Biocompatible three-dimensional network and use thereof as a cell support

Abstract

An infusible three-dimensional network of crosslinked acrylic-type polymer fibers, where the diameter of the fibers is between 0.1 and 1.5 ?m, the size of the interstices between the fibers is between 0.1 and 50 ?m.sup.2 and the stiffness of the network includes an elastic modulus between 0.01 and 10,000 kPa.

Claims

1. An infusible three-dimensional network of crosslinked acrylic-type polymer fibers, wherein: the diameter of said fibers is comprised between 0.1 and 1.5 ?m; the size of the interstices between said fibers is comprised between 0.1 and 50 ?m.sup.2; and the stiffness of said network includes an elastic modulus comprised between 0.01 and 10,000 kPa.

2. The three-dimensional network according to claim 1, wherein: the polymer of said fibers is obtained by thermal crosslinking of polyacrylonitrile (PAN), and the stiffness of the network includes an elastic modulus between 0.01 and 10,000 kPa.

3. The three-dimensional network according to claim 2, wherein: the polymer of said fibers is obtained by thermal crosslinking of PAN; said crosslinked polymer comprises nanofillers, at a concentration comprised between 0.00001 and 5% said percentage being expressed in weight percent of the polymer solution before crosslinking; the stiffness of the network includes an elastic modulus between 0.1 and 1,000 kPa; and the surface of the fibers is coated with laminin.

4. A process for preparing a three-dimensional network of crosslinked acrylic-type polymer fibers according to claim 3, said process comprising the following successive steps: a) a step of synthesizing said network by electrospinning of a PAN solution the concentration of which is comprised between 8 and 12%, expressed in weight percent of said PAN solution, in order to obtain a three-dimensional network of fibers; and b) a step of heat treatment under oxidizing atmosphere and at a temperature comprised between 40? C. and 400? C. of the three-dimensional network of fibers obtained during step a); wherein in said step of synthesis by electrospinning: the PAN solution is extruded from a needle the displacement amplitude of which is comprised between 30 and 50 mm, and the displacement rate of which is comprised between 2 and 10 mm/second; the distance between the polymer source and the collecting electrode is comprised between 1 and 50 cm, and the electrical field applied in the extrusion field is comprised between 16 and 24 kV; and the flow rate of the polymer solution during supply of the syringe is comprised between 0.5 and 8.6 mL/h.

5. The process according to claim 4, wherein the PAN solution is extruded onto a rotating collecting electrode, with a diameter comprised between 12 and 18 cm, the speed of rotation of said electrode being comprised between 1 and 100,000 g.

6. The process according to claim 4, wherein said polymer solution comprises carbon nanotubes at a concentration comprised between 0.00001 and 5% expressed in weight percent of said polymer solution before crosslinking.

7. The process according to claim 4, wherein said heat treatment step is followed by a step of treating the surface of said fibers, comprising bringing said network into the presence of a solution of at least one protein of the extracellular matrix or hyaluronic acid.

8. A device for cell support, comprising at least one network according to claim 1.

9. The infusible three-dimensional network of crosslinked polymer fibers according to claim 1, wherein the diameter of said fibers is comprised between 0.3 and 1 ?m.

10. The infusible three-dimensional network of crosslinked polymer fibers according to claim 1, wherein the size of the interstices between said fibers is comprised between 0.5 and 10 ?m.sup.2.

11. The infusible three-dimensional network of crosslinked polymer fibers according to claim 1, wherein the stiffness of said network includes an elastic modulus comprised between 0.1 and 10,000 kPa.

12. The three-dimensional network according to claim 1, wherein the surface of the fibers is coated with at least one protein of the extracellular matrix.

13. The three-dimensional network according to claim 1, wherein the stiffness of the network includes an elastic modulus between 0.1 and 10,000 kPa.

14. The three-dimensional network according to claim 2, wherein said crosslinked polymer comprises nanofillers are carbon nanotubes.

15. The process for preparing a three-dimensional network of crosslinked polymer fibers according to claim 3, wherein in step a) the PAN solution is at a concentration of 10%, expressed in weight percent of said PAN solution.

16. The process for preparing a three-dimensional network of crosslinked polymer fibers according to claim 3, wherein in step b) said heat treatment under oxidizing atmosphere is at a temperature comprised between 200? C. and 300? C.

17. The process according to claim 4, wherein the PAN solution is extruded onto a drum.

18. The process according to claim 4, wherein said polymer solution comprises carbon nanotubes at a concentration comprised between 0.00001 and 1%.

19. The process according to claim 4, wherein said heat treatment step is followed by a step of treating the surface of said fibers, comprising bringing said network into the presence of a solution of at least one protein of the extracellular matrix, said bringing it into the presence being preceded by a prior treatment of the surface of the fibers of said network.

20. The process according to claim 4, wherein said at least one protein of the extracellular matrix is selected from: laminin, fibronectin, vitronectin and collagen.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIGS. 1A to 1C illustrate the physical characterization of a 3D network of fibres according to the invention, according to Example 1: FIG. 1A: scanning electron microscopy images (scale bar: 50 ?m) of aligned (left-hand image) or non-aligned (right-hand image) PAN fibres; FIG. 1B: immunostaining image showing laminin deposits on the fibres (scale bar: 20 ?m), FIG. 1C: 3D reconstruction from a z-stack corresponding to the acquisition of the image in FIG. 1B, the discontinuous deposits of laminin on the fibres are indicated by arrows (scale bar: 20 ?m).

(2) FIGS. 2A to 2F illustrate the adherence and the penetration of the neurospheres (NSs) within the network of fibres, according to Example 2: FIG. 2A: image showing the adherence and the penetration of the neurospheres in the 3D network (scale bar: 500 ?m), at the moment of deposition of a neurosphere containing 5,000 cells; FIG. 2B: image showing the neurospheres of Gli4 cells 5 hours after deposition on the network of fibres (scale bar: 200 ?m); FIG. 2C: image showing the neurospheres of Gli4 cells 6 days after deposition on the network of fibres (scale bar: 200 ?m), FIG. 2D: side view of the distribution of the migratory Gli4 cells, moving away from the neurospheres and at a depth in the network of fibres (scale bar: 20 ?m), FIG. 2E: immunostaining image showing the adhesion of the Gli4 cells to the fibres (scale bar: 10 ?m), the arrows indicate the Gli4 cells; FIG. 2F: 3D reconstruction from a z-stack corresponding to the acquisition of the image of the Gli4 cells on the fibres (FIG. 2E).

(3) FIGS. 3A to 3D illustrate the individual or collective migration of cells deposited on a 3D network of fibres, according to Example 2, after 6 days of culture under differentiation conditions, in the absence (FIGS. 3A and 3B) or in the presence (FIGS. 3C and 3D) of laminin; FIGS. 3A and 3C: visualization of the Gli4 cells by scanning electron microscopy (scale bar: 500 ?m), FIGS. 3B and 3D: visualization of the Gli4 cells after marking the actin cytoskeleton with phalloidin green (scale bar: 20 ?m). In FIGS. 3A and 3B, the arrows indicate the lamellipodial extensions of the peripheral cells and the round cells situated at the centre of a group of cells which are migrating collectively, in the absence of laminin on the fibres. In FIGS. 3C and 3D, the arrows indicate the cells migrating individually, in the presence of laminin on the fibres.

(4) FIG. 4 illustrates the number of collective migrations (y-axis) quantified in the presence (left-hand histogram) or in the absence (right-hand histogram) of laminin on the aligned fibres, the clusters formed by at least 2 cells and physically separated from the neurospheres are considered as a collective migration, the results are representative of at least 3 different experiments, ****p<0.0001 (Student's t-test).

(5) FIGS. 5A to 5D illustrate the direction of the migration of the cells on aligned or non-aligned fibres, according to Example 2, after 6 days of culture under differentiation conditions on a 3D network of fibres: the actin skeleton was stained with phalloidin and the nucleus with Hoechst 3342 stain (scale bar 200 ?m), the direction of the fibres is indicated by the arrows, FIGS. 5A and 5C: in the absence of laminin, 5B and 5D: in the presence of laminin, FIGS. 5A and 5B: non-aligned fibres, FIGS. 5C and 5D: aligned fibres.

(6) FIG. 6 represents the quantification (y-axis) of the total number of Gli4 cells migrating in a parallel (bars A and C) or perpendicular (bars B and D) direction of the aligned fibres, in the absence (bars A and B) or presence (bars C and D) of laminin. The number of cells is counted at a distance comprised between 200 ?m and 2,000 ?m of the outer edge of the neurospheres. This field was chosen to exclude migration due to expansive growth of the cells near to the neurospheres. Between bars A and B: *p<0.1, between bars C and D: **p<0.01 (Student's t-test). All the results are representative of two independent experiments.

(7) FIGS. 7A to 7C represent scanning electron microscopy images (scale bar: 200 ?m) of crosslinked polymer fibres, said polymer is obtained by crosslinking of 5% PMMA+5% PAN (FIG. 7A), 5% PMMA+10% PAN (FIG. 7B) or 20% PMMA (FIG. 7C) solutions.

(8) FIG. 8 (MTT assay) is a histogram illustrating on the y-axis the absorbance of formazan produced by reduction of MTT tetrazolium salt (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) by living and metabolically active cells on the polymer fibres, and on the x-axis, from left to right, 10% PAN; 5% PMMA +5% PAN; 5% PMMA+10% PAN and 20% PMMA.

(9) FIGS. 9A to 9C represent the direction of the migration of Gli4 cells on the 10% PAN (FIG. 9A), 5% PMMA+5% PAN (FIG. 9B) or 20% PMMA (FIG. 9C) fibres after 5 days of culture under differentiation conditions on a 3D network of fibres: the actin skeleton was stained with phalloidin and the nucleus with Hoechst 3342 stain.

(10) FIG. 10 represents a histogram indicating, on the y-axis, the surface area of the migration area (in pmt) of the Gli4 cells deposited on the polymer fibres, and on the x-axis, from left to right, of 10% PAN, 5% PAN+5% PMMA, or 20% PMMA.

EXAMPLES

Example 1: Synthesis of a 3D Network of Polyacrylonitrile Fibres and Physical Characterization

1.1. Materials and Methods

Synthesis of the 3D Network of PAN Fibres

(11) A 10% by weight solution of polyacrylonitrile (Sigma Aldrich) is prepared in dimethylformamide. For 10 g of 10% PAN solution: weighed out to a value of 1 g PAN for 9 g DMF, followed by heating to 75? C. under stirring until complete dissolution of the PAN in the DMF.

(12) This solution is formed by electrospinning. The collector is a rotating collector 15 cm in diameter. A 20-kV electrical field is applied between the needle distributing the polymer and the collector. 3D network with aligned fibres: The needle is located at a distance of 15 cm from the rotating collector. The collector around which the fibres are wound has a speed of rotation of 2,000 rpm making it possible to obtain a network the fibres of which are aligned. 3D network with fibres called tangled fibres: The needle is located at a distance of 15 cm from a flat, non-rotating collector.

(13) In both cases, the polymer is conveyed to the needle by a syringe pump with a pushing force of 2.4 ml/h in this case. During electrospinning, the needle performs displacements with an amplitude of 40 mm at a displacement rate of 5 mm/sec. The electrospinning lasts at least 20 minutes. The tissue of fibres then undergoes a heat treatment before being sterilized.

Heat Treatment of the 3D Network

(14) The network obtained by electrospinning is heat-treated under air at a temperature of 250? C., with a heating rate of 120? C./hour, a plateau at 250? C. for 2 hours, the cooling rate is 300? C./h. During treatment, the colour of the network passes from white to brown, reflecting the aromatization of the fibres.

(15) Before use, the network undergoes sterilization in 70? ethanol or in an autoclave.

Synthesis of a 3D Network of PAN Fibres and Carbon Nanotubes

(16) During production of fibres containing multi-walled carbon nanotubes (Nanocyl?, 95% purity, product reference NC3101 or NC3151) and to increase the dispersion of the MWCTs in the PAN solution, the surfactant triton 100? is added at 2.5% of the final solution for 0.05% WT of MWCTs.

(17) For a preparation of a 0.05% WT PAN solution, the following steps are carried out:

(18) a) Preparation of 10 g of a 0.05% solution (weight percent) of MWCTs in 10% PAN: Weighed out to a value of 1 g PAN, 0.05 g MWCTs and 8.95 g DMF, Heating to 75? C. under stirring until complete dissolution of the PAN in the DMF, Sonication of the solution

(19) b) Producing a 10% PAN solution according to section 1.1.1.

(20) c) Mixing 1 g of 10% PAN +0.05% WT (weight percent) MWCT solution for 0.75 g Triton 100? and 8.25 g 10% PAN solution

(21) d) Sonication of the mixed solution. The polymer solution is then treated by electrospinning, then the network obtained is heat-treated as indicated above.

(22) By dilution with 10% PAN, a network constituted by PAN fibres and 0.0016% (weight percent) MWCTs is synthesized.

Coating the Surface of the Fibres with Laminin

(23) For their functionalization by laminin, the culture dishes and the 3D network of biocompatible electrospun fibres are incubated overnight with 25 ?g/m poly-D-lysine in borate buffer at 37%, then washed twice in sterile water and incubated with 5.2 ?g/ml laminin in sterile water for 4 h at 37? C. It is washed twice with water.

Inclusion of the Network of Fibres in Wells

(24) This inclusion can be carried out by cutting them out using a punch with a size corresponding to the size of the well intended to accommodate the fibres. The cut-out fibres are then deposited at the bottom of the well and ready for use.

(25) It is also possible to position the cut-out fibres in inserts, of a size corresponding to the size of the wells, making it possible to make it easier for them to enter and be removed from the wells or making it possible to produce concentration gradients on both sides of the fibres.

1.2. Results

Characterization of a 3D Network of PAN Fibres

(26) The diameter of the fibres, estimated at 0.68+/?0.28 ?m, is similar to the diameter of the axons present in the corpus callosum, estimated at 0.64+/?0.42 ?m (Liewald et al., 2014, Biol. Cybern. 108, 541-557). Depending on the electrospinning process implemented, the network of fibres generated comprises fibres aligned or organized randomly (FIG. 1A). The network of aligned fibres has a general orientation rather than fibres that are strictly parallel.

(27) Evaluation of the mechanical properties is carried out by means of atomic force microscopy. The elasticity of the network constituted by PAN fibres with no carbon nanotubes added is characterized by an elastic modulus of 0.16 kPa. The experiments are carried out in contact mode on an Asylum MFP-3D device (Asylum Research, Santa Barbara, California, USA) mounted on an inverted microscope of the Olympus brand, using a silicon nitride triangular cantilever system (MLCTAUHW, Veeco) and with a stiffness constant of 10 pN/nm. The measurements are conducted by applying a maximum force of 1 nN. The stiffness is measured as an average value over all of the image recorded. For a network constituted by PAN fibres and 0.0016% (weight percent) MWCTs, the elasticity is characterized by an elastic modulus of 62 kPa.

(28) For a network constituted by PAN fibres and 0.05% (weight percent) MWCTs, the elasticity is characterized by an elastic modulus of 250 kPa.

(29) The interstices are such that the mesh of fibres is permissive but nevertheless constrains the cells to deform in order to enter into the matrix. The interstices are comprised between 0.5 ?m.sup.2 and 9 ?m.sup.2, which corresponds to a confinement location of the cells.

Fluorescence Properties

(30) When the network is subjected to a light energy, the fibres appear as fluorescent (FIG. 1B). They are autofluorescent in the green (488 nm) and red (594 nm), and to a lesser extent in the blue (350 nm) and in the infrared (647 nm). The functionalization of the fibres with laminin makes it possible to modify the microenvironment.

Characterization of the Network Coated with Laminin

(31) The laminin deposits are distributed discontinuously on the fibres (FIG. 1C).

Conclusion

(32) The 3D network thus obtained is non-cytotoxic, and can constitute a 3D support for cells, allowing their proliferation, their migration and their differentiation.

Example 2: Characterization of the Migration of Glioblastoma Cells

2.1. Materials and Methods

Culture of Glioblastoma Cells

(33) Obtaining and isolating glioblastoma cells (GBM) as well as the cultures are carried out using the protocol drawn up by Guichet et al. (Guichet et al., 2013, Glia, 61, 225-239), according to Dromard et al. (Dromard et al., 2008, J. Neurosci. Res. 86, 1916-1926) for non-adherent neurospheres. The Gli4 and GliT cells are primary glioblastoma cultures derived from two different patients. These GBM cells are cultivated under two different conditions in DMEM/F12 medium, supplemented with glucose, glutamine, insulin, N2 and ciproflaxin.

(34) Under non-adherent conditions, called proliferation conditions, the culture dishes are pre-treated with poly-2 hydroxyethyl methacrylate (poly-HEMA, SIGMA), the medium is also supplemented with epidermal growth factor (EGF) and fibroblast growth factor (FGF), gentamicin, heparin, fungizone, fungin and B27. Under these conditions, the GBM cells form neurospheres (NSs) reminiscent of in vitro neural stem cells (NSCs), and express neural progenitors and stem cell markers (nestin, olig2, sox2, etc.), self-renew and propagate tumours in immunocompromised animals. After confluency, the NSs are mechanically dissociated using HBSS (without calcium or magnesium) and reseeded.

(35) Under conditions called differentiation conditions, the DMEM/F12 contains foetal bovine serum (0.5%) without growth factor and without heparin. In this case, the GBM cells are cultivated adhering to a flat (2D) surface or on a 3D network prepared according to Example 1, without poly-HEMA.

Culture of Cells on the 3D Network

(36) Before deposition of the cells, the 3D networks of biocompatible electrospun fibres (denoted fibres), prepared as described in Example 1, are sterilized in 70% alcohol, autoclaved and functionalized, or not, with poly-D-lysine/laminin, as described in Example 1. For the functionalization, the 2D culture dishes and the fibres are incubated overnight with 25 ?g/ml poly-D-lysine in borate buffer at 37%, then washed twice in sterile water and incubated with 5.2 ?g/ml laminin in sterile water for 4 h at 37? C. The dishes and the fibres are washed twice in sterile water and the neurospheres, or the dissociated cells, of Gli4 and GliT are seeded thereon. The cells are kept under culture conditions for 6 days at 37? C. and 5% CO.sub.2.

(37) In order to obtain neurospheres, 96-well Corning microplates are used (Corning 7007). The dissociated GBM neurospheres are cultivated under proliferation conditions, and seeded at 5,000 cells per well. The cells sediment and the NSs are harvested 24 hours after seeding.

Orthotopic Xenotransplantation of GBM

(38) The Gli4 and GliT cells are enzymatically dissociated using 0.25% trypsin and resuspended in PBS at 0.5?10.sup.5 cells per microliter. 3 microliters (1.5?10.sup.5 cells) are injected into the striatum (1 mm rostral, 2 mm lateral and 2.5 mm depth) of 6-week old female NMRI nude mice (Janvier laboratories) under anaesthesia with isoflurane. A Hamilton syringe connected to a pump is used to inject the cell suspension with a flow of 0.3 ml/min. At the end of the surgery, the remaining cells will be seeded to verify the cell viability. After 3 months, the animals are sacrificed under anaesthesia with pentobarbital and fixed with an intracardiac perfusion of PFA. The brains are recovered and post-fixed overnight in 4% PFA then immersed successively in 7%, 15% and 30% sucrase. Then, these brains are included in OCT (optimal cutting temperature), frozen in liquid nitrogen and preserved at ?20? C. Coronal section of the brain 14 ?m thick are produced in a cryostat.

Cryoselection of the 3D Networks

(39) The 3D networks of fibres are included in an OCT compound. The thickness of the lateral sections is 14 ?m.

Immunofluorescence and 3D Reconstruction

(40) After 6 days of culture, the GSCs cultivated in NF or in dishes are fixed with 4% PFA. The 3D networks, the dishes and the sections of brain are blocked and permeabilized using 0.5% PBS-triton-5% horse serum. The primary antibodies are incubated overnight at 4? C. The secondary antibodies coupled to a fluorochrome are incubated for 2 h at ambient temperature at the dilution of 1/500. These antibodies are: N-cadherin (Abcam ab12221), calpain-2 (Abcam ab155666) and human nuclei (Millipore MAB 1281). The sections are mounted with Fluoromount and dried before observation. The actin cytoskeleton is highlighted with (green) phalloidin and the nuclei with Hoechst 33342. The images are taken with a Z-stack acquisition using Confocal 2 Zeiss LSM 5 Live DUO and Widefield 1-Zeiss Axio Imager Z1/Zen (equipped with an Apotome) microscopes. Imaris X64 8.1.2 software is used for reconstructing 3D images.

(41) The quantifications are carried out with ZEN 2012 software.

(42) The Gli4 cells cultivated on the fibres are fixed with glutaraldehyde 2.5% in PHEM buffer for one hour at ambient temperature then overnight at 4? C. The cells are then dehydrated with alcohol at 70%, 96% and 100% successively, then incubated in HMDS for drying.

Western Blot

(43) The proteins are extracted from cultures on fibres in RIPA buffer (supplemented with phosphatase and protease inhibitors). 20 ?g protein lysate are separated by SDS-PAGE and transferred onto PVDF membranes, which will be subsequently blocked with TBS-0.1% Tween-5% skimmed milk. The primary antibodies used are: Galectin-3 (abcam ab2785), Integrin ?1 (Millipore AB 1952), Integrin ?6 (abcam ab75737), FAK (abcam ab40794), phospho-FAK Y397 (abcam ab 80298), Talin1/2 (abcam ab11188), calpain-2 (abcam ab155666) and GAPDH as control (Millipore MAB374). Horseradish peroxidase coupled with secondary antibodies is incubated for 2 h at ambient temperature. The ChemiDoc XRS+ imager is used for detecting the chemiluminescence. The quantifications of pixels are carried out with Image Lab software.

Characterization of the Cell Migration

(44) The direction and the distance of migration of the Gli4 cells are analysed on aligned or non-aligned fibres, coated or not coated with laminin. At T=0, neurospheres having 5,000 cells are seeded. The number of cells per migration distance is quantified with AXIO-IMAJEUR software. Essentially, concentric arcs are defined from the centre of the sphere at regular intervals and the number of cells present between 2 successive arcs is counted, thus defining a number of cells per migration distance interval. The migration arcs are comprised between 200 and 2,000 ?m from the edge of the neurosphere. This space is chosen to exclude migration associated with the expansion of NS growth. This test is carried out in 2D and 3D.

Statistical Analysis

(45) At least 3 replicates are produced per experiment. The values are expressed as an average +/?SD. The statistical tests are carried out with GraphPad software.

2.2. Results

(46) The aligned fibres constitute a 3D environment for the adhesion and the migration of glioma cells

(47) The network of fibres makes it possible to better understand, characterize and target the cells migrating in the corpus callosum. When they are deposited on a 2D surface and cultivated in the absence of growth factors and in the presence of serum (differentiation medium), the neurospheres (NSs) adhere to the support and the Gli4 cells differentiate themselves and migrate by moving away from the NSs (Guichet et al., Cell death and neuronal differentiation of glioblastoma stem-like cells induced by neurogenic transcription factors, Glia. February; 61(2):225-39, 2013). When they are seeded on the 3D network of fibres and cultivated in the differentiation medium, the Gli4 NSs adhere to the surface and penetrate into the network of fibres (FIG. 2A). The Gli4 cells proliferate and migrate by moving away from the NSs (FIGS. 2A-2C). Analysis of the distribution of the Gli4 cells within the network of fibres shows that the cells migrate deeply inside the network (FIG. 2D). In this environment, the Gli4 cells form cell extensions in different directions in order to attach to several fibres, in a three-dimensional manner (FIGS. 2E and 2F). The Gli4 cells have a diameter of approximately 40 times that of the fibres (20 ?m versus approximately 0.5 ?m) and surround the fibres in order to adhere and migrate (FIGS. 2E and 2F, arrows). This surrounding makes it possible for the Gli4 cells to interact in a ventral, lateral and dorsal manner within the network. In addition, the heterogenous interstices between the fibres are distributed between 0.1 and 10 ?m.sup.2, with a maximum distribution between 1 and 2 ?m.sup.2. These spaces force the cells to deform in order to penetrate into the network of fibres (FIG. 2D), which is reminiscent of the natural cellular confinement observed in vivo (Friedl and Alexander Cancer invasion and the microenvironment: plasticity and reciprocity, Cell, 147, 992-1009, 2011). These results show that the PAN fibres are permissive for the adhesion and the migration of the glioblastoma cells in a 3D microenvironment. Dynamics of the cellular adhesion to the proteins of the extracellular matrix and the focal adhesion of the Gli4 cells with regard to flat (2D) conventional surfaces and aligned (3D) fibres

(48) In order to compare the interactions between the Gli4 cells and the extracellular medium (ECM) and their focal adhesion (AF) on the flat surfaces and in the 3D network of fibres, functionalized or not with laminin (+or ?LN), the expression of integrin ?1, integrin ?6 and galectin-3 was analysed. Between the dishes coated with laminin (PS+LN) and the fibres coated with laminin (NF+LN), the level of expression of galectin-3 and integrin ?1 was increased by 6 and 2.6 times, respectively, and the level of integrin ?6 was reduced by 2.5 times. Between the flat surfaces and the networks not coated with laminin, no difference between the levels of expression of integrins ?1 and ?6 is observed. By immunofluorescence, integrin ?1 is located on the membrane of the Gli4 cells migrating on the 3D network coated with laminin, when it is distributed in the cytoplasm and around the nucleus in the Gli4 cells deposited on the flat surfaces coated with laminin. In addition, on the networks of fibres coated with laminin, the staining of the integrin ?1 is located on the points of attachment with the fibres. In addition, the phosphorylation of the focal adhesion kinase (FAK), a major actor in the transduction of the integrin-mediated signal, increases when the flat surfaces or the 3D network are coated with laminin.

(49) The expression of talin ?, vinculin and calpain-2 in the Gli4 cells on the PS+LN and NF+LN supports was measured. The intact and cleaved forms were detected in the Gli4 cells on the flat surfaces with and without laminin, with the highest rate of cleaving of the talin on the PS+LN support. Conversely, talin ? is not cleaved in the Gli4 cells on fibres, with or without laminin. The level of expression of vinculin does not vary under different conditions. The expression of calpain-2 is reduced by a factor of 2 for the Gli4 cells deposited on the fibres coated with laminin compared with the flat surfaces coated with laminin.

(50) These results show that the adhesion mediated by integrin ?1 and galectin-3 to ECM is increased on the electrospun fibres. The dynamics of the focal adhesion are regulated by the expression of calpain-2 and the cleaving of the talin differs between the flat surface and the 3D network. Adherence of the GliT cells and their interaction with the cells of the extracellular matrix are not improved on the fibres

(51) The GliT cells are another primary line of neuroblastoma cells, less invasive than Gli4 in vivo. GliT and Gli4 differ in the expression of numerous proteins linked with the extracellular matrix, in particular the different expression of galectin-3, integrin ?1 and integrin ?6, in the different systems studied (flat surface or neurofibres, with or without laminin). Adhesion of the GliT cells via galectin-3, integrin ?1 and integrin ?6 is not improved in the presence of the network of fibres. Integrin ?1 and galectin-3 are overexpressed in Gli4 cells in the corpus callosum while calpain-2 is underexpressed in invasive Gli4 and GliT cells in vivo

(52) The results obtained indicate that the Gli4 cells are more invasive than the GliT cells, and that the expression of integrin ?1 and galectin-3 in the Gli4 cells is dependent on the cerebral microenvironment and that the expression of calpain-2 seems to be inversely correlated with the invasive potential of the invasive glioblastoma cells (GICs) in vivo. The Gli4 cells migrate individually or collectively in the presence or in the absence of laminin on aligned PAN fibres.

(53) As observed using fluorescence microscopy and 3D image reconstruction by electron microscopy (FIGS. 3A to 3D), when they are seeded on a 3D network of fibres not coated with laminin, the Gli4 cells migrate collectively forming clusters composed of cells that are strongly combined with one another. Conversely, in the presence of a network of fibres coated with laminin, the Gli4 cells separate from one another and migrate individually. The cells thus adopt two different migration modes, collective mode or individual mode, depending on the functionalization of the fibres. In addition, on the fibres without laminin, the staining of the actin of the cytoskeleton with phalloidin shows that the majority of the cells are round, within the clusters the actin cytoskeleton is continuous between the cells, at the centre of the cell mass the cells are round while on the outside of the cluster they are bipolar and have a lamellipodium. Conversely, on the network of fibres coated with laminin, the Gli4 cells are individual, bipolar, asymmetrical and each has a lamellipodium on the front face. In addition, the cells express N-cadherin on their membrane.

(54) Moreover, the number of collective migration events by neurospheres was quantified, on the 3D network functionalized with laminin or not. To this end, neurospheres of the same size and containing the same number of cells were seeded on the two networks. A significant reduction (****p<0.0001) in the number of collective migrations by neurospheres is observed after the functionalization of the fibres with laminin. The number of units of collective migration per neurosphere decreases from 80 to 20 when the fibres are functionalized. The Gli4 cells respond to a topoinduction signal resulting from the orientation and organization of the biocompatible electrospun fibres.

(55) The direction of the migration of the Gli4 cells on the aligned fibres (AF) or non-aligned fibres (N-AF), functionalized by laminin or not, is compared. The results show that the presence of laminin increases the number of cells migrating outside the NSs, on aligned and non-aligned fibres. On the non-aligned fibres, the Gli4 cells migrate by moving away from the neurospheres in all directions, in the absence or in the presence of laminin. On the aligned fibres, the Gli4 cells migrate predominantly in the direction of the fibres, rather than in the perpendicular direction, and this effect is observed mainly in the presence of laminin. The number of migratory cells, as a function of the distance of migration on the fibres was quantified. The length of the migration region was delimited between the edge of the neurospheres and a distance of 2 mm in the direction of migration. The results show a significant increase in the number of migratory cells on the non-aligned fibres in the presence of laminin (right-hand histogram), in comparison with the non-aligned fibres without laminin (left-hand histogram) (FIG. 4). The addition of laminin increases the number of Gli4 cells migrating in a direction parallel to the aligned fibres, and in a perpendicular direction. In a direction parallel to the fibres and in the presence of laminin, the number of Gli4 cells increases significantly at a distance between 200 and 800 ?m from the edge of the NSs, by comparison with the non-functionalized fibres. The same result is observed in the direction perpendicular to the aligned fibres, in which the number of cells also increases significantly at a distance between 200 and 600 ?m from the edge of the NSs in the presence of laminin.

(56) Conversely, no significant difference is observed in the number of migratory cells near to the edge of the NSs (from 0 to 200 ?m), in parallel with and perpendicular to the fibres, in the presence and in the absence of laminin. The presence of cells adjacent to the NSs results from proliferation more than migration. A significant increase in the number of cells migrating in a direction parallel to the aligned fibres is observed compared with the perpendicular direction, in the absence and in the presence of laminin. In the model described, it is therefore not necessary for the fibres to be aligned in order to create interstitial spaces that are permissive for cell infiltration.

(57) Together, these results show that the presence of laminin increases the migration of Gli4 cells on the aligned and non-aligned fibres. In addition, the orientation of the fibres dictates the direction of the migration of the Gli4 cells. On the non-oriented fibres the Gli4 cells migrate in all directions, while on the aligned fibres they migrate more in a direction parallel to the fibres than in a perpendicular direction.

(58) In conclusion, the 3D matrix of electrospun fibres allows the adhesion of glioblastoma cells and their migration in a 3D microenvironment. This model shows the importance of cell-cell adhesion via N-cadherin in the collective migration process and the role of laminin in the accelerated migration of glioblastoma cells. This matrix of fibres represents an important tool, in particular for studying the role of the extra-cellular matrix in the migration of glioblastoma cells.

Example 3: Synthesis of 3D Networks of Acrylic-Type Polymer FIBRES AND PHYSICAL CHARACTERIZATION

3.1. Materials and Methods

3.1.1. 3D Network of Aligned Fibres of 5% PMMA, 5% PAN Polymer

(59) A 5% by weight solution of polyacrylonitrile (Sigma Aldrich) and 5% poly(methyl methacrylate) (PMMA, Sigma-Aldrich) is prepared in dimethylformamide. For 10 g polymer solution: weighed out to a value of 0.5 g PAN and 0.5 g PMMA for 9 g DMF, followed by heating to 75? C. under stirring until complete dissolution of the polymers in the DMF.

(60) This solution is formed by electrospinning. The collector is a rotating collector 15 cm in diameter. A 20-kV electrical field is applied between the needle distributing the polymer and the collector. The needle is located at a distance of 15 cm from the rotating collector. The collector around which the fibres are wound has a speed of rotation of 2,000 rpm, making it possible to obtain a network the fibres of which are aligned. The polymer is conveyed to the needle by a syringe pump with a pushing force of 3.4 ml/h in this case. During electrospinning, the needle performs displacements with an amplitude of 40 mm at a displacement rate of 5 mm/sec. The electrospinning lasts at least 20 minutes. The tissue of fibres then undergoes a heat treatment before being sterilized.

(61) The network obtained by electrospinning is heat-treated under air at a temperature of 110? C., with a heating rate of 120? C./hour, a plateau at 110? C. for 1 hour, the cooling rate is 300? C./h. During treatment, the colour of the network passes from white to brown, reflecting the aromatization of the fibres. Before use, the network undergoes sterilization in 70? ethanol or in an autoclave.

3.1.2. 3D Network of Fibres of 5% PMMA, 10% PAN Polymer

(62) A 10% by weight solution of polyacrylonitrile (Sigma Aldrich) and 5% poly(methyl methacrylate) (PMMA, Sigma-Aldrich) is prepared in dimethylformamide. For 10 g polymer solution: weighed out to a value of 1.0 g PAN and 0.5 g PMMA for 8.5 g DMF, followed by heating to 75? C. under stirring until complete dissolution of the polymers in the DMF.

(63) This solution is formed by electrospinning. The collector is a rotating collector 15 cm in diameter. A 20-kV electrical field is applied between the needle distributing the polymer and the collector. The needle is located at a distance of 15 cm from the rotating collector. The collector around which the fibres are wound has a speed of rotation of 2,000 rpm, making it possible to obtain a network the fibres of which are aligned. The polymer is conveyed to the needle by a syringe pump with a pushing force of 2.4 ml/h in this case. During electrospinning, the needle performs displacements with an amplitude of 40 mm at a displacement rate of 5 mm/sec. The electrospinning lasts at least 20 minutes. The tissue of fibres then undergoes a heat treatment before being sterilized.

(64) The network obtained by electrospinning is heat-treated under air at a temperature of 110? C., with a heating rate of 120? C./hour, a plateau at 110? C. for 1 hour, the cooling rate is 300? C./h. During treatment, the colour of the network passes from white to brown, reflecting the aromatization of the fibres.

(65) Before use, the network undergoes sterilization in 70? ethanol or in an autoclave.

3.1.3. 3D Network of 20% PMMA Polymer Fibres,+DMF +THF

(66) A 20% by weight solution of poly(methyl methacrylate) (PMMA, Sigma-Aldrich) is prepared in a mixture of dimethylformamide and tetrahydrofuran (THF). For 10 g polymer solution: weighed out to a value of 2.0 g PMMA for 4 g DMF and 4 g THF, followed by stirring until complete dissolution of the polymers in the solvent. This solution is formed by electrospinning. The collector is a rotating collector 15 cm in diameter. A 20-kV electrical field is applied between the needle distributing the polymer and the collector. The needle is located at a distance of 15 cm from the rotating collector. The collector around which the fibres are wound has a speed of rotation of 2,000 rpm, making it possible to obtain a network the fibres of which are aligned. The polymer is conveyed to the needle by a syringe pump with a pushing force of 3.8 ml/h in this case. During electrospinning, the needle performs displacements with an amplitude of 40 mm at a displacement rate of 5 mm/sec. The electrospinning lasts at least 20 minutes. The tissue of fibres then undergoes a heat treatment before being sterilized.

(67) The network obtained by electrospinning is heat-treated under air at a temperature of 110? C., with a heating rate of 120? C./hour, a plateau at 110? C. for 1 hour, the cooling rate is 300? C./h. During treatment, the colour of the network passes from white to brown, reflecting the aromatization of the fibres.

(68) Before use, the network undergoes sterilization in 70? ethanol or in the autoclave.

3.2. Results

Characterization of the 3D Networks of Polymer Fibres

(69) FIGS. 7A to 7C represent the 3D networks according to the invention obtained by crosslinking of the polymer solutions comprising respectively: 5% PMMA, 5% PAN in DMF: FIG. 7A 5% PMMA, 10% PAN in DMF: FIG. 7B 20% PMMA, in DMF+THF: FIG. 7C

(70) The scanning electron microscopy images show that the average diameter of the fibres is 300 nm in the case of the 5% PMMA, 5% PAN mixture, 600 nm in the case of the 5% PMMA, 10% PAN mixture and 1.2 ?m in the case of 20% PMMA.

Example 4: Characterization of the Viability of Glioblastoma Cells

4.1. Materials and Methods

Culture of Gli4 Glioblastoma Cells

Culture of Cells on the 3D Network

(71) Before deposition of the cells, the 3D networks of biocompatible electrospun fibres (denoted fibres), prepared as described in Example 3, are sterilized in autoclaved 70% alcohol. The dishes and the fibres are washed twice in sterile water.

(72) In order to obtain neurospheres, 96-well Corning microplates are used (Corning 7007). The dissociated Gli4 neurospheres are cultivated under proliferation conditions, and seeded at 5,000 cells per well. The cells sediment and the NSs are harvested 24 hours after seeding.

4.2. Results

(73) FIG. 8 (MTT assay) is a histogram illustrating on the y-axis the absorbance of formazan produced by reduction of MTT tetrazolium salt (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) by living and metabolically active cells on the polymer fibres, and on the x-axis, from left to right, 10% PAN; 5% PMMA+5% PAN; 5% PMMA+10% PAN and 20% PMMA. This MTT assay, well known to a person skilled in the art, shows that the 3D network constituted by fibres derived from acrylics or obtained from a mixture of acrylic-type polymers do not show cytotoxicity. This test was carried out on 30,000 dissociated Gli4 cells deposited on each network of fibres. The incubation time is 5 days in differentiation medium.

Example 5: Characterization of the Migration of Glioblastoma CELLS

5.1. Materials and Methods

(74) Before deposition of the cells, the 3D networks of biocompatible electrospun fibres (denoted fibres), prepared as described in Example 3, are sterilized in autoclaved 70% alcohol. The dishes and the fibres are washed twice in sterile water.

(75) In order to obtain neurospheres, 96-well Corning microplates are used (Corning 7007). 7500 Gli4 cells are deposited in each well and cultivated under proliferation conditions so as to form Gli4 neurospheres of the same size. The NSs are harvested 48 hours after seeding and deposited on the different fibres. The neurospheres are left to migrate for 5 days in a differentiation medium before fixing and staining as described in Example 1.

5.2. Results

(76) The fluorescence microscopy images of Gli4 in neurospheres of the same size (7500 cells) seeded on the fibres derived from acrylic or obtained from a mixture of acrylic-type polymers show a permissivity of said networks for cell migration as well as an adherence of the cells. The Gli4 migrate in parallel with the preferred orientation of the network of fibres. The actin cytoskeleton of the Gli4 is marked with phalloidin and the nucleus is marked with Hoechst. The migration areas are then calculated using Zen software, by subtracting the initial area of the neurosphere, making it possible to evaluate the amplitude of the migration on the fibres. The results (FIGS. 9A to 9C, FIG. 10) show the influence of the physical and chemical characteristics of the fibres on the migration kinetics.