Method for preparing a hydrogel matrix by photopolymerization

Abstract

The invention relates to a method for preparing a hydrogel type matrix comprising at least two contiguous zones with distinct stiffnesses, comprising a step to photopolymerize a solution comprising one or several polymerizable compounds and a photopolymerization initiator, by application of light onto the entire solution and at a different intensity in at least two zones of the solution, as a result of which a hydrogel matrix is obtained comprising at least two contiguous zones with distinct stiffnesses, characterized in that the photopolymerization step is done in the presence of a polymerization catalyst.

Claims

1. A method for preparing a hydrogel type matrix comprising at least two contiguous zones with distinct stiffnesses comprising photopolymerizing a solution in the presence of a polymerization catalyst, said solution comprising one or several polymerizable compounds and a photopolymerization initiator, by applying light onto the entire solution and at a different intensity in at least two zones of the solution, thereby obtaining a hydrogel matrix comprising at least two contiguous zones with distinct stiffnesses.

2. A method according to claim 1, wherein the hydrogel type matrix is made from a material selected from the group consisting of: polyacrylamides; polyethyleneglycols containing repetitive patterns derived from polymerization of acrylate or methacrylate compounds; polysaccharides, optionally modified; (co)polymers derived from polymerization of diacrylate and/or (meth)acrylate compounds; polyvinyl alcohols containing repetitive patterns derived from polymerization of acrylate or methacrylate compounds; dextranes containing repetitive patterns derived from polymerization of (meth)acrylate compounds; polypropylene fumarates and derivatives thereof; and combinations thereof.

3. A method according to claim 1, wherein the photopolymerization initiator is selected from the group consisting of aromatic ketones, acridine compounds and fluorone compounds.

4. A method according to claim 1, wherein the polymerization catalyst is an amine compound.

5. A method according to claim 1, wherein the solution further comprises one or several ingredients selected from the group consisting of stains, cross-linking agents, water and mixtures thereof.

6. A method according to claim 1, wherein light is applied to the solution through a stencil, delimiting zones with distinct light intensity.

7. A method according to claim 1, wherein light is applied according to a distribution with spatially modulated intensity.

8. A method according to claim 1, wherein the hydrogel type matrix comprises at least two contiguous zones with distinct stiffnesses with a stiffness gradient of the order of 1 kPa/μm.

9. A method according to claim 1, further comprising a matrix functionalization step after the photopolymerizing, using a protein inducing cell adhesion through integrins.

10. An in vitro cell adhesion method comprising: a) preparing a hydrogel type matrix according to claim 1; b) depositing cells of one or several types on the matrix obtained in step a), such that the cells of at least one of the types adhere entirely or partly to some zones of the matrix with higher stiffness than contiguous zones.

11. An in vitro cell shaping method comprising: a) preparing a hydrogel type matrix according to claim 1; b) depositing cells of one or several types on the matrix obtained in step a), such that the cells of at least one of the types are concentrated entirely or partly on some zones of the matrix with higher stiffness than contiguous zones and adopt the shape of said higher stiffness zones.

12. A method for preparing a hydrogel type matrix comprising at least two contiguous zones with distinct stiffnesses comprising photopolymerizing a solution in the presence of a polymerization catalyst, said solution comprising one or several polymerizable compounds and a photopolymerization initiator, by application of light onto the entire solution and at a different intensity in at least two zones of the solution, wherein a hydrogel matrix is obtained comprising at least two contiguous zones with distinct stiffnesses and, wherein the solution comprises acrylamide, a photopolymerization initiator with the following formula: ##STR00004## where Ph denotes a phenyl group, n-propylamine as the polymerization catalyst, N,N′-methylenebisacrylamide as the cross-linking agent and water.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows a view of the device used for example 1.

(2) FIG. 2a shows a chart illustrating the variation in the height h (in μm) of the matrix obtained for example 1 as a function of the distance d (in μm) (0 corresponding to the delimitation between the zone illuminated through the transparent zone of the stencil and the zone illuminated through the grey level zone).

(3) FIG. 2b shows a chart illustrating the variation of the stiffness Y (in kPa) of the matrix obtained for example 1 as a function of the distance d (in μm) (0 corresponding to the delimitation between the zone illuminated through the transparent zone of the stencil and the zone illuminated through the grey level zone).

(4) FIG. 2c shows a photograph of the matrix obtained for the example 1 after cell seeding.

(5) FIG. 3 shows a view of the device used for example 2.

(6) FIG. 4 shows a chart illustrating the variation of the stiffness Y (in kPa) of the matrix obtained for example 2 as a function of the distance d (in μm).

