background of the invention

In technical literature, e.g.: Anne J. Meinel: Optimization strategies for electrospun silk fibroin tissue engineering scaffolds, Biomaterials 30 (2009) 3058- 3067, or Schofer .D., Boudriot U., Wack C, Leifeld I., Grabedunkel C, Dersch R., Rudisile M., Wendorff J.H., Greiner A., Paletta J.R., Fuchs- Winkelmann S. J.: Influence of nanofibers on the growth and osteogenic differentiation of stem cells: a comparison of biological collagen nanofibers and synthetic PLLA fibers, Mater Sci Mater Med. 2009 Mar;20(3):767-74.), as well as in many patent documents, e.g. US 2010233807, several versions of nanofiber cell culture substrates are described, which are intended, according to the specific cell type used, e.g. to cover flesh wounds, implantation in the patient's body, etc. Regarding to the fact that these substrates are formed only by a separate layer of nanofibers, their insufficient mechanical strength in all stress directions is their disadvantage that considerably complicates not only the application and the cultivation of the cells,, but especially any handling of these substrates and so also their practical use. Another disadvantage is that the separate nanofiber layer is not stable in shape and dimensions while using in a humid environment, and just after getting wet it shrinks spontaneously and/or rolls up.

To remove these disadvantages the entombment of the nanofiber layer was designed on the bearing stratum formed e.g. by a membrane from hollow fibers according to CN 101050418, by a woven textile according to CN 101264349 or by a microporous layer according to WO 2010136983. These solutions remove disadvantages of the substrate formed by a separate nanofiber layer partially though, nevertheless they bring other disadvantages. The most substantial disadvantages thereof are especially impossibility to watch and check the cells deposited on the nanofiber layer during their cultivation by means of common inverted, i.e. optical microscopes without destruction, as the bearing stratum prevents the light passage. The only possibility in these cases is to stop the cultivation of the deposited cells, to kill them and to examine them subsequently by means of immunohistochemical methods. Nevertheless, only a small sample of substrates can be examined in this way, which is not representative enough.

Another common problem of all substrates containing the nanofiber layer known up to now is the fact, that due to the stateoftheart in the field of nanofiber production, wherein the nanofibers are formed mostly by means of an equipment comprising a spinning electrode formed by a nozzle or capillary, these substrates can be produced only as single parts or in very small series essentially, whereas individual pieces differ in the uniformity of the nanofiber layer, in its planar weight and in other parameters relatively considerably from one another. Due to the fact that these substrates cannot be produced with a repeatable quality and version it is not possible to test, to compare them additionally or to put them to prescribed trials e.g. to use them as medicaments or to develop and improve them further. An objective of the present invention is to design a cell culture substrate having appropriate mechanical properties for handling and practical use, appropriate biological properties for cell cultivation, and which enables at the same time to watch the quality and/or amount of these cells during cultivation. Furthermore, the objective of the invention is to design a method for producing such substrate that enables industrial production thereof.

Detailed description of the invention

The objective of the invention will be achieved by the substrate according to the invention, the subject matter of which consists in that the nanofiber layer from a biologically compatible polymer is deposited on a bearing stratum formed by a reticule from a biologically compatible material, whereas the nanofiber layer covers and fills up the space of the reticule meshes. The reticule provides the substrate with required mechanical properties, while the nanofiber layer represents an environment suitable for placement and subsequent cultivation of cells essentially of any type. Due to this arrangement the deposited cells can be observed during the cultivation by means of common inverted microscopes without destruction as the reticule does not prevent the light from passage to the nanofiber layer.

In case that a sufficiently tight and resistant coupling is not reached by means of surface (intermolecular) forces during placing the nanofiber layer on the bearing stratum, both layers can be interconnected by a suitable biologically compatible binding agent.

In a preferred embodiment of the substrate the bearing stratum is formed by a monofilament warp knitted fabric from fair polyester silk, which provides the substrate with suitable mechanical properties but which at the same time does not limit its elasticity and shape adaptability in any way.

To be able to place and cultivate two different and mutually separated cell types on one substrate or to achieve a double amount of cells of one type, in one embodiment the nanofiber layer from a biologically compatible polymer is deposited from both of the substrate sides. In addition, the connection of both nanofiber layers in the spaces of the meshes of the bearing stratum by means of surface forces increases the strength and resistance of their placement on the bearing layer significantly.

Suitable nanofiber materials are preferably biologically compatible polymers from the group consisting of gelatin, chitosan, polyvinyl alcohol (PVA), polycaprolactone (PCL), polylactid (PLA), polyamide (PA), polyurethane (PUR), poly- (lactide-co-glycolic) acid, their mixture or their copolymers because they are relatively easily spinnable polymers. The nanofiber layer may contain at least one active substance when necessary that prevents the undesirable microorganisms from penetrating from outside into the substrate or its surroundings, such as e.g. antibiotic, antiseptic, silver, or a substance supporting the cultivation and/or activity of cells deposited on the nanofiber layer as e.g. a growth factor, vitamin, vegetable extract, hormone, corticoid, cytokine, etc.

To prevent the substrate, especially the nanofiber layer, from mechanical damages, it is preferred to cover this layer with a protective layer before using the substrate. It can be e.g. a layer of a polyethylene (PE) foil or a non-woven textile of the spunbond type.

Based on the selection of the substrate, e.g. the cells from the group of keratinocytes, fibroblasts, epithelial cells, endothelial cells, osteocytes, mesenchymal stem cells, or combinations thereof can be deposited on the nanofiber layer of said substrate and cultivated.

The objective of the invention will be further achieved by the method for producing the substrate, in which the nanofiber layer from a biologically compatible polymer is deposited on the bearing stratum formed by a reticule from a biologically compatible material by means of an electrostatic spinning of the polymer system in an electric field formed between a collecting electrode and an oblong spinning electrode arranged against said electrode, whereas the nanofiber layer covers and fills up also the reticule meshes. At the same time this guarantees not only a high uniformity of the deposited nanofiber layer but it enables to produce the substrate according to the invention in industrial scale. To increase the uniformity of the nanofiber layer more, especially while using the bearing stratum with a low electric conductivity, it is suitable to increase its electric conductivity before placing the nanofibers by means of application of a conductivity increasing agent on the surface and/ or in the structure of said stratum. A suitable conductivity increasing agent is e.g. an aqueous solution of NaCI, Et₄NBr or betaine.

Optionally a biologically compatible binding agent can be applied on the bearing stratum before and/or during depositing the nanofiber layer and/or after depositing the nanofiber layer, which assures their tight and resistant coupling Then a suitable bearing stratum is formed by a monofilament warp knitted fabric from fair polyester silk. Brief description of the figures

In Fig.1 the attached drawing illustrates a photo of mutually separated layers of the cell culture substrate according to the invention in the embodiment wherein the bearing stratum is formed by a monofilament warp knitted fabric and the nanofiber layer is covered by the protective layer formed by a non-woven textile of the spunbond type, and a section through the substrate with layers deposited on each other in Fig.2.

Examples The cell culture substrate according to the invention contains a nanofiber layer from a biologically compatible polymer deposited on a bearing stratum wherein the bearing stratum is formed by a reticule, whereas said nanofiber layer fills up the spaces of its meshes. The polymer nanofiber layer is formed from a biologically compatible spinnable polymer, e.g. from gelatin, chitosan, polyvinyl alcohol (PVA), polycaprolactone (PCL), polylactide (PLA), polyamide (PA), polyurethane (PUR), poly-(lactide-co-glycolic) acid (PLGA), etc., their mixture or their copolymers, and due to its structure, which in fact reproduces the intercellular structure of a live organism, it represents a suitable environment to cultivate both human and animal cells. Its planar weight before placing the cells is preferably of about 0.03 up to 5 g/m², when it supports already these cells sufficiently and is relatively resistant to abrasion or rupture, but it is still permeable for light so that it enables to watch the amount and/or quality of cells deposited during their cultivation by means of common inverted (optical, light) microscopes. Optionally the planar weight of the polymer nanofiber layer can be even bigger, but its permeability for light decreases considerably in this way.

If required the polymer nanofiber layer may contain at least one substance, which stimulates the cultivation and/or activity of the cells deposited on it, as e.g. a growth factor, vitamin, vegetable extract, hormone, corticoid, cytokine or another substance preventing the undesired microorganisms from penetrating from outside into the substrate or its surroundings, as e.g. antibiotic, antiseptic, silver etc. Then this substance is deposited in the polymer nanofiber layer as an integral part of nanofibers and/or as nanoparticles and/or microparticles, which are fixed in the nanofibers and get out penetrate on their surface, and/or as nanoparticles and/or nanofibers deposited free on and/or among its nanofibers and/or joined to them by means of a physical and/or chemical bond, and/or as its impregnation, and/or a layer and/or film on its surface. To implement this substance directly in the polymer nanofibers the substance and/or its antecedent is, e.g. according to the procedure described in EP 1869232, added or fully or partially solved in a liquid solution or a polymer melt, of which polymer nanofibers are formed subsequently by means of the electrostatic spinning.

The substrate bearing stratum is formed by a reticule from a biologically compatible material. The specific material as well as the reticule parameters are chosen according to the intended use of the substrate and so the reticule can be formed e.g. from metal, plastic or textile or textile fibers for different applications, and can be formed as regular or irregular one. Then this bearing stratum provides the substrate with required mechanical properties, it supports and stiffens the polymer nanofiber layer, whereby it prevents from its self-convolution or shrinkage if getting wet, and at the same time it enables the light to pass through in this layer by means of its meshes and to observe the cells deposited on it. In addition to it, in the embodiments, wherein the bearing stratum is formed by a reticule from textile fibers, in principle the stratum does not limit the elasticity and shape adaptability of the substrate in any way, and so it does not limit possibilities of its use. The reticule mesh diameter is preferably of 0.5 mm up to 25 mm, wherein the reticule does not hinder the light from passage in any way, nevertheless the polymer nanofiber layer is stiffened also in the space of its meshes. The reticule mesh diameter can be bigger in some applications, but in such cases the polymer nanofiber layer is susceptible to a rupture or a break due to its relatively small area and minimum mechanical properties especially in the space of reticule meshes.

If the cultivation of two different and mutually separated cell types is required on one substrate or when a double amount of cells of one type shall be cultivated, the bearing stratum of the substrate has a polymer nanofiber layer from a biologically compatible polymer on each of its sides. In this way the strength and resistance of the entombment of both polymer nanofiber layers increase on the bearing stratum as these layers cling together in the space of their meshes and due to their large surface they join by means of relative big surface (intercellular) forces.

To protect the polymer nanofiber layer from mechanical damages, the substrate preferably has a protective layer at least on one of its sides, which is formed e.g. by a transparent polyester (PE) foil, a non-woven textile layer of the spunbond type, etc. This protective layer fulfills its function especially while stocking the substrate and handling it and it is removed before depositing the cells on the polymer nanofiber layer or before the substrate application.

The enclosed drawing contains a photo of the cell culture substrate in Fig.1 and 2 according to the invention in the version, when its bearing stratum 1 is formed by a monofilament warp knitted fabric. Specifically the fabric is a warp knitted fabric CHS 100 formed by fair polyester silk, which was approved by the Institut pro testovani a certifikaci, a.s., Zlin, CR, in the Czech Republic to be used as a net for surgery purposes. Vyzkumny ustav pletafsky, a.s., Brno, Czech Republic, is its producer. This warp knitted fabric has meshes sized 1 mm, the mean value of its thickness is of 0.34 mm, and its tear strength in the warp direction is of 230 N/width of 5 cm. The structure and material of the knitted fabric enable a reverse deformation at the relative extension up to 5 %. The polymer fabric layer 2 in this version is formed by nanofibers of poiycaprolactone (PLC) having the molecular weight of 100 000, the supplier of which is the company Scientific Polymer Products, Inc., Ontario, USA, under the catalogue number 25 1031. Its planar weight is of 0.3 g/m². The substrate formed in this way is covered by a protective layer 3 from a non-woven textile of the spunbond type from the side of the bearing stratum 1. To spread out and cultivate the cells on the substrate according to the invention equally in the all surface of it also the polymer nanofiber layer 2 must be sufficiently equal. Nowadays the highest uniformity is achieved by creating a polymer nanofiber layer 2 by means of an electrostatic spinning of the polymer system - a liquid solution or a polymer melt with possible addition of at least one active substance, which shall be incorporated in the electric field formed between a collecting electrode and an oblong spinning electrode— e.g. a cylinder (see e.g. EP 1673493) or a string (see e.g. EP 2059630 or EP 2173930), etc. In case that the polymer nanofibers are deposited on an electrically non-conductive base or on a base with a low electrical conductivity during the electrostatic spinning, it is further advantageous to get a higher uniformity of their layer, if the electrical conductivity of this base increases (see e.g. WO 2007054039). Due to it, it is possible to lead the electric charge applied on it by means of loaded polymer nanofibers away from the base, so that it is not concentrated and does not repel next polymer nanofibers. Either directly the bearing stratum 1 of the substrate or another material can be used as a base, from which the deposited nanofiber layer 2 is taken off and transferred on the bearing stratum ±.

In producing the substrate in Fig.1 and Fig.2 the bearing stratum formed by a monofilament warp knitted fabric is used as a base for the polymer nanofiber layer 2, whereas said layer is deposited or led in the electric field between the collecting electrode and the spinning electrode. Regarding to its negligible electrical conductivity its electrical conductivity increases before laying the polymer nanofiber layer 2 preferably to the value of 5.3 x 10¹⁰Ω by applying a suitable conductivity increasing agent, e.g. aqueous solution of NaCI, EtiNBr or Betain, on its surface and/or in its internal structure. During the electrostatic spinning the polymer nanofibers are deposited preferably on the warp knitted fibers, which create a physical barrier in their motion to the collecting electrode, but they fill up and cover also the spaces of their meshes step-by-step. In so doing they join together with the warp knitted fibers relatively firmly due to their large surface and so also big surface (intracellular) forces, and eventually also due to the impact on it in a not totally solidified state. Nevertheless when the connection formed in this way is not sufficient to use the intended substrate, it is possible to apply a suitable biologically compatible bond on the bearing stratum Λ before and/or during and/or after application of the polymer nanofiber layer 2, eventually to interconnect both layers in other suitable way, e.g. according to CZ PV 2010-373.

The substrate according to the invention is either produced directly in a required size or shape or a textile composite is prepared, which contains a bearing stratum 1 and a polymer nanofiber layer 2 e.g. as a band, from which individual substrates are cut out, cut or separated in other way in the required size and shape.

Due to a long-term stable and equal process of the electrostatic spinning while using the technology Nanospider™ of the company Elmarco it is possible to prepare cell culture substrates by means of these both procedures in contrast to the state-of-the-date solutions repeatedly in the same quality, version and with the same planar weight of the polymer nanofiber layer 2. Due to it, it is not only possible to produce these substrates industrially or to put them to trials for being used as medicament, but also to compare their different, variants and develop them further.

The substrate according to the invention can be used to cover flesh wounds, whereas it is profitable if there are keratinocytes (i.e. main epidermal cells), fibroblasts (fibrous tissue cells), mesenchymal stem cells, etc., eventually a combination of these cells, on its nanofiber layer. Besides it is possible to use this substrate also as an implant to be implanted in the recipient^'s body, whereas in this case it is advantageous, if there are osteocytes (elementary cells of bones), endothelial cells, mesenchymal stem cells, etc., eventually their combination, on its nanofiber layer, whereas these cells can be both autologous, when the implant recipient is their donor, and allogeneic, when another person is their donor. Besides, the substrate can be used also without cells deposited before, when it shapes a space to be settled by the patient's cells. Before its application and eventually before laying the cells it is necessary to sterilize the substrate in a convenient way, which does not evoke material degradation of the bearing stratum 1 and/or of the nanofiber layer 2 and/or active substances in it, namely e.g. by means of the UV radiation, gamma radiation, eventually in other way. In the substrate embodiment according to the invention with applied cells the cells are applied on the polymer nanofiber layer 2 e.g. as their suspension in a nutrient solution of a convenient type, which is instilled by means of a pipette equally on the surface of the polymer nanofiber layer 2. After depositing these cells cling to the polymer nanofibers and copy the structure of the nanofiber layer 1 , whereas they form one or more layers according to their nature and way of cultivation. Neither the type of deposited cells nor the concentration of their layer is limited by any substrate parameters, but only by its intended use. At the same time the material used assure that during the cultivation and while using the substrate no substances release, which would affect the cultivation of cells deposited, with the exception of the substances targeted implemented in the polymer nanofiber layer 2, or which would encumber the patient's organisms. Before the substrate application it is advantageous when the cells deposited on the polymer nanofiber layer 2 are cultivated in the required culture medium with defined conditions, which supports and/or verifies their viability after the substrate is applied. The cultivation duration is chosen according to the fact, whether the intended use of the substrate requires further reproduction of cells on the polymer nanofiber layer 2 or not. The culture medium matches the type of cells used advantageously, whereas several different media may be used subsequently. Before using the substrate according to the invention it is advantageous then if the surplus culture medium/ media drain away.

Claims

1 CLAIMS

1. The cell culture substrate containing a nanofiber layer (2) from a biologically compatible polymer deposited on a bearing stratum (1), wherein the bearing stratum (1) is formed by a reticule from a biologically compatible material and the nanofiber layer (2) fills up the space of meshes of said reticule.

2. The substrate of claim 1 , wherein the bearing stratum (1) is connected with the nanofiber layer (2) by means of a biologically compatible binding agent.

3. The substrate of claim 1 or 2, wherein the bearing stratum (1) is formed by a monofilament warp knitted fabric from fair polyester silk.

4. The substrate of any of claims 1 - 3, wherein the nanofiber layer (2) from a biologically compatible material is deposited on both sides of the bearing stratum (1).

5. The substrate of any of claims 1 - 4, wherein the nanofiber layer (2) is formed from a polymer selected from the group of gelatin, chitosan, polyvinyl alcohol (PVA), polycaprolactone (PCL), polylactide (PLA), polyamide (PA), polyurethane (PUR), poly-(lactide-co-glycolic) acid, and mixtures or copolymers thereof.

6. The substrate of any of claims 1 - 5, wherein the nanofiber layer (2) contains an active substance at least from the group of antibiotics, antiseptics, silver growth factors, vitamins, plant extracts, hormones, corticoids and cytokines.

7. The substrate of any of the preceding claims, wherein the nanofiber layer (2) is covered with a protective layer (3) from outside.

8. The substrate of claim 7, wherein the protective layer (3) is formed by a polyethylene foil or a non-woven textile of the spunbond type. 2

9. The substrate of any of the preceding claims, wherein the cells deposited on the nanofiber layer (2) are selected from the group of keratinocytes, fibroblasts, epithelial cells, endothelial cells, osteocytes, mesenchymal stem cells, or combinations thereof.

10. A method for producing of the cell culture substrate of any of the preceding claims, wherein the nanofiber layer (2) from a biologically compatible material is deposited on a bearing stratum (1) formed by a reticule from a biologically compatible material by means of an electrostatic spinning of the polymer system in an electric field formed between a collecting electrode and an oblong spinning electrode arranged against said electrode, wherein the nanofiber layer (2) being deposited covers also the reticule meshes.

1 . The method of claim 10, wherein the electric conductivity of the bearing stratum (1) is increased before the application of fibers by means of applicating a conductivity increasing agent on the surface and/or into the structure of said stratum (1).

12. The method of claim 11 , wherein the conductivity increasing agent is an aqueous solution of NaCI, Et^NBr or betaine.

13. The method of any of claims 9 - 12, wherein a biologically compatible binding agent is applied on the bearing stratum (1) before and/or during depositing the nanofiber layer (2) and/or after depositing the nanofiber layer (2).

14. The process of any of claims 9 - 13, wherein the bearing stratum (1) is formed by a monofilament warp knitted fabric from fair polyester silk.