(7) FIG. 5 shows a photograph of the matrix obtained for example 2 after cell seeding.

(8) FIG. 6 shows a view of the device used for example 3.

(9) FIG. 7 shows a chart illustrating the variation of the stiffness Y (in kPa) of the matrix obtained in example 3 as a function of a distance d (in μm) relative to the centre of the channel of this matrix (this distance to the centre being equal to 0 at the abscissa).

(10) FIG. 8 shows a photograph of the matrix obtained for example 3 after cell seeding.

(11) FIG. 9 shows a view of the device used for example 4.

(12) FIG. 10 shows a chart illustrating the variation of the stiffness Y (in kPa) of the matrix obtained for example 4 as a function of the distance d (in μm).

(13) FIG. 11 shows a photograph of a top view of the matrix obtained for example 4.

(14) FIG. 12 shows a view of the device used for example 5.

(15) FIG. 13 is a photograph illustrating the cell positioning phenomenon with conformation of the cell geometry with the geometry of the pattern with higher stiffness than the zones surrounding this pattern, in accordance with example 5.

(16) FIG. 14 shows two enlarged views (a and b) of the matrix seen from above after cell seeding for example 6.

(17) FIG. 15 shows an enlarged view (with right and left inserts taken at different cell seeding durations) of the matrix seen from above after cell seeding for example 7.

(18) FIG. 16 shows an enlarged view of the matrix seen from above after cell seeding for example 8.

(19) FIG. 17 shows an enlarged view of the matrix seen from above after cell seeding for example 9.

DETAILED PRESENTATION OF PARTICULAR EMBODIMENTS

Example 1

(20) This example illustrates the preparation of a hydrogel matrix comprising two zones with different stiffnesses involving the following steps: a step to prepare a glass cover glass that will be used as a support for the hydrogel matrix (a); a step in which a stencil is prepared (b); a step in which the hydrogel matrix is prepared (c).

(21) a) Preparation of the Cover Glass

(22) A bottom cover glass is cleaned with a 0.1 mol/L soda solution for 10 minutes. It is then rinsed intensively with water and then ethanol. It is then immersed for 5 minutes in a 10% solution of APTMS (3-aminopropyltrimethoxysilane) in ethanol (5 ml APTMS for 45 mL ethanol), while stirring slowly. The cover glass is then rinsed with ethanol and kept for 10 minutes while stirring slowly in an ethanol bath. After simple rinsing with de-ionized water, the cover glass is immersed in a 0.5% glutaraldehyde solution in water for 25 minutes. It is then rinsed intensively with water and strongly dried. The result obtained is a cover glass with aldehyde functions on its surface, which will enable covalent grafting of hydrogel in polyacrylamide, which will be prepared subsequently.

(23) b) Preparation of a Stencil

(24) An optical microscopy slide (26 mm×76 mm) is washed in a solution of oxygenated water/concentrated sulfuric acid in proportion 1:1, for 20 minutes. A cleaved silicon wafer is fixed onto the left half of the slide. This wafer hides the transparent part before the deposit is made. 1 nm of titanium and then 4 nm of chromium are deposited by a Plassys type electron gun evaporator.

(25) The slide is then made hydrophobic by an Optool treatment (Daikin DSX): immersion for 1 minute in Optool diluted to 1:1000 in perfluorohexane. The slide is then left for 1 hour in steam at 80° C. It is finally immersed in perfluorohexane for 10 minutes while stirring slowly.

(26) The slide obtained is shown in FIG. 1 as reference 1, this slide comprising two zones: a transparent zone 3; a zone 5 covered with a 5 nm layer (1 nm of titanium and 4 nm of chromium) (called the grey level zone) contiguous with the transparent zone,

(27) this slide forming a stencil that will be inserted between a light flux (shown by an arrow in the figure indicating that it is a UV-A flux at 2 W/cm.sup.2) and the solution (shown as reference 7) deposited on the glass slide 9 mentioned above and that will be photocrosslinked in order to form the target matrix, namely a matrix with two contiguous zones with distinct stiffness, these zones corresponding to the geometry of the stencil.

(28) A second stencil is prepared in the same way as defined above, except that the so-called grey level zone is composed of a 1 nm layer of titanium on which 19 nm of chromium is deposited.

(29) c) Preparation of the Hydrogel Matrix

(30) Irgacure 819® (1.7 mg) is weighed in a flask opaque to ultraviolet rays. n-propylamine (10 μL) is added. The mixture is heated for 2 minutes at 50° C. After heating, the result obtained is a homogeneous and transparent solution. De-ionized water (490 μL), acrylamide (250 μL of a 40% solution) and N,N′-methylenebisacrylamide (250 μL of a 2% solution) are added. The complete mixture is homogenized gently with a pipette, to limit incorporation of oxygen.

(31) 30 μL of the solution obtained is placed on the 30 mm glass cover glass prepared according to the protocol described above. The cover glass is placed on a sample holder on which shims are placed to maintain a space of 40 μm between the cover glass and the previously prepared stencil deposited on the shims.

(32) The assembly (stencil, solution, cover glass) is illuminated by an Eleco UVP281 fiber lamp (2 W/cm.sup.2) for 20 seconds, as shown in FIG. 1. This assembly is then immersed in water to detach the stencil from the hydrogel matrix thus formed using pliers. The hydrogel matrix is rinsed 3 times with de-ionized water.

(33) Two tests were carried out using the same methods: a first test with the stencil comprising a grey level zone (composed of a layer composed of 1 nm of titanium and 4 nm of chromium); and a second test with a stencil comprising a grey level zone (composed of a layer of 1 nm of titanium and 19 nm of chromium).

(34) The surface topography and the stiffness of the matrix obtained were determined for both tests.

(35) The surface topography was determined by measuring the height of the matrix as a function of the distance d relative to the delimitation between the zone illuminated through the transparent zone (this delimitation being equal to 0 at the abscissa) and the zone illuminated through the grey level zone of the stencil, the results being shown in FIG. 2a that shows the variation in the height h (in μm) as a function of the distance d (in μm), this distance being defined in the upper part of the graph by the representation of zones of the stencil through which the illumination was made.

(36) A substantial reduction in the height of the matrix is observed both for the first test (curve a) and for the second test (curve b), when moving from the zone illuminated through the transparent zone of the stencil to the zone illuminated through the grey level zone of the stencil.

(37) The stiffness of the matrix obtained was determined by measuring the local stiffness of the hydrogel by atomic force microscopy (AFM) in aqueous medium (brand JPK). The resistance of the matrix to penetration of a point is recorded. A 400 μm scan is made through the boundary. The scans are made at a pitch of 10 μm. The result is a series of indentation curves. Each curve is processed using the manufacturer's protocol with an elastic indentation model. Each point on the graph is the average of 3 measurements made on 3 points at a spacing of 10 μm along a direction parallel to the boundary.

(38) The results are shown in FIG. 2b representing the variation of the stiffness Y (in kPa) as a function of the distance d (in μm) relative to the delimitation between the zone illuminated through the transparent zone (this delimitation being equal to the value 0 at the abscissa) and the zone illuminated through the grey level zone of the stencil.

(39) It is found that there is a substantial reduction in the stiffness both for the first test (curve a) and the second test (curve b) when moving from the zone illuminated through the transparent zone of the stencil (this illuminated zone can be qualified as a stiff zone) to the zone illuminated through the grey level zone of the stencil (this illuminated zone can be qualified as a soft zone), the stiffness gradient being of the order of 1 kPa/μm, particularly for the second test.

(40) A cell culture was also made on the matrices obtained above.

(41) Before starting, the matrix was functionalized with fibronectin. This was done by gluing the 30 mm diameter cover glass on which the hydrogel is polymerized to the bottom of a Petri dish in which a 35 mm diameter hole is drilled. Water is drawn out of the dish, 1 mg of Sulfo SDA (Pierce) (corresponding to succinimidyl-ester diazirine) is dissolved in 2.27 mL of sterile de-ionized water. The solution obtained is deposited on the matrix and is left for 2 hours in the dark. After this time, it is drawn in with the pipette and 2 mL of a 25 mg/mL fibronectin solution in sterile PBS is deposited for 1 hour. The dish is always kept in the dark. This solution is drawn in with the pipette and the dish is placed under the Eleco UVP281 lamp (2 W/cm.sup.2) for 3 min. The matrix is rinsed 3 times with sterile PBS (phosphate buffer salt).

(42) HUVEC cells (that correspond to mature human endothelial cells isolated from the umbilical cord vein) are seeded at a density of 2000 cells/cm.sup.2 in medium M199, 20% FCS (foetal calf serum), 2% LSGS (low serum growth supplement), 1% ATAM (antibiotics (penicillin, streptomycin)+antimycotic, Invitrogen).

(43) As can be seen in FIG. 2c which is a photograph taken by phase contrast microscopy, with 20× magnification on an inverted microscope 24 hours after seeding, there is a crossing of a delimitation cell between the soft zone and the stiff zone.

(44) The topographic profile does not influence the cell organization. Cells pass through the stiffness boundary by positioning themselves perpendicular to the boundary (as shown in FIG. 2c). If topography were to affect cell behavior, the cells should align themselves along the boundary, which is obviously not the case.

Example 2

(45) This example illustrates the preparation of a matrix comprising zones with distinct stiffness in the form of 10 μm lines alternated with 40 μm lines involving the following steps: a step to prepare a glass cover glass that will be used as a support for the matrix (a); a step in which a stencil is prepared (b); a step in which the hydrogel matrix is prepared (c).

(46) a) Preparation of the Cover Glass

(47) The cover glass is prepared in the same way as in example 1 described above.

(48) b) Preparation of a Stencil

(49) An optical microscopy slide (26 mm×76 mm) is washed in a 1:1 solution of oxygenated water/sulfuric acid for 20 minutes. 1 nm of titanium and then 9 nm of chromium are deposited on this slide, using a Plassys type electron gun evaporator. An AZ1512HS type resin (available from Clariant) diluted to 50% in its AZ-EB solvent (which is a propyleneglycolmonomethylether acetate solvent) is deposited on the chrome-plated side of the slide using a whirler, as a result of which a resin thickness of 600 nm is obtained. It is annealed for 2 minutes at 120° C. It is illuminated through a Sodalime stencil with the required pattern for 2 seconds (MJB4 aligner at 30 mW/cm.sup.2). After development (AZ-Developer+de-ionized water 1:1, 1 minute), the slide is rinsed intensively with de-ionized water.

(50) The slide is then placed in a DPS type etching reactor and is etched for 30 seconds using a chlorine treatment (⅔Cl.sub.2:⅓O.sub.2) at a pressure of between 10 and 25 mTorrs. The resin is removed by an O.sub.2 plasma for 30 seconds in the DPS reactor. The slide is then made hydrophobic by an Optool treatment (Daikin DSX): immersion for 1 minute in Optool diluted to 1:1000 in perfluorohexane. The slide is then left for 1 hour in steam at 80° C. It is finally immersed in perfluorohexane for 10 minutes while stirring slowly.

(51) The stencil is shown in FIG. 3 as reference 11, this stencil including an alternation between transparent zones 13 (not covered with titanium and chromium) consisting of 10 μm wide lines and zones 15 consisting of 40 μm wide lines covered with a 10 nm thick layer (1 nm of titanium and 9 nm of chromium) (these zones possibly being qualified as grey level zones).

(52) This stencil will be inserted between a light flux (shown by an arrow in the figure indicating that it is a UV-A flux at 2 W/cm.sup.2) and the solution (shown as reference 17) deposited on the glass slide 19 mentioned above and that will be photocrosslinked to form the target matrix, namely a matrix with contiguous zones with a distinct stiffness, namely 10 μm wide lines alternating with 40 μm wide lines.

(53) c) Preparation of the Hydrogel Matrix

(54) The hydrogel matrix is prepared in the same way as in example 1 described above.

(55) The stiffness of the matrix obtained was determined.

(56) This was done by measuring the local stiffness of the gel using an atomic force microscope in an aqueous medium (brand JPK). The resistance of the gel to penetration of the point is recorded. A 100 μm scan is made from the centre of the network of lines spaced at 40 μm. The scans are made at a pitch of 5 μm. The result is a series of indentation curves. Each curve is processed using the manufacturer's protocol with an elastic indentation model. Each point of the graph is obtained by taking the average of the measurements on two curves.

(57) The results are shown in FIG. 4 representing the variation of the stiffness Y (in kPa) as a function of a distance d (in μm). Abscissa point 0 corresponds approximately to the centre of the network.

(58) The shape of the curve confirms the presence of stiffness channels at a separation of about 40 μm, these stiffness channels corresponding to the zones illuminated through transparent zones in the above-mentioned stencil.

(59) A cell culture was also made on the matrices mentioned above.

(60) Before starting, the matrix was functionalized with fibronectin. This was done by gluing the 30 mm diameter cover glass on which the hydrogel is polymerized to the bottom of a Petri dish in which a 35 mm diameter hole is drilled. Water is drawn out of the dish, 1 mg of Sulfo SDA (Pierce) (corresponding to succinimidyl-ester diazirine) is dissolved in 2.27 mL of sterile de-ionized water. The solution obtained is deposited on the matrix and is left for 2 hours in the dark. After this time, it is drawn in with the pipette and 2 mL of a 25 mg/mL fibronectin solution in sterile PBS is deposited for 1 hour. The dish is always kept in the dark. This solution is drawn in with the pipette and the dish is placed under the Eleco UVP281 lamp (2 W/cm.sup.2) for 3 min. The matrix is rinsed 3 times with sterile PBS (phosphate buffer salt).

(61) LN229 cells (glioblastoma tumor cells extracted from the right parieto-occiputal frontal cortex) are seeded at a density of 5000 cells/cm.sup.2 in a DMEM medium (Eagle minimum essential medium modified by Dubelcco) to which 10% FBS and 1% ATAM are added.

(62) As can be seen in FIG. 5, that is a photograph made by phase contrast microscopy with magnification 20× on an inverted microscope 2 hours after seeding, there is a concentration of cells along the zones illuminated through transparent zones of the stencil, these zones corresponding to rigid zones (and corresponding to the above-mentioned stiffness channels).

Example 3

(63) This example illustrates preparation of a matrix comprising different stiffness zones involving the following steps: a step to prepare a glass cover glass that will be used as a support for the matrix (a); a step in which a stencil is prepared (b); a step in which the hydrogel matrix is prepared (c).

(64) a) Preparation of the Cover Glass

(65) The cover glass is prepared in the same way as in example 1 described above.

(66) b) Preparation of a Stencil

(67) An optical microscopy slide (26 mm×76 mm) is washed in a 1:1 solution of oxygenated water/sulfuric acid for 20 minutes. On this slide, an AZ1512HS type resin (available from Clariant) is deposited using a whirler at a rate of 2500 revolutions/min for 120 seconds. It is annealed for 2 minutes at 100° C. It is illuminated for 35 seconds with a 6 mW/cm.sup.2 UV lamp filtered on the 365 nm wavelength through a PDF stencil printed on a transparency and having a 100 μm wide black line. The pattern is developed using the AZ-EBR developer for 30 seconds. After development, the cover glass is once again annealed for 2 minutes at 120° C. 1 nm of titanium and then 4 nm of chromium are deposited using a Plassys type electron gun evaporator. The cover glass is then immersed for 1 minute in acetone to remove the resin line. The result is a 100 μm channel transparent to the medium of the chrome-plated cover glass.

(68) The cover glass is then made hydrophobic by an Optool treatment (Daikin DSX): immersion for 1 minute in Optool diluted to 1:1000 in perfluorohexane. The cover glass is then left for 1 hour in steam at 80° C. It is finally immersed in perfluorohexane for 10 minutes while stirring slowly.

(69) The stencil obtained is shown in FIG. 6 as reference 21, this stencil comprising a transparent zone 23 (not covered with titanium and chromium) consisting of a 100 μm wide line and two zones 25 surrounding the zone 23, these two zones being covered by a 5 nm thick layer (1 nm titanium and 4 nm chromium) (these zones possibly being qualified as grey level zones).

(70) This stencil will subsequently be inserted between a light flux (shown by an arrow in the figure indicating that it is a UV-A flux at 2 W/cm.sup.2) and the solution (shown as reference 27) deposited on the above-mentioned cover glass 29 and that will be photocrosslinked in order to form the target matrix, namely a matrix with contiguous zones with a distinct stiffness, namely in this case a central rigid zone surrounded by two soft end zones.

(71) c) Preparation of the Hydrogel Matrix

(72) The hydrogel matrix is prepared in the same was as in example 1 described above, except that the illumination duration is only 8 s.

(73) The stiffness of the matrix obtained was determined.

(74) This was done by measuring the local stiffness of the gel using an atomic force microscope in an aqueous medium (brand JPK). The resistance of the gel to penetration of the point is recorded. A 200 μm scan is made through the channel. The scans are made at a pitch of 10 μm. The result is a series of indentation curves. Each curve is processed using the manufacturer's protocol with an elastic indentation model. Each point on the graph is obtained by taking the average of two measurements made on two separate points at a distance of 10 μm parallel to the channel.

(75) The results are shown in FIG. 7 representing the variation of the stiffness Y (in kPa) as a function of a distance d (in μm) from the centre of the channel (the value of this distance at the centre being equal to 0 at the abscissa).

(76) Considering the shape of the curve, it can be seen that there is a stiffness channel corresponding to the zone illuminated through the transparent zone of the stencil.

(77) A cell culture was also made on the matrices obtained above.

(78) Preferably, the matrix is functionalized with fibronectin. This was done by gluing the 30 mm diameter cover glass on which the hydrogel is polymerized to the bottom of a Petri dish in which a 35 mm diameter hole is drilled. Water is drawn out of the dish, 1 mg of Sulfo SDA (Pierce) (corresponding to succinimidyl-ester diazirine) is dissolved in 2.27 mL of sterile de-ionized water. The solution obtained is deposited on the matrix and is left for 2 hours in the dark. After this time, it is drawn in with the pipette and 2 mL of a 25 mg/mL fibronectin solution in sterile PBS is deposited for 1 hour. The dish is always kept in the dark. This solution is drawn in with the pipette and the dish is placed under the Eleco UVP281 lamp (2 W/cm.sup.2) for 3 min. The matrix is rinsed 3 times with sterile PBS.

(79) Hela cells (immortal cells extracted from a malignant tumor of the cervix of the uterus) are seeded at a density of 5000 cells/cm.sup.2 in a DMEM medium to which 10% FBS and 1% ATAM are added.

(80) As can be seen in FIG. 8 that is a photograph made by phase contrast microscopy at a magnification of 20× on an inverted microscope 2 hours after seeding, there is a concentration of cells along the above-mentioned channel, this channel corresponding to the zone in the matrix with the highest stiffness.

Example 4

(81) This example illustrates the preparation of a matrix comprising zones with different stiffnesses in the form of a first network of 6 μm wide lines alternating with 2 μm wide lines and a second network of 4 μm wide lines alternating with 2 μm wide lines involving the following steps: a step to prepare a glass cover glass that will be used as a support for the matrix (a); a step in which a stencil is prepared (b); a step in which the hydrogel matrix is prepared (c).

(82) a) Preparation of the Cover Glass

(83) The cover glass is prepared in the same way as in example 1 described above.

(84) b) Preparation of a Stencil

(85) An optical microscopy slide (26 mm×76 mm) is cleaned in a 1:1 solution of oxygenated water/sulfuric acid for 20 minutes. 1 nm of titanium and then 4 nm of chromium are deposited on this cover glass, using a Plassys type electron gun evaporator. An AZ1512HS type resin (available from Clariant) diluted to 50% in its AZ-EBR solvent (which is a propyleneglycolmonomethylether acetate solvent) is deposited on the chrome-plated side of the slide using a whirler, as a result of which a resin thickness of 600 nm is obtained. It is annealed for 2 minutes at 120° C. It is illuminated through a Sodalime stencil with the required pattern for 2 seconds (MJB4 aligner at 30 mW/cm.sup.2). After development (AZ-Developer+de-ionized water 1:1, 1 minute), the slide is rinsed intensively with de-ionized water.

(86) The slide is then placed in a DPS type etching reactor and is etched for 30 seconds using a chlorine treatment (⅔Cl.sub.2:⅓O.sub.2) at a pressure of between 10 and 25 mTorrs. The resin is removed by an O.sub.2 plasma for 30 seconds in the DPS reactor. The slide is then made hydrophobic by an Optool treatment (Daikin DSX): immersion for 1 minute in Optool diluted to 1:1000 in perfluorohexane. The slide is then left for 1 hour in steam at 80° C. It is finally immersed in perfluorohexane for 10 minutes while stirring slowly.

(87) The stencil is shown in FIG. 9 as reference 31, this stencil comprising a first network of covered 6 μm wide lines 33 (called grey level lines) alternating with 2 μm wide lines 35 (transparent lines) and a second network of covered 4 μm wide lines 37 (called grey level lines) alternating with 2 μm wide lines 39 (transparent lines, in other words not covered with titanium and chromium).

(88) This stencil will be inserted between a light flux (shown by an arrow in the figure indicating that it is a UV-A flux at 2 W/cm.sup.2) and the solution (shown as reference 41) deposited on the glass slide 43 mentioned above and that will be photocrosslinked to form the target matrix, namely a matrix with a first network of 6 μm wide lines (soft lines) alternating with 2 μm wide lines (stiff lines) and a second network of 4 μm wide lines (soft lines) alternating with 2 μm wide lines (stiff lines).

(89) c) Preparation of the Hydrogel Matrix

(90) The hydrogel matrix is prepared in the same way as in example 1 described above.

(91) The stiffness of the matrix obtained was determined.

(92) This was done by measuring the local stiffness of the gel using an atomic force microscope in an aqueous medium (brand JPK). The resistance of the gel to penetration of the point is recorded. A 30 μm scan is made from the centre of the network of lines spaced at 6 μm. In exactly the same way, a 20 μm scan is made from the centre of the network of lines at a spacing of 4 μm. The scans are made at a pitch of 1 μm. The result is a series of indentation curves. Each curve is processed using the manufacturer's protocol with an elastic indentation model. Each point on the graph is the average of two measurements made on two points at a distance of 10 μm parallel to the lines.

(93) The results are shown in FIG. 10 representing the variation of the stiffness Y (in kPa) as a function of a distance d (in μm). The origin d=0 on each curve corresponds to the centers of the networks of lines spaced at 4 μm (grey dashed lines) and 6 μm (continuous black line) respectively. Two curves are shown: one curve a) for the first network of lines (alternation of soft 6 μm lines alternating with 2 μm rigid lines) and a curve b (alternation of 4 μm soft lines alternating with 2 μm rigid lines).

(94) The shape of these two curves confirms the presence of stiffness channels located at the rigid lines, these stiffness channels being at a spacing of 6 μm (curve a) or 4 μm (curve b), the stiffness gradient being very steep particularly for the first network of lines between rigid lines and soft lines, this arrangement being confirmed by FIG. 11 that shows a photograph of the matrix obtained seen from above.

Example 5

(95) This example illustrates the preparation of a hydrogel matrix comprising zones with distinct stiffness comprising the following patterns from left to right respectively: two adjacent lines each comprising three patterns in the shape of anchors two adjacent lines each comprising three patterns in the shape of triangles; and two adjacent lines each comprising three patterns in the form of dunce caps,

(96) said method including the following steps: a step to prepare a cover glass that will act as a support for the hydrogel matrix (a); a step in which a stencil is prepared (b); a step in which the hydrogel matrix is prepared (c).

(97) a) Preparation of the Cover Glass

(98) The cover glass is prepared in the same way as in example 1 described above.

(99) b) Preparation of a Stencil

(100) An optical microscopy slide (26 mm×76 mm) is washed in a 1:1 solution of oxygenated water/concentrated sulfuric acid for 20 minutes. 1 nm of titanium and then 9 nm of chromium are deposited on this slide using a Plassys type electron gun evaporator. An AZ1512HS type resin (available from Clariant) diluted to 50% in its AZ-EBR solvent (which is a propyleneglycolmonomethylether acetate solvent) is deposited on the chrome-plated side of the slide using a whirler turning at 3000 rpm for 30 seconds, as a result of which a resin thickness of 600 nm is obtained. It is illuminated through a Sodalime stencil with the required patterns. After development, the slide is placed in a DPS type etching reactor and is etched for 30 seconds using a chlorine treatment (⅔Cl.sub.2:⅓O.sub.2) at a pressure of between 10 and 25 mTorrs. The resin is removed by an O.sub.2 plasma for 30 seconds in the DPS reactor. The slide is then made hydrophobic by an Optool treatment (Daikin DSX): immersion for 1 minute in Optool diluted to 1:1000 in perfluorohexane. The slide is then left for 1 hour in steam at 80° C. It is finally immersed in perfluorohexane for 10 minutes while stirring slowly.

(101) The stencil is shown in FIG. 12 as reference 45, this stencil comprising from left to right, a first network of lines 47 each comprising three anchor shaped patterns (the dimensions and spacing of which are shown to the left of the stencil), a second network of lines 49 each comprising three right-angled triangle shaped patterns (the dimensions and spacing of which are shown in the lower part of the figure) and a third network of lines 51 each comprising three dunce cap shaped patterns (the dimensions and spacing of which are shown to the right of the stencil), the patterns in the first network, the second network and the third network being transparent, in other words not covered with titanium and chromium.

(102) This stencil will be inserted between a light flux (shown by an arrow on the figure indicating that it is a UV-A flux at 2 W/cm.sup.2) and the solution (shown as reference 53) deposited on the above-mentioned slide 55 that will be photocrosslinked in order to form the target matrix, namely a matrix with a first network of lines each comprising three anchor shaped patterns, a second network of lines each comprising three right angle triangle shaped patterns and a third network of lines each comprising three dunce cap shaped patterns.

(103) c) Preparation of the Hydrogel Matrix

(104) The hydrogel matrix is prepared in the same way as in example 1 described above.

(105) A cell culture was prepared on the matrices described above.

(106) REF52 cells were seeded uniformly on the above-mentioned matrix at a cell density of 5000 cells/cm.sup.2. Two hours after seeding, the cells were fixed using paraformaldehyde (PAF) at 4% containing 0.5% of triton for 10 minutes and then with PAF at 4% for 20 minutes. Actin is marked with phalloidin-Cy5 on the cells thus fixed and permeabilized, vinculin is marked with a monoclonal anti-vinculin antibody and then with a mouse anti-IdG antibody coupled with Alexa 488, while the nuclei are marked with Hoechst stain. After these fluorescent markings, cells are observed in phase contrast and fluorescence microscopy using a *60 lens on an inverted microscope.

(107) As shown in FIG. 13, the cells are localized on the above-mentioned patterns with the highest stiffness (in other words the anchor, right-angle triangle and dunce cap shaped patterns).

Example 6

(108) This example illustrates the preparation of a hydrogel matrix comprising zones with distinct stiffness with a network of 2 μm diameter pads at a spacing of 6 μm or a network of 5 μm diameter pads at a spacing of 7.5 μm, these pads having a stiffness of 15 kPa, while the space between the pads is composed of a soft matrix with a stiffness of 6 kPa.

(109) a) Preparation of the Cover Glass

(110) The cover glass is prepared in the same way as in example 1 described above.

(111) b) Preparation of a Stencil

(112) The stencil is prepared in the same way as in the examples described above.

(113) c) Preparation of the Hydrogel Matrix

(114) The hydrogel matrix is prepared in the same way as in the previous examples.

(115) A cell culture was made on the matrices obtained above.

(116) REF52 cells overexpressing paxiline coupled to YFP (symbolized as REF52-Pax-YFP) were seeded uniformly on the above-mentioned matrix at a cell density of 5000 cells/cm.sup.2. Two hours after seeding, the cells were fixed using paraformaldehyde (PAF) at 4% containing 0.5% triton for 10 minutes and then with PAF at 4% for 20 minutes. Actin is marked with phalloidin-Cy5 on the cells thus fixed and permeabilized, vinculin is marked with a monoclonal anti-vinculin antibody and then with a mouse anti-IdG antibody coupled with Alexa 488, while the nuclei are marked with Hoechst stain. After these fluorescent markings, cells are observed in phase contrast and fluorescence microscopy using a *60 lens on an inverted microscope.

(117) As shown in FIG. 14 illustrating an enlarged view of the matrices (part (a) for the matrix with 2 μm diameter pads or part (b) for the matrix with 5 μm diameter pads), the adhesion of cells takes place at focal contacts at which vinculin is collected, these focal contacts being produced only on the rigid pads (in other words the zones with the highest stiffness). When the distance between the pads increases, the cellular shape becomes increasingly constrained by the position of the focal contacts.

Example 7

(118) This example 7 is a variant of example 6 described above under similar conditions, except that the matrix contains pads with a stiffness of 12 kPa and a diameter of 6 μm, these pads being at a spacing of 9 μm, the space between the pads being composed of a soft matrix with a stiffness of 4 kPa.

(119) Images were taken every fifteen minutes as shown in FIG. 15 attached in the appendix. The adhesion of cells takes place at focal contacts that are set up only at the rigid pads at which paxiline-YFP collects.

(120) The left and right inserts show how the two focal contacts change with time. The focal contact in the left insert disintegrates progressively, while the focal contact in the right insert is reinforced. These effects result in a cellular migration from the left towards the right.

Example 8

(121) This example is aimed at highlighting the modulation of the cell shape by varying the spacing of rigid pads.

(122) It forms a variant of example 6 mentioned above and performed under similar conditions, except that the matrix comprises pads with a stiffness of 6 kPa and a diameter of 10 μm, these pads being at a spacing of 10 μm, the space between the pads being composed of a soft matrix with a stiffness of 1 kPa (stiffness value less than the cell survival threshold).

(123) As shown in FIG. 16, a REF52 cell has a triangular shape defined by the distribution of pads. Note that only the cells that were directly deposited on the patterns or were at a distance less than the cell size (of the order of 10 μm) survived and got organized on the rigid pads.

Example 9

(124) This example forms a variant of example 6 mentioned above performed under similar conditions, except that the matrix comprises pads with a stiffness of 15 kPa and a diameter of 3 μm, these pads being at a spacing of 1 μm, the space between the pads consisting of a soft matrix with a stiffness of 5 kPa.

(125) As shown in FIG. 17, the cell shape is completely deformed.

(126) It can be deduced from examples 7 to 9 mentioned above that illustrate matrices with rigid patterns with a size smaller than the cell size, that the pattern size limits the growth of adhesive sites and the pattern density influences the cell geometry. Consequently, the choice of the size and spacing of patterns influence firstly the geometry of the cell and secondly contractile forces applied by the cell